• Don't forget to check out Neurotransmitter.net's research index for a list of dozens of other resources created by Shawn Thomas.
• The abstracts are retrieved from PubMed Central and PubMed on a daily basis.
• Free full text articles are also available for the following subjects: biology, neuroscience, genetics and proteomics, psychiatry, pharmacology, endocrinology, microbiology, cancer, biotechnology, informatics, and miscellaneous.
  
• You can jump directly to articles from Molecular Biology of the Cell, Molecular and Cellular Biology, Journal of Cell Science, Journal of Cellular and Molecular Medicine, Molecules and Cells, Cell Research, Cellular & Molecular Biology Letters, BMC Cell Biology, Cell Communication and Signaling, Eukaryotic Cell, The Journal of Biological Chemistry, Bioscience, Biotechnology, Biochemistry, Experimental & Molecular Medicine, BMC Biochemistry, BMC Chemical Biology, and BMC Molecular Biology.
• You can view earlier articles from these journals as well: skip 0, skip 60, skip 90, or skip 120 of the most recent articles.
• Confused by the terminology? Try searching The On-Line Medical Dictionary (OMD).

Web www.neurotransmitter.net

---

Recent Articles in Molecular Biology of the Cell

---

Destaing O, Sanjay A, Itzstein C, Horne WC, Toomre D, Camilli PD, Baron R
The Tyrosine Kinase Activity of c-Src Regulates Actin Dynamics and Organization of Podosomes in Osteoclasts.
Mol Biol Cell. 2007 Oct 31; .
Monitoring Editor: Joan Brugge Podosomes are dynamic actin-rich structures composed of a dense F-actin core surrounded by a cloud of more diffuse F-actin. Src performs one or more unique functions in osteoclasts (OCLs), and podosome belts and bone resorption are impaired in the absence of Src. Using Src(-/-) OCLs, we investigated the specific functions of Src in the organization and dynamics of podosomes. We found that podosome number and the podosome-associated actin cloud were decreased in Src(-/-) OCLs. Videomicroscopy and fluorescence recovery after photobleaching (FRAP) analysis revealed that the life span of Src(-/-) podosomes was increased fourfold and that the rate of actin flux in the core was decreased by 40%. Thus, Src regulates the formation, structure, life span and rate of actin polymerization in podosomes and in the actin cloud. Rescue of Src(-/-) OCLs with Src mutants showed that both the kinase activity and either the SH2 or the SH3 binding domain are required for Src to restore normal podosome organization and dynamics. Moreover, inhibition of Src family kinase activities in Src(-/-) OCLs by Src inhibitors or by expressing dominant-negative Src(K295M) induced the formation of abnormal podosomes. Thus, Src is an essential regulator of podosome structure, dynamics and organization. [Abstract/Link to Full Text]

Matzat LH, Berberoglu S, Lévesque L
Formation of a Tap/NXF1 Homotypic Complex Is Mediated Through the Amino-Terminal Domain of Tap and Enhances Interaction with Nucleoporins.
Mol Biol Cell. 2007 Oct 31;
Monitoring Editor: Karsten Weis Nuclear export of mRNAs is mediated by the Tap/Nxt1 pathway. Tap moves its RNA cargo through the NPC by direct interaction with nucleoporin FG repeats. This interaction is strengthened by the formation of a Tap/Nxt1 heterodimer. We now present evidence that Tap can form a multimeric complex with itself and with other members of the NXF family. We also show that the homotypic Tap complex can interact with both Nxt1 and nucleoporins in vitro. The region mediating this oligomerization is localized to the first 187 amino acids of Tap, which overlaps with its RNA-binding domain. Removal of this domain greatly reduces the ability of Tap to bind nucleoporins in vitro and in vivo. This is the first report showing that the Tap amino terminus modulates the interaction of Tap with nucleoporins. We speculate that this mechanism has a regulatory role for RNA export independent of RNA-binding. [Abstract/Link to Full Text]

Strickfaden SC, Pryciak PM
Distinct Roles for Two G{alpha}-G{beta} Interfaces in Cell Polarity Control by a Yeast Heterotrimeric G Protein.
Mol Biol Cell. 2007 Oct 31;
Monitoring Editor: Daniel Lew S. cerevisiae mating pheromones trigger dissociation of a heterotrimeric G protein (Galphabetagamma) into Galpha-GTP and Gbetagamma. The Gbetagamma dimer regulates both MAP kinase cascade signaling and cell polarization. Here, by independently activating the MAP kinase pathway, we studied the polarity role of Gbetagamma in isolation from its signaling role. MAP kinase signaling alone could induce cell asymmetry but not directional growth. Surprisingly, active Gbetagamma, either alone or with Galpha-GTP, could not organize a persistent polarization axis. Instead, following pheromone gradients (chemotropism) or directional growth without pheromone gradients (de novo polarization) required an intact receptor-Galphabetagamma module and GTP hydrolysis by Galpha. Our results indicate that chemoattractant-induced cell polarization requires continuous receptor-Galphabetagamma communication but not modulation of MAP kinase signaling. To explore regulation of Gbetagamma by Galpha, we mutated Gbeta residues in two structurally-distinct Galpha-Gbeta binding interfaces. Polarity control was disrupted only by mutations in the N-terminal interface, and not the Switch interface. Incorporation of these mutations into a Gbeta-Galpha fusion protein, which enforces subunit proximity, revealed that Switch interface dissociation regulates signaling, whereas the N-terminal interface may govern receptor-Galphabetagamma coupling. These findings raise the possibility that the Galphabetagamma heterotrimer can function in a partially-dissociated state, tethered by the N-terminal interface. [Abstract/Link to Full Text]

Gurda GT, Guo L, Lee SH, Molkentin JD, Williams JA
Cholecystokinin (CCK) Activates Pancreatic Calcineurin-NFAT Signaling In Vitro and In Vivo.
Mol Biol Cell. 2007 Oct 31;
Monitoring Editor: J. Silvio Gutkind Elevated endogenous CCK release induced by protease inhibitors leads to pancreatic growth. This response has been shown to be mediated by the phosphatase calcineurin, but its downstream effectors are unknown. Here we examined activation of calcineurin-regulated Nuclear Factor of Activated T-cells (NFATs) in isolated acinar cells, as well as in an in vivo model of pancreatic growth. Western blotting of endogenous NFATs and confocal imaging of NFATc1-GFP in pancreatic acini showed that CCK dose-dependently stimulated NFAT translocation from the cytoplasm to the nucleus within 0.5-1 h. This shift in localization correlated with CCK-induced activation of NFAT-driven luciferase reporter and was similar to that induced by a calcium ionophore and constitutively active calcineurin. The effect of CCK was dependent on calcineurin, as these changes were blocked by immunosuppressants FK506 and CsA and by overexpression of the endogenous protein inhibitor CAIN. Parallel NFAT activation took place in vivo. Pancreatic growth was accompanied by an increase in nuclear NFATs and subsequent elevation in expression of NFAT-luciferase in the pancreas, but not in organs unresponsive to CCK. The changes also required calcineurin, as they were blocked by FK506. We conclude that CCK activates NFATs in a calcineurin-dependent manner, both in vitro and in vivo. [Abstract/Link to Full Text]

Jin M, Cai M
A Novel Function of Arp2p in Mediating Prk1p-specific Regulation of Actin and Endocytosis in Yeast.
Mol Biol Cell. 2007 Oct 31;
Monitoring Editor: Howard Riezman The yeast protein Pan1p plays essential roles in actin cytoskeleton organization and endocytosis. It couples endocytosis with actin polymerization through its dual function in endocytic complex assembly and activation of the actin polymerization initiation complex Arp2/3p. Phosphorylation of Pan1p and other components of the endocytic complex by the kinase Prk1p leads to disassembly of the coat complex and the termination of vesicle-associated actin polymerization. A homologous kinase, Ark1p, has also been implicated in this regulatory process. In this study, we investigated the distinct roles of Prk1p and Ark1p. We found that the nonkinase domains determined the functional specificity of the two kinases. A short region located adjacent to the kinase domain unique to Prk1p was found to be required for the kinase to interact with Arp2p. Further studies demonstrated that the Prk1p-Arp2p interaction is critical for down-regulation of Pan1p. These findings reveal that, in addition to its role in the nucleation of actin polymerization, Arp2p also mediates what appears to be an auto-regulatory mechanism possibly adapted for efficient coordination of actin assembly and disassembly during endocytosis. [Abstract/Link to Full Text]

Wang H, Dictenberg J, Ku L, Li W, Bassell GJ, Feng Y
Dynamic Association of the Fragile X Mental Retardation Protein as an mRNP between Microtubules and Polyribosomes.
Mol Biol Cell. 2007 Oct 31;
Monitoring Editor: Marvin P. Wickens The fragile X mental retardation protein (FMRP) is a selective RNA-binding protein that regulates translation and plays essential roles in synaptic function. FMRP is bound to specific mRNA ligands, actively transported into neuronal processes in a microtubule-dependent manner, and associated with polyribosomes engaged in translation elongation. However, the biochemical relationship between FMRP-microtubule association and FMRP-polyribosome association remains elusive. Here we report that although the majority of FMRP is incorporated into elongating polyribosomes in the soluble cytoplasm, microtubule-associated FMRP is predominantly retained in translationally dormant, polyribosome-free messenger ribonucleoprotein (mRNP) complexes. Interestingly, FMRP-microtubule association is increased when mRNPs are dynamically released from polyribosomes as a result of inhibiting translation initiation. Furthermore, the I304N mutant FMRP that fails to be incorporated into polyribosomes is associated with microtubules in mRNP particles and transported into neuronal dendrites in a microtubule-dependent, DHPG-stimulated manner with similar kinetics to that of wild type FMRP. Hence, polyribosome-free FMRP-mRNP complexes travel on microtubules and wait for activity-dependent translational de-repression at the functional destination. The dual participation of FMRP in dormant mRNPs and polyribosomes suggests distinct roles of FMRP in dendritic transport and translational regulation, two distinct phases that control local protein production to accommodate synaptic plasticity. [Abstract/Link to Full Text]

Sakaguchi M, Sonegawa H, Murata H, Kitazoe M, Futami JI, Kataoka K, Yamada H, Huh NH
S100A11, an Dual Mediator for Growth Regulation of Human Keratinocytes.
Mol Biol Cell. 2007 Oct 31;
Monitoring Editor: M. Bishr Omary We previously revealed a novel signal pathway involving S100A11 for inhibition of the growth of normal human keratinocytes (NHK) caused by high Ca(++) or TGFbeta (ProNAS USA, 102:13921-13926, 2005). Exposure to either agent resulted in transfer of S100A11 to nuclei, where it induced p21(WAF1). On the other hand, S100A11 has been shown to be overexpressed in many human cancers. To address this apparent discrepancy, we analyzed possible new functions of S100A11 and provide herewith evidence that 1) S100A11 is actively secreted by NHK, 2) extracellular S100A11 acts on NHK to enhance the production of EGF family proteins, resulting in growth stimulation, 3) RAGE (receptor for advanced glycation endproducts), NF-kappaB, Akt, and CREB are involved in the S100A11-triggered signal transduction, and 4) production and secretion of S100A11 are markedly enhanced in human squamous cancer cells. These findings indicate that S100A11 plays an dual role in growth regulation of epithelial cells. [Abstract/Link to Full Text]

Chan NC, Lithgow T
The Peripheral Membrane Subunits of the SAM Complex Function Co-dependently in Mitochondrial Outer Membrane Biogenesis.
Mol Biol Cell. 2007 Oct 31;
Monitoring Editor: Thomas Fox The SAM complex functions in the assembly of beta-barrel proteins into the mitochondrial outer membrane. It is related to the Omp85/YaeT machinery in bacterial outer membranes, but the eukaryotic SAM complex is distinguished by two peripheral subunits, Sam37 and Sam35, that sit on the cytosolic face of the complex. The function of these subunits in beta-barrel protein assembly is currently unclear. By screening a library of sam35 mutants, we show that thirteen distinct alleles were each specifically suppressed by overexpression of SAM37. Two of these mutants, sam35-409 and sam35-424, show distinct phenotypes that enable us to distinguish the function of Sam35 from that of Sam37. Sam35 is required in order for the SAM complex to bind outer membrane substrate proteins: destabilisation of Sam35 inhibits substrate binding by Sam50. Sam37 acts later than Sam35, apparently to assist release of substrates from the SAM complex. Very different environments surround bacteria and mitochondria, and we discuss the role of Sam35 and Sam37 in terms of the problems peculiar to mitochondrial protein substrates. [Abstract/Link to Full Text]

Müller JM, Milenkovic D, Guiard B, Pfanner N, Chacinska A
Precursor Oxidation by Mia40 and Erv1 Promotes Vectorial Transport of Proteins into the Mitochondrial Intermembrane Space.
Mol Biol Cell. 2007 Oct 31;
Monitoring Editor: Donald Newmeyer The mitochondrial intermembrane space contains chaperone complexes that guide hydrophobic precursor proteins through this aqueous compartment. The chaperones consist of hetero-oligomeric complexes of small Tim proteins with conserved cysteine residues. The precursors of small Tim proteins are synthesized in the cytosol. Import of the precursors requires the essential intermembrane space proteins Mia40 and Erv1 that were proposed to form a relay for disulfide formation in the precursor proteins. However, experimental evidence for a role of Mia40 and Erv1 in the oxidation of intermembrane space precursors has been lacking. We have established a system to directly monitor the oxidation of precursors during import into mitochondria and dissected distinct steps of the import process. Reduced precursors bind to Mia40 during translocation into mitochondria. Both Mia40 and Erv1 are required for formation of oxidized monomers of the precursors that subsequently assemble into oligomeric complexes. While the reduced precursors can diffuse back into the cytosol, the oxidized precursors are retained in the intermembrane space. Thus oxidation driven by Mia40 and Erv1 determines vectorial transport of the precursors into the mitochondrial intermembrane space. [Abstract/Link to Full Text]

Rapoport I, Boll W, Yu A, Böcking T, Kirchhausen T
A Motif in the Clathrin Heavy Chain Required for the Hsc70/Auxilin Uncoating Reaction.
Mol Biol Cell. 2007 Oct 31;
Monitoring Editor: Sandra Schmid The Hsc70 chaperone is an ATP-dependent "disassembly enzyme" for many subcellular structures, including clathrin-coated vesicles where it functions as an uncoating ATPase. Hsc70, and its cochaperone, auxilin, together catalyze coat disassembly. Like other members of the Hsp70 chaperone family, it is believed that ATP-bound Hsc70 recognizes the clathrin triskelion through an unfolded exposed hydrophobic segment. The best candidate is the unstructured C-terminus (residues 1631-1675) of the heavy chain at the foot of the tripod below the hub, containing a sequence motif, QLMLT, closely related to the sequence bound preferentially by the substrate groove of Hsc70 (Fotin et al., 2004b). To test this hypothesis, we generated in insect cells recombinant mammalian triskelions that in vitro form clathrin cages and clathrin/AP-2 coats exactly like those assembled from native clathrin. We show that coats assembled from recombinant clathrin are good substrates for ATP- and auxilin-dependent, Hsc70 catalyzed uncoating. Finally, we show that this uncoating reaction proceeds normally when the coats contain recombinant heavy chains truncated C-terminal to the QLMLT motif, but very inefficiently when the motif is absent. Thus, the QLMLT motif is required for Hsc-70 facilitated uncoating, consistent with the proposal that this sequence is a specific target of the chaperone. [Abstract/Link to Full Text]

Kollman JM, Zelter A, Muller EG, Fox B, Rice LM, Davis TN, Agard DA
The Structure of the {gamma}-Tubulin Small Complex: Implications of Its Architecture and Flexibility for Microtubule Nucleation.
Mol Biol Cell. 2007 Oct 31;
Monitoring Editor: Tim Stearns The gamma-tubulin small complex (gamma-TuSC) is an evolutionarily conserved heterotetramer essential for microtubule nucleation. We have determined the structure of the S. cerevisiae gamma-TuSC at 25 A resolution by electron microscopy. gamma-TuSC is Y-shaped, with an elongated body connected to two arms. Gold labeling showed that the two gamma-tubulins are located in lobes at the ends of the arms, and the relative orientations of the other gamma-TuSC components were determined by in vivo FRET. The structures of different subpopulations of gamma-TuSC indicate flexibility in the connection between a mobile arm and the rest of the complex, resulting in variation of the relative positions and orientations of the gamma-tubulins. In all of the structures the gamma-tubulins are distinctly separated, a configuration incompatible with the microtubule lattice. The separation of the gamma-tubulins in isolated gamma-TuSC likely plays a role in suppressing its intrinsic microtubule nucleating activity, which is relatively weak until the gamma-TuSC is incorporated into higher-order complexes or localized to microtubule organizing centers. We propose that further movement of the mobile arm is required to bring the gamma-tubulins together in microtubule-like interactions, and provide a template for microtubule growth. [Abstract/Link to Full Text]

Roberts TM, Waris Zaidi I, Vaisica JA, Peter M, Brown GW
Regulation of Rtt107 Recruitment to Stalled DNA Replication Forks by the Cullin Rtt101 and the Rtt109 Acetyltransferase.
Mol Biol Cell. 2007 Oct 31;
Monitoring Editor: Wendy Bickmore RTT107 (ESC4, YHR154W) encodes a BRCA1 C-terminal-domain protein that is important for recovery from DNA damage during S phase. Rtt107 is a substrate of the checkpoint kinase Mec1 and forms complexes with DNA repair enzymes including the nuclease subunit Slx4, but the role of Rtt107 in the DNA damage response remains unclear. We find that Rtt107 interacts with chromatin when cells are treated with compounds that cause replication forks to arrest. This damage-dependent chromatin binding requires the acetyltransferase Rtt109, but does not require acetylation of the known Rtt109 target, histone H3-K56. Chromatin binding of Rtt107 also requires the cullin Rtt101, which appears to play a direct role in Rtt107 recruitment as the two proteins are found in complex with each other. Finally, we provide evidence that Rtt107 is bound at or near stalled replication forks in vivo. Together these results indicate that Rtt109, Rtt101, and Rtt107, which genetic evidence suggests are functionally related, form a DNA damage response pathway that recruits Rtt107 complexes to damaged or stalled replication forks. [Abstract/Link to Full Text]

Goulimari P, Knieling H, Engel U, Grosse R
LARG and mDia1 Link G{alpha}12/13 to Cell Polarity and Microtubule Dynamics.
Mol Biol Cell. 2007 Oct 24;
Monitoring Editor: Martin A. Schwartz Regulation of cell polarity is a process observed in all cells. During directed migration, cells orientate their microtubule cytoskeleton and the microtubule-organizing-center (MTOC), which involves integrins and downstream Cdc42 and GSK-3beta activity. However, the contribution of G protein-coupled receptor signal transduction for MTOC polarity is less well understood. Here we report that the heterotrimeric Galpha12 and Galpha13 proteins are necessary for MTOC polarity as well as microtubule dynamics based on studies using Galpha12/13-deficient mouse embryonic fibroblasts. Cell polarization involves the Galpha12/13-interacting leukemia-associated Rho-GEF (LARG) and the actin nucleating diaphanous formin mDia1. Interestingly, LARG associates with pericentrin and localizes to the MTOC and along microtubule tracks. We propose that Galpha12/13 proteins exert essential functions linking extracellular signals to microtubule dynamics and cell polarity via RhoGEF and formin activity. [Abstract/Link to Full Text]

Stevens TL, Rogers EM, Koontz LM, Fox DT, Homem CC, Nowotarski SH, Artabazon NB, Peifer M
Using Bcr-Abl to Examine Mechanisms by which Abl Kinase Regulates Morphogenesis in Drosophila.
Mol Biol Cell. 2007 Nov 7;
Monitoring Editor: Jean Schwarzbauer Signaling by the nonreceptor tyrosine kinase Abelson (Abl) plays key roles in normal development, while its inappropriate activation helps trigger the development of several forms of leukemia. Abl is best known for its roles in axon guidance but Abl and its relatives also help regulate embryonic morphogenesis in epithelial tissues. Here we explore the role of regulation of Abl kinase activity during development. We first compare the subcellular localization of Abl protein and of active Abl, using a phospho-specific antibody, providing a catalogue of places where Abl is activated. Next we explore the consequences for morphogenesis of overexpressing wild-type Abl or expressing the activated form found in leukemia, Bcr-Abl. We find dose-dependent effects of elevating Abl activity on morphogenetic movements such as head involution and dorsal closure, on cell shape changes, on cell protrusive behavior, and on the organization of the actin cytoskeleton. Most of the effects of Abl activation parallel those caused by reduction in function of its target Enabled. Abl activation leads to changes in Enabled phosphorylation and localization, suggesting a mechanism of action. These data provide new insights into how regulated Abl activity helps direct normal development and into possible biological functions of Bcr-Abl. [Abstract/Link to Full Text]

Gehrig K, Cornell RB, Ridgway ND
Expansion of the Nucleoplasmic Reticulum Requires the Coordinated Activity of Lamins and CTP:Phosphocholine Cytidylyltransferase (CCT) {alpha}
Mol Biol Cell. 2007 Oct 31;
Monitoring Editor: Jennifer Lippincott-Schwartz The nucleoplasmic reticulum (NR), a nuclear membrane network implicated in signaling and transport, is formed by the biosynthetic and membrane curvature-inducing properties of the rate-limiting enzyme in phosphatidylcholine synthesis, CTP:phosphocholine cytidylyltransferase (CCT)alpha. The NR is formed by invagination of the nuclear envelope and has an underlying lamina that may contribute to membrane tubule formation or stability. In this study we investigated the role of lamins A and B in NR formation in response to expression and activation of endogenous and fluorescent protein-tagged CCTalpha. Similarly to endogenous CCTalpha, CCT-green fluorescent protein (GFP) reversibly translocated to nuclear tubules projecting from the NE in response to oleate, a lipid promoter of CCT membrane binding. Coexpression and RNA interference experiments revealed that both CCTalpha and lamin A and B were necessary for NR proliferation. Expression of CCT-GFP mutants with compromised membrane binding affinity produced fewer nuclear tubules, indicating that the membrane-binding function of CCTalpha promotes the expansion of the NR. Proliferation of atypical bundles of nuclear membrane tubules by a CCTalpha mutant that constitutively associated with membranes revealed that expansion of the double-bilayer NR requires the coordinated assembly of an underlying lamin scaffold and induction of membrane curvature by CCTalpha. [Abstract/Link to Full Text]

Fong KW, Choi YK, Rattner JB, Qi RZ
CDK5RAP2 Is a Pericentriolar Protein That Functions in Centrosomal Attachment of the {gamma}-Tubulin Ring Complex.
Mol Biol Cell. 2007 Oct 24;
Monitoring Editor: Stephen Doxsey Microtubule nucleation and organization by the centrosome require gamma-tubulin, a protein that exists in a macromolecular complex called the gamma-tubulin ring complex (gammaTuRC). We report characterization of CDK5RAP2, a novel centrosomal protein whose mutations have been linked to autosomal recessive primary microcephaly. In somatic cells, CDK5RAP2 localizes throughout the pericentriolar material in all stages of the cell cycle. When overexpressed, CDK5RAP2 assembled a subset of centrosomal proteins including gamma-tubulin onto the centrosomes or under the microtubule-disrupting conditions into microtubule-nucleating clusters in the cytoplasm. CDK5RAP2 associates with the gammaTuRC via a short conserved sequence present in several related proteins found in a range of organisms from fungi to mammals. The binding of CDK5RAP2 is required for gammaTuRC attachment to the centrosome but not for gammaTuRC assembly. Perturbing CDK5RAP2 function delocalized gamma-tubulin from the centrosomes and inhibited centrosomal microtubule nucleation, thus leading to disorganization of interphase microtubule arrays and formation of anastral mitotic spindles. Together, CDK5RAP2 is a pericentriolar structural component that functions in gammaTuRC attachment and therefore in the microtubule organizing function of the centrosome. Our findings suggest that centrosome malfunction due to the CDK5RAP2 mutations may underlie autosomal recessive primary microcephaly. [Abstract/Link to Full Text]

Lladó A, Timpson P, Vilŕ de Muga S, Moretó J, Pol A, Grewal T, Daly RJ, Enrich C, Tebar F
PKC{delta} and Calmodulin Regulate EGF Receptor Recycling from Early Endosomes through Arp2/3 Complex and Cortactin.
Mol Biol Cell. 2007 Oct 24;
Monitoring Editor: Adam Linstedt The intracellular trafficking of the epidermal growth factor receptor (EGFR) is regulated by a cross-talk between calmodulin (CaM) and protein kinase C-delta (PKCdelta). On inhibition of CaM, PKCdelta promotes the formation of enlarged early endosomes and blocks EGFR recycling and degradation. Here we show that PKCdelta impairs EGFR trafficking due to the formation of an F-actin coat surrounding early endosomes. The PKCdelta-induced polymerization of actin is orchestrated by the Arp2/3 complex and requires the interaction of cortactin with PKCdelta. Accordingly, inhibition of actin polymerization utilizing cytochalasin D or by overexpression of active cofilin, restored the normal morphology of the organelle and the recycling of EGFR. Similar results were obtained after down-regulation of cortactin and the sequestration of the Arp2/3 complex. Furthermore we demonstrate an interaction of cortactin with CaM and PKCdelta, the latter being dependent on CaM inhibition. In summary, this study provides the first evidence that CaM and PKCdelta organize actin dynamics in the early endosomal compartment, thereby regulating the intracellular trafficking of EGFR. [Abstract/Link to Full Text]

Shrivastava-Ranjan P, Faundez V, Fang G, Rees H, Lah JJ, Levey AI, Kahn RA
Mint3/X11{gamma} Is an Arf-dependent Adaptor that Regulates the Traffic of the Alzheimer's Precursor Protein from the TGN.
Mol Biol Cell. 2007 Oct 24;
Monitoring Editor: Vivek Malhotra beta-amyloid peptides (Abeta) are the major component of plaques in brains of Alzheimer's patients and are derived from the proteolytic processing of the beta-amyloid precursor protein (APP). The movement of APP between organelles is highly regulated and is tightly connected to its processing by secretases. We proposed previously that transport of APP within the cell is mediated in part through its sorting into Mint/X11-containing carriers. To test our hypothesis, we purified APP-containing vesicles from human neuroblastoma SH-SY5Y cells and show that Mint2/3 are specifically enriched and that Mint3 and APP are present in the same vesicles. Increasing cellular APP levels increased the amounts of both APP and Mint3 in purified vesicles. Additional evidence supporting an obligate role for Mint3 in traffic of APP from the TGN to the plasma membrane include the observations that depletion of Mint3 by siRNA or mutation of the Mint binding domain of APP changes the export route of APP from the basolateral to the endosomal/lysosomal sorting route. Finally, we show that increased expression of Mint3 decreased and siRNA mediated knockdowns increased the secretion of the neurotoxic beta-amyloid peptide, Abeta1-40. Taken together, our data implicate Mint3 activity as a critical determinant of post-Golgi APP traffic. [Abstract/Link to Full Text]

Kohn KW, Aladjem MI, Weinstein JN, Pommier Y
Chromatin Challenges during DNA Replication: A Systems Representation.
Mol Biol Cell. 2007 Oct 24;
Monitoring Editor: Gerard Evan In a recent review, Groth et al. (Groth et al., 2007) presented a comprehensive account of nucleosome disassembly in front of a DNA replication fork, assembly behind the replication fork, and the copying of epigenetic information onto the replicated chromatin. Understanding those processes however would be enhanced by a comprehensive graphical depiction analogous to a circuit diagram. Accordingly, we have constructed a molecular interaction map (MIM) (Kohn et al., 2006b) that preserves in essentially complete detail the processes described by Groth et al. The MIM organizes and elucidates the information presented by Groth et al. on the complexities of chromatin replication, thereby providing a tool for system-level comprehension of the effects of genetic mutations, altered gene expression, and pharmacologic intervention. [Abstract/Link to Full Text]

Wälchli S, Skĺnland SS, Gregers TF, Lauvrak SU, Torgersen ML, Ying M, Kuroda S, Maturana A, Sandvig K
The MAP Kinase p38 Links Shiga Toxin Dependent Signaling and Trafficking.
Mol Biol Cell. 2007 Oct 24;
Monitoring Editor: Jennifer Lippincott-Schwartz Shiga toxin (Stx) binds to the cell and is transported via endosomes and the Golgi apparatus to the endoplasmic reticulum and cytosol, where it exerts its toxic effect. We have recently shown that Stx activates the tyrosine kinase Syk, which in turn induces clathrin phosphorylation and upregulates Stx uptake. Here, we show that toxin-induced signaling can also regulate another step in intracellular Stx transport. We demonstrate that transport of Stx to the Golgi apparatus is dependent on the MAP kinase p38. Treatment of cells with chemical inhibitors or siRNA targeting p38, inhibited Stx transport to the Golgi and reduced Stx toxicity. This p38 dependency is specific to Stx since transport of the related toxin ricin was not affected by p38 inhibition. Stx rapidly activated p38, and recruited it to early endosomes in a Ca(2+)-dependent manner. Furthermore, agonist-induced oscillations in cytosolic Ca(2+) levels were inhibited upon Stx stimulation, possibly reflecting Stx-dependent local alterations in cytosolic Ca(2+) levels. Intracellular transport of Stx is Ca(2+)-dependent, and we provide evidence that Stx activates a signaling cascade involving cross-talk between Ca(2+) and p38, to regulate its trafficking to the Golgi apparatus. [Abstract/Link to Full Text]

Alder NN, Sutherland J, Buhring AI, Jensen RE, Johnson AE
Quaternary Structure of the Mitochondrial TIM23 Complex Reveals Dynamic Association between Tim23p and Other Subunits.
Mol Biol Cell. 2007 Oct 24;
Monitoring Editor: Reid Gilmore Tim23p is an essential channel-forming component of the multi-subunit TIM23 complex of the mitochondrial inner membrane that mediates protein import. Radiolabeled Tim23p monocysteine mutants were imported in vitro, incorporated into functional TIM23 complexes, and subjected to chemical cross-linking. Three regions of proximity between Tim23p and other subunits of the TIM23 complex were identified: Tim17p and the first transmembrane segment of Tim23p; Tim50p and the C-terminal end of the Tim23p hydrophilic region; and the entire hydrophilic domains of Tim23p molecules. These regions of proximity reversibly change in response to changes in membrane potential across the inner membrane, and also when a translocating substrate is trapped in the TIM23 complex. These structural changes reveal that the macromolecular arrangement within the TIM23 complex is dynamic and varies with the physiological state of the mitochondrion. [Abstract/Link to Full Text]

Segrelles C, Moral M, Lorz C, Santos M, Lu J, Cascallana JL, Lara MF, Carbajal S, Martínez-Cruz AB, García-Escudero R, Beltran L, Segovia JC, Bravo A, Digiovanni J, Paramio JM
Constitutively Active Akt Induces Ectodermal Defects and Impaired BMP Signaling.
Mol Biol Cell. 2007 Oct 24;
Monitoring Editor: M. Bishr Omary Aberrant activation of the Akt pathway has been implicated in several human pathologies including cancer. However, current knowledge on the involvement of Akt signaling in development is limited. Previous data have suggested that Akt-mediated signaling may be an essential mediator of epidermal homeostasis through cell autonomous and noncell autonomous mechanisms. Here we report the developmental consequences of deregulated Akt activity in the basal layer of stratified epithelia, mediated by the expression of a constitutively active Akt1 (myrAkt) in transgenic mice. Contrary to mice overexpressing wild type Akt1 (Akt(wt)), these myrAkt mice display, in a dose-dependent manner, altered development of ectodermally derived organs such as hair, teeth, nails and epidermal glands. To identify the possible molecular mechanisms underlying these alterations, gene profiling approaches were used. We demonstrate that constitutive Akt activity disturbs the bone morphogenetic protein (BMP)-dependent signaling pathway. In addition, these mice also display alterations in adult epidermal stem cells. Collectively, we show that epithelial tissue development and homeostasis is dependent on proper regulation of Akt expression and activity. [Abstract/Link to Full Text]

Brauer MJ, Huttenhower C, Airoldi EM, Rosenstein R, Matese JC, Gresham D, Boer VM, Troyanskaya OG, Botstein D
Coordination of Growth Rate, Cell Cycle, Stress Response, and Metabolic Activity in Yeast.
Mol Biol Cell. 2007 Oct 24;
Monitoring Editor: Thomas Fox We studied the relation between growth rate and genome-wide gene expression, cell cycle progression, and glucose metabolism in 36 steady state continuous cultures limited by one of six different nutrients (glucose, ammonium, sulfate, phosphate, uracil or leucine). The expression of more than a quarter of all yeast genes is linearly correlated with growth rate, independently of the limiting nutrient. The subset of negatively growth-correlated genes is most enriched for peroxisomal functions, whereas positively correlated genes mainly encode ribosomal functions. Many (not all) genes associated with stress response are strongly correlated with growth rate, as are genes that are periodically expressed under conditions of metabolic cycling. We confirmed a linear relationship between growth rate and the fraction of the cell population in the G0/G1 cell cycle phase, independent of limiting nutrient. Cultures limited by auxotrophic requirements wasted excess glucose, whereas those limited on phosphate, sulfate or ammonia did not; this phenomenon (reminiscent of the "Warburg effect" in cancer cells) was confirmed in batch cultures. Using an aggregate of gene expression values, we predict (in both continuous and batch cultures) an "instantaneous growth rate". This concept is useful in interpreting the system-level connections among growth rate, metabolism, stress and the cell cycle. [Abstract/Link to Full Text]

Zhou M, Wang YL
Distinct Pathways for the Early Recruitment of Myosin II and Actin to the Cytokinetic Furrow.
Mol Biol Cell. 2007 Oct 24;
Monitoring Editor: Fred Chang Equatorial organization of myosin II and actin has been recognized as a universal event in cytokinesis of animal cells. Current models for the formation of equatorial cortex favor either directional cortical transport toward the equator, or localized de novo assembly. However, this process has never been analyzed directly in dividing mammalian cells at a high resolution. Here we applied total internal reflection fluorescence microscope (TIRF-M), coupled with spatial temporal image correlation spectroscopy (STICS) and a new imaging approach termed temporal differential microscopy (TDM), to image the dynamics of myosin II and actin during the assembly of equatorial cortex. Our results indicated distinct and at least partially independent mechanisms for the early equatorial recruitment of myosin and actin filaments. Cortical myosin showed no detectable directional flow during early cytokinesis. In addition to equatorial assembly, we showed that localized inhibition of disassembly contributed to the formation of the equatorial myosin band. In contrast to myosin, actin filaments underwent a striking flux toward the equator. Myosin motor activity was required for the actin flux, but not for actin concentration in the furrow, suggesting that there was a flux-independent, de novo mechanism for actin recruitment along the equator. Our results indicate that cytokinesis involves signals that regulate both assembly and disassembly activities, and argue against mechanisms that are coupled to global cortical movements. [Abstract/Link to Full Text]

Panaretakis T, Hjortsberg L, Tamm KP, Björklund AC, Joseph B, Grandér D
IFN{alpha} Induces Nucleus-independent Apoptosis by Activating ERK1/2 and JNK Downstream of PI3K and mTOR.
Mol Biol Cell. 2007 Oct 17;
Monitoring Editor: Gerard Evan Interferon-alpha (IFNalpha) induces apoptosis via Bak and Bax and the mitochondrial pathway. Here we investigated the role of known IFNalpha-induced signaling cascades upstream of Bak activation. By pharmacological and genetic inhibition of the kinases PKCdelta, ERK and JNK in U266-1984- and RHEK-1 cells we could demonstrate that all three enzymes are critical for the apoptosis associated mitochondrial events and apoptotic cell death induced by IFNalpha, at a step downstream of PI3K and mTOR. Furthermore, the activation of JNK was found to occur in a PKCdelta/ERK dependent manner. Inhibition of these kinases did not affect the canonical IFNalpha-stimulated JAK-STAT signaling or expression of IFN-responsive genes. Therefore enucleated cells (cytoplasts) were examined for IFNalpha-induced apoptosis, to test directly whether this process depends on gene transcription. Cytoplasts were found to undergo apoptosis following IFNalpha treatment, as analyzed by several apoptosis markers using flow cytometry, live cell imaging and biochemical analysis of flow-sorted cytoplasts. Furthermore, inhibition of mTOR, ERK and JNK blocked IFNalpha-induced apoptosis in cytoplasts. In conclusion, IFNalpha-induced apoptosis requires activation of ERK1/2, PKCdelta and JNK downstream of PI3K and mTOR, and can occur in a nucleus-independent manner, thus demonstrating for the first time that IFNalpha induces apoptosis in the absence of de novo transcription. [Abstract/Link to Full Text]

Kanada M, Nagasaki A, Uyeda TQ
Novel Functions of Ect2 in Polar Lamellipodia Formation and Polarity Maintenance during "Contractile Ring-Independent" Cytokinesis in Adherent Cells.
Mol Biol Cell. 2007 Oct 17;
Monitoring Editor: Yu-Li Wang Some mammalian cells are able to divide via both the classic contractile ring-dependent method (cytokinesis A) and a contractile ring-independent, adhesion-dependent method (cytokinesis B). Cytokinesis A is triggered by RhoA, which, in HeLa cells, is activated by the guanine nucleotide-exchange factor Ect2 localized at the central spindle and equatorial cortex. Here, we show that in HT1080 cells undergoing cytokinesis A, Ect2 does not localize in the equatorial cortex, though RhoA accumulates there. Moreover, Ect2 depletion resulted in only modest multinucleation of HT1080 cells, enabling us to establish cell lines in which Ect2 was constitutively depleted. Thus, RhoA is activated via an Ect2-independent pathway during cytokinesis A in HT1080 cells. During cytokinesis B, Ect2-depleted cells showed narrower accumulation of RhoA at the equatorial cortex, accompanied by compromised pole-to-equator polarity, formation of ectopic lamellipodia in regions where RhoA normally would be distributed, and delayed formation of polar lamellipodia. Furthermore, C3 exoenzyme inhibited equatorial RhoA activation and polar lamellipodia formation. Conversely, expression of dominant active Ect2 in interphase HT1080 cells enhanced RhoA activity and suppressed lamellipodia formation. These results suggest that equatorial Ect2 locally suppresses lamellipodia formation via RhoA activation, which indirectly contributes to restricting lamellipodia formation to polar regions during cytokinesis B. [Abstract/Link to Full Text]

Lee CW, Peng HB
The Function of Mitochondria in Presynaptic Development at the Neuromuscular Junction.
Mol Biol Cell. 2007 Oct 17;
Monitoring Editor: Thomas Pollard Mitochondria with high membrane potential (DeltaPsim) are enriched in the presynaptic nerve terminal at vertebrate neuromuscular junctions, but the exact function of these localized synaptic mitochondria remains unclear. Here, we investigated the correlation between mitochondrial DeltaPsim and the development of synaptic specializations. Using mitochondrial DeltaPsim-sensitive probe JC-1, we found that DeltaPsim in Xenopus spinal neurons could be reversibly elevated by creatine and suppressed by FCCP. Along naďve neurites, preexisting synaptic vesicle (SV) clusters were positively correlated with mitochondrial DeltaPsim, suggesting a potential regulatory role of mitochondrial activity in synaptogenesis. Indicating a specific role of mitochondrial activity in presynaptic development, mitochondrial ATP synthase inhibitor oligomycin, but not mitochondrial Na(+)/Ca(2+) exchanger inhibitor CGP-37157, inhibited the clustering of SVs induced by growth-factor coated beads. Local F-actin assembly induced along spinal neurites by beads was suppressed by FCCP or oligomycin Our results suggest that a key role of presynaptic mitochondria is to provide ATP for the assembly of actin cytoskeleton involved in the assembly of the presynaptic specialization including the clustering of SVs and mitochondria themselves. [Abstract/Link to Full Text]

Satyanarayana A, Hilton MB, Kaldis P
p21 Inhibits Cdk1 in the Absence of Cdk2 to Maintain the G1/S Phase DNA Damage Checkpoint.
Mol Biol Cell. 2007 Oct 17;
Monitoring Editor: Daniel Lew Cdk1 was proposed to compensate for the loss of Cdk2. Here we present evidence that this is possible due to premature translocation of Cdk1 from the cytoplasm to the nucleus in the absence of Cdk2. We also investigated the consequence of loss of Cdk2 on the maintenance of the G1/S DNA damage checkpoint. Cdk2(-/-) mouse embryonic fibroblasts in vitro as well as regenerating liver cells after partial hepatectomy (PH) in Cdk2(-/-) mice, arrest promptly at the G1/S checkpoint in response to gamma-irradiation due activation of p53 and p21 inhibiting Cdk1. Furthermore reentry into S phase after irradiation was delayed in Cdk2(-/-) cells due to prolonged and impaired DNA repair activity. In addition, Cdk2(-/-) mice were more sensitive to lethal irradiation compared with wild type and displayed delayed resumption of DNA replication in regenerating liver cells. Our results suggest that the G1/S DNA damage checkpoint is intact in the absence of Cdk2, but Cdk2 is important for proper repair of the damaged DNA. [Abstract/Link to Full Text]

Pagé EL, Chan DA, Giaccia AJ, Levine M, Richard DE
Hypoxia-inducible Factor-1{alpha} Stabilization in Nonhypoxic Conditions: Role of Oxidation and Intracellular Ascorbate Depletion.
Mol Biol Cell. 2007 Oct 17;
Monitoring Editor: J. Silvio Gutkind Hypoxia-inducible factor-1 (HIF-1) is a decisive element for the transcriptional regulation of many genes induced under low oxygen conditions. Under normal oxygen conditions, HIF-1alpha, the active subunit of HIF-1, is hydroxylated on proline residues by specific prolyl-hydroxylases (PHD) leading to ubiquitination and degradation by the proteasome. In hypoxia, hydroxylation and ubiquitination are blocked and HIF-1alpha accumulates in cells. Recent studies have shown that in normal oxygen conditions G-protein-coupled receptor agonists, including Ang II and thrombin, potently induce and activate HIF-1 in vascular smooth muscle cells. The current study identifies HIF-1alpha protein stabilization as a key mechanism for HIF-1 induction by Ang II. We show that hydroxylation on proline 402 is altered by Ang II, decreasing pVHL binding to HIF-1alpha and allowing HIF-1alpha protein to escape subsequent ubiquitination and degradation mechanisms. We show that HIF-1alpha stability is mediated through the Ang II-mediated generation of hydrogen peroxide and a subsequent decrease in ascorbate levels leading to decreased HIF prolyl-hydroxylase activity and HIF-1alpha stabilization. These findings identify novel and intricate signaling mechanisms involved in HIF-1 complex activation and will lead to the elucidation of the importance of HIF-1 in different Ang II-related cell responses. [Abstract/Link to Full Text]

Piasecki BP, Lavoie M, Tam LW, Lefebvre PA, Silflow CD
The Uni2 Phosphoprotein is a Cell-Cycle Regulated Component of the Basal Body Maturation Pathway in Chlamydomonas reinhardtii.
Mol Biol Cell. 2007 Oct 17;
Monitoring Editor: Stephen Doxsey Mutations in the UNI2 locus in Chlamydomonas reinhardtii result in a "uniflagellar" phenotype in which flagellar assembly occurs preferentially from the older basal body and ultrastructural defects reside in the transition zones. The UNI2 gene encodes a protein of 134 kDa that shares 20.5% homology with a human protein. Immunofluorescence microscopy localized the protein on both basal bodies and probasal bodies. The protein is present as at least two molecular-weight variants that can be converted to a single form with phosphatase treatment. Synthesis of Uni2 protein is induced during cell division cycles; accumulation of the phosphorylated form coincides with assembly of transition zones and flagella at the end of the division cycle. Using the Uni2 protein as a cell-cycle marker of basal bodies, we observed migration of basal bodies before flagellar resorption in some cells, indicating that flagellar resorption is not required for mitotic progression. We observed the sequential assembly of new probasal bodies beginning at prophase. The uni2 mutants may be defective in the pathways leading to flagellar assembly and to basal body maturation. [Abstract/Link to Full Text]

---

Recent Articles in Molecular and Cellular Biology

---

Greig KT, Antonchuk J, Metcalf D, Morgan PO, Krebs DL, Zhang JG, Hacking DF, Bode L, Robb L, Kranz C, de Graaf C, Bahlo M, Nicola NA, Nutt SL, Freeze HH, Alexander WS, Hilton DJ, Kile BT
Agm1/Pgm3-mediated sugar nucleotide synthesis is essential for hematopoiesis and development.
Mol Cell Biol. 2007 Aug;27(16):5849-59.
Carbohydrate modification of proteins includes N-linked and O-linked glycosylation, proteoglycan formation, glycosylphosphatidylinositol anchor synthesis, and O-GlcNAc modification. Each of these modifications requires the sugar nucleotide UDP-GlcNAc, which is produced via the hexosamine biosynthesis pathway. A key step in this pathway is the interconversion of GlcNAc-6-phosphate (GlcNAc-6-P) and GlcNAc-1-P, catalyzed by phosphoglucomutase 3 (Pgm3). In this paper, we describe two hypomorphic alleles of mouse Pgm3 and show there are specific physiological consequences of a graded reduction in Pgm3 activity and global UDP-GlcNAc levels. Whereas mice lacking Pgm3 die prior to implantation, animals with less severe reductions in enzyme activity are sterile, exhibit changes in pancreatic architecture, and are anemic, leukopenic, and thrombocytopenic. These phenotypes are accompanied by specific rather than wholesale changes in protein glycosylation, suggesting that while universally required, the functions of certain proteins and, as a consequence, certain cell types are especially sensitive to reductions in Pgm3 activity. [Abstract/Link to Full Text]

Lang T, Dalal S, Chikova A, DiMaio D, Sweasy JB
The E295K DNA polymerase beta gastric cancer-associated variant interferes with base excision repair and induces cellular transformation.
Mol Cell Biol. 2007 Aug;27(15):5587-96.
Approximately 30% of human tumors examined for mutations in polymerase beta (pol beta) appear to express pol beta variant proteins (D. Starcevic, S. Dalal, and J. B. Sweasy, Cell Cycle 3:998-1001, 2004). Many of these variants result from a single amino acid substitution. We have previously shown that the K289M and I260M colon and prostate cancer variants, respectively, induce cellular transformation most likely due to sequence-specific mutator activity (S. Dalal et al., Biochemistry 44:15664-15673, 2005; T. Lang et al., Proc. Natl. Acad. Sci. USA 101:6074-6079, 2004; J. B. Sweasy et al., Proc. Natl. Acad. Sci. USA 102:14350-14355, 2005). In the work described here, we show that the E295K gastric carcinoma pol beta variant acts in a dominant-negative manner by interfering with base excision repair. This leads to an increase in sister chromatid exchanges. Expression of the E295K variant also induces cellular transformation. Our data suggest that unfilled gaps are channeled into a homology-directed repair pathway that could lead to genomic instability. The results indicate that base excision repair is critical for maintaining genome stability and could therefore be a tumor suppressor mechanism. [Abstract/Link to Full Text]

Li H, Zhang Z, Wang B, Zhang J, Zhao Y, Jin Y
Wwp2-mediated ubiquitination of the RNA polymerase II large subunit in mouse embryonic pluripotent stem cells.
Mol Cell Biol. 2007 Aug;27(15):5296-305.
Ubiquitination and the degradation of the large subunit of RNA polymerase II, Rpb1, is not only involved in DNA damage-induced arrest but also in other transcription-obstructing events. However, the ubiquitin ligases responsible for DNA damage-independent processes in mammalian cells remain to be identified. Here, we identified Wwp2, a mouse HECT domain ubiquitin E3 ligase, as a novel ubiquitin ligase of Rpb1. We found that Wwp2 specifically interacted with mouse Rpb1 and targeted it for ubiquitination both in vitro and in vivo. Interestingly, the interaction with and ubiquitination of Rpb1 was dependent neither on its phosphorylation state nor on DNA damage. However, the enzymatic activity of Wwp2 was absolutely required for its ubiquitin modification of Rpb1. Furthermore, our study indicates that the interaction between Wwp2 and Rpb1 was mediated through WW domain of Wwp2 and C-terminal domain of Rpb1, respectively. Strikingly, downregulation of Wwp2 expression compromised Rpb1 ubiquitination and elevated its intracellular steady-state protein level significantly. Importantly, we identified six lysine residues in the C-terminal domain of Rpb1 as ubiquitin acceptor sites mediated by Wwp2. These results indicate that Wwp2 plays an important role in regulating expression of Rpb1 in normal physiological conditions. [Abstract/Link to Full Text]

Mohammad-Qureshi SS, Haddad R, Hemingway EJ, Richardson JP, Pavitt GD
Critical contacts between the eukaryotic initiation factor 2B (eIF2B) catalytic domain and both eIF2beta and -2gamma mediate guanine nucleotide exchange.
Mol Cell Biol. 2007 Jul;27(14):5225-34.
Diverse guanine nucleotide exchange factors (GEFs) regulate the activity of GTP binding proteins. One of the most complicated pairs is eukaryotic initiation factor 2B (eIF2B) and eIF2, which function during protein synthesis initiation in eukaryotes. We have mutated conserved surface residues within the eIF2B GEF domain, located at the eIF2Bepsilon C terminus. Extensive genetic and biochemical characterization established how these residues contribute to GEF activity. We find that the universally conserved residue E569 is critical for activity and that even a conservative E569D substitution is lethal in vivo. Several mutations within residues close to E569 have no discernible effect on growth or GCN4 expression, but an alanine substitution at the adjacent L568 is cold sensitive and deregulates GCN4 activity at 15 degrees C. The mutation of W699, found on a separate surface approximately 40 A from E569, is also lethal. Binding studies show that W699 is critical for interaction with eIF2beta, while L568 and E569 are not. In contrast, all three residues are critical for interaction with eIF2gamma. These data show that multiple contacts between eIF2gamma and eIF2Bepsilon mediate nucleotide exchange. [Abstract/Link to Full Text]

Mo JS, Kim MY, Han SO, Kim IS, Ann EJ, Lee KS, Seo MS, Kim JY, Lee SC, Park JW, Choi EJ, Seong JY, Joe CO, Faessler R, Park HS
Integrin-linked kinase controls Notch1 signaling by down-regulation of protein stability through Fbw7 ubiquitin ligase.
Mol Cell Biol. 2007 Aug;27(15):5565-74.
Integrin-linked kinase (ILK) is a scaffold and protein kinase that acts as a pivotal effector in integrin signaling for various cellular functions. In this study, we found that ILK remarkably reduced the protein stability of Notch1 through Fbw7. The kinase activity of ILK was essential for the inhibition of Notch1 signaling. Notably, the protein level and transcriptional activity of the endogenous Notch1 intracellular domain (Notch1-IC) were higher in ILK-null cells than in ILK wild-type cells, and the level of endogenous Notch1-IC was increased by the blocking of the proteasome, suggesting that ILK enhances the proteasomal degradation of Notch1-IC. ILK directly bound and phosphorylated Notch1-IC, thereby facilitating proteasomal protein degradation through Fbw7. Furthermore, we found down-regulation of Notch1-IC and up-regulation of ILK in basal cell carcinoma and melanoma patients but not in squamous cell carcinoma patients. These results suggest that ILK down-regulated the protein stability of Notch1-IC through the ubiquitin-proteasome pathway by means of Fbw7. [Abstract/Link to Full Text]

Xiao R, Sun Y, Ding JH, Lin S, Rose DW, Rosenfeld MG, Fu XD, Li X
Splicing regulator SC35 is essential for genomic stability and cell proliferation during mammalian organogenesis.
Mol Cell Biol. 2007 Aug;27(15):5393-402.
The members of the SR family of splicing regulators were initially characterized for their critical roles in constitutive and regulated splicing. They are implicated in different aspects of gene expression processes, including transcription, RNA stability, mRNA transport, and translational control. While knockout studies have demonstrated their essential functions during animal development, the pathway(s) leading to a specific cellular phenotype remains poorly understood. We report here that the SR protein SC35 controls cell proliferation during pituitary gland development but is completely dispensable in terminal differentiated mature cardiomyocytes in mice. We show that loss of SC35 in mouse embryonic fibroblasts induces G2/M cell cycle arrest and genomic instability, resulting at least in part from p53 hyperphosphorylation and hyperacetylation. While p53 hyperphosphorylation appears related to ATM activation, its hyperacetylation has been attributed to the increased expression of the acetyltransferase gene p300 and the aberrant splicing of the deacetylase gene SirT1. These findings reveal the involvement of SC35 in specific pathways in regulating cell proliferation and genomic stability during mammalian organogenesis and suggest its potential function in tumorigenesis. [Abstract/Link to Full Text]

Niu H, Li X, Job E, Park C, Moazed D, Gygi SP, Hollingsworth NM
Mek1 kinase is regulated to suppress double-strand break repair between sister chromatids during budding yeast meiosis.
Mol Cell Biol. 2007 Aug;27(15):5456-67.
Mek1 is a meiosis-specific kinase in budding yeast which promotes recombination between homologous chromosomes by suppressing double-strand break (DSB) repair between sister chromatids. Previous work has shown that in the absence of the meiosis-specific recombinase gene, DMC1, cells arrest in prophase due to unrepaired DSBs and that Mek1 kinase activity is required in this situation to prevent repair of the breaks using sister chromatids. This work demonstrates that Mek1 is activated in response to DSBs by autophosphorylation of two conserved threonines, T327 and T331, in the Mek1 activation loop. Using a version of Mek1 that can be conditionally dimerized during meiosis, Mek1 function was shown to be promoted by dimerization, perhaps as a way of enabling autophosphorylation of the activation loop in trans. A putative HOP1-dependent dimerization domain within the C terminus of Mek1 has been identified. Dimerization alone, however, is insufficient for activation, as DSBs and Mek1 recruitment to the meiosis-specific chromosomal core protein Red1 are also necessary. Phosphorylation of S320 in the activation loop inhibits sister chromatid repair specifically in dmc1Delta-arrested cells. Ectopic dimerization of Mek1 bypasses the requirement for S320 phosphorylation, suggesting this phosphorylation is necessary for maintenance of Mek1 dimers during checkpoint-induced arrest. [Abstract/Link to Full Text]

Terzian T, Wang Y, Van Pelt CS, Box NF, Travis EL, Lozano G
Haploinsufficiency of Mdm2 and Mdm4 in tumorigenesis and development.
Mol Cell Biol. 2007 Aug;27(15):5479-85.
The tumor suppressor p53 is inactivated by multiple mechanisms that include mutations of the p53 gene itself and increased levels of the p53 inhibitors MDM2 and MDM4. Mice lacking Mdm2 or Mdm4 exhibit embryo-lethal phenotypes that are completely rescued by concomitant deletion of p53. Here we show that Mdm2 and Mdm4 haploinsufficiency leads to increased p53 activity, exhibited as increased sensitivity to DNA damage and decreased transformation potential. Moreover, in in vivo tumor development, Emu-myc Mdm4+/- mice show a delayed onset of B-cell lymphomas compared to Emu-myc mice. Additionally, Mdm2+/- Mdm4+/- double-heterozygous mice are not viable and exhibit defects in hematopoiesis and cerebellar development. The defects in Mdm2+/- Mdm4+/- mice are corrected by deletion of a single p53 allele. These findings highlight the exquisite sensitivity of p53 to Mdm2 and Mdm4 levels and suggest that some cell types may be more sensitive to therapeutic drugs that inhibit the Mdm-p53 interaction. [Abstract/Link to Full Text]

Kato J, Zhu J, Liu C, Moss J
Enhanced sensitivity to cholera toxin in ADP-ribosylarginine hydrolase-deficient mice.
Mol Cell Biol. 2007 Aug;27(15):5534-43.
Cholera toxin (CT) produced by Vibrio cholerae causes the devastating diarrhea of cholera by catalyzing the ADP-ribosylation of the alpha subunit of the intestinal Gs protein (Gsalpha), leading to characteristic water and electrolyte losses. Mammalian cells contain ADP-ribosyltransferases similar to CT and an ADP-ribosyl(arginine)protein hydrolase (ADPRH), which cleaves the ADP-ribose-(arginine)protein bond, regenerating native protein and completing an ADP-ribosylation cycle. We hypothesized that ADPRH might counteract intoxication by reversing the ADP-ribosylation of Gsalpha. Effects of intoxication on murine ADPRH-/- cells were greater than those on wild-type cells and were significantly reduced by overexpression of wild-type ADPRH in ADPRH-/- cells, as evidenced by both ADP-ribose-arginine content and Gsalpha modification. Similarly, intestinal loops in the ADPRH-/- mouse were more sensitive than their wild-type counterparts to toxin effects on fluid accumulation, Gsalpha modification, and ADP-ribosylarginine content. Thus, CT-catalyzed ADP-ribosylation of cell proteins can be counteracted by ADPRH, which could function as a modifier gene in disease. Further, our study demonstrates that enzymatic cross talk exists between bacterial toxin ADP-ribosyltransferases and host ADP-ribosylation cycles. In disease, toxin-catalyzed ADP-ribosylation overwhelms this potential host defense system, resulting in persistence of ADP-ribosylation and intoxication of the cell. [Abstract/Link to Full Text]

Gonzalez D, Bowen AJ, Carroll TS, Conlan RS
The transcription corepressor LEUNIG interacts with the histone deacetylase HDA19 and mediator components MED14 (SWP) and CDK8 (HEN3) to repress transcription.
Mol Cell Biol. 2007 Aug;27(15):5306-15.
Transcription corepressors are general regulators controlling the expression of genes involved in multiple signaling pathways and developmental programs. Repression is mediated through mechanisms including the stabilization of a repressive chromatin structure over control regions and regulation of Mediator function inhibiting RNA polymerase II activity. Using whole-genome arrays we show that the Arabidopsis thaliana corepressor LEUNIG, a member of the GroTLE transcription corepressor family, regulates the expression of multiple targets in vivo. LEUNIG has a role in the regulation of genes involved in a number of different physiological processes including disease resistance, DNA damage response, and cell signaling. We demonstrate that repression of in vivo LEUNIG targets is achieved through histone deacetylase (HDAC)-dependent and -independent mechanisms. HDAC-dependent mechanisms involve direct interaction with HDA19, a class 1 HDAC, whereas an HDAC-independent repression activity involves interactions with the putative Arabidopsis Mediator components AtMED14/SWP and AtCDK8/HEN3. We suggest that changes in chromatin structure coupled with regulation of Mediator function are likely to be utilized by LEUNIG in the repression of gene transcription. [Abstract/Link to Full Text]

Chang YL, King B, Lin SC, Kennison JA, Huang DH
A double-bromodomain protein, FSH-S, activates the homeotic gene ultrabithorax through a critical promoter-proximal region.
Mol Cell Biol. 2007 Aug;27(15):5486-98.
More than a dozen trithorax group (trxG) proteins are involved in activation of Drosophila HOX genes. How they act coordinately to integrate signals from distantly located enhancers is not fully understood. The female sterile (1) homeotic (fs(1)h) gene is one of the trxG genes that is most critical for Ultrabithorax (Ubx) activation. We show that one of the two double-bromodomain proteins encoded by fs(1)h acts as an essential factor in the Ubx proximal promoter. First, overexpression of the small isoform FSH-S, but not the larger one, can induce ectopic expression of HOX genes and cause body malformation. Second, FSH-S can stimulate Ubx promoter in cultured cells through a critical proximal region in a bromodomain-dependent manner. Third, purified FSH-S can bind specifically to a motif within this region that was previously known as the ZESTE site. The physiological relevance of FSH-S is ascertained using transgenic embryos containing a modified Ubx proximal promoter and chromatin immunoprecipitation. In addition, we show that FSH-S is involved in phosphorylation of itself and other regulatory factors. We suggest that FSH-S acts as a critical component of a regulatory circuitry mediating long-range effects of distant enhancers. [Abstract/Link to Full Text]

Hakim ZS, DiMichele LA, Doherty JT, Homeister JW, Beggs HE, Reichardt LF, Schwartz RJ, Brackhan J, Smithies O, Mack CP, Taylor JM
Conditional deletion of focal adhesion kinase leads to defects in ventricular septation and outflow tract alignment.
Mol Cell Biol. 2007 Aug;27(15):5352-64.
To examine a role for focal adhesion kinase (FAK) in cardiac morphogenesis, we generated a line of mice with a conditional deletion of FAK in nkx2-5-expressing cells (herein termed FAKnk mice). FAKnk mice died shortly after birth, likely resulting from a profound subaortic ventricular septal defect and associated malalignment of the outflow tract. Additional less penetrant phenotypes included persistent truncus arteriosus and thickened valve leaflets. Thus, conditional inactivation of FAK in nkx2-5-expressing cells leads to the most common congenital heart defect that is also a subset of abnormalities associated with tetralogy of Fallot and the DiGeorge syndrome. No significant differences in proliferation or apoptosis between control and FAKnk hearts were observed. However, decreased myocardialization was observed for the conal ridges of the proximal outflow tract in FAKnk hearts. Interestingly, chemotaxis was significantly attenuated in isolated FAK-null cardiomyocytes in comparison to genetic controls, and these effects were concomitant with reduced tyrosine phosphorylation of Crk-associated substrate (CAS). Thus, it is possible that ventricular septation and appropriate outflow tract alignment is dependent, at least in part, upon FAK-dependent CAS activation and subsequent induction of polarized myocyte movement into the conal ridges. Future studies will be necessary to determine the precise contributions of the additional nkx2-5-derived lineages to the phenotypes observed. [Abstract/Link to Full Text]

Li L, Zhang L, Zhang X, Yan Q, Minamishima YA, Olumi AF, Mao M, Bartz S, Kaelin WG
Hypoxia-inducible factor linked to differential kidney cancer risk seen with type 2A and type 2B VHL mutations.
Mol Cell Biol. 2007 Aug;27(15):5381-92.
Clear cell carcinoma of the kidney is a major cause of mortality in patients with von Hippel-Lindau (VHL) disease, which is caused by germ line mutations that inactivate the VHL tumor suppressor gene. Biallelic VHL inactivation, due to mutations or hypermethylation, is also common in sporadic clear cell renal carcinomas. The VHL gene product, pVHL, is part of a ubiquitin ligase complex that targets the alpha subunits of the heterodimeric transcription factor hypoxia-inducible factor (HIF) for destruction under well-oxygenated conditions. All VHL mutations linked to classical VHL disease compromise this pVHL function although some missense mutations result in a low risk of kidney cancer (type 2A VHL disease) while others result in a high risk (type 2B VHL disease). We found that type 2A mutants were less defective than type 2B mutants when reintroduced into VHL-/- renal carcinoma cells with respect to HIF regulation. A stabilized version of HIF2alpha promoted tumor growth by VHL-/- cells engineered to produce type 2A mutants, while knock-down of HIF2alpha in cells producing type 2B mutants had the opposite effect. Therefore, quantitative differences with respect to HIF deregulation are sufficient to account for the differential risks of kidney cancer linked to VHL mutations. [Abstract/Link to Full Text]

Durant M, Pugh BF
NuA4-directed chromatin transactions throughout the Saccharomyces cerevisiae genome.
Mol Cell Biol. 2007 Aug;27(15):5327-35.
Two of the major histone acetyltransferases in Saccharomyces cerevisiae are NuA4 and SAGA, which acetylate histones H4 and H3, respectively. Acetylated H3 and H4 tails have been implicated in binding bromodomain proteins, including Bdf1. Bdf1 interacts with the general transcription factor TFIID, which might promote preinitiation complex (PIC) assembly. Bdf1 also interacts with the SWR complex (SWR-C). SWR-C is responsible for the deposition of the histone H2A variant H2A.Z. The placement of these interactions into a connected pathway of PIC assembly has not been fully established. Moreover, it is not known how widespread and how variable such a pathway might be on a genomic scale. Here we provide genomic evidence for S. cerevisiae that PIC assembly (TFIID occupancy) and chromatin remodeling (SWR-C and H2A.Z occupancy) are linked in large part to NuA4-directed H4 acetylation and subsequent Bdf1 binding, rather than through SAGA-directed H3 acetylation. Bdf1 and its homolog Bdf2 tend to have distinct locations in the genome. However, the deletion of BDF1 leads to the accumulation of Bdf2 at Bdf1-vacated sites. Thus, while Bdf1 and Bdf2 are at least partially redundant in function, their functions in the genome are geographically distinct. [Abstract/Link to Full Text]

Dobi KC, Winston F
Analysis of transcriptional activation at a distance in Saccharomyces cerevisiae.
Mol Cell Biol. 2007 Aug;27(15):5575-86.
Most fundamental aspects of transcription are conserved among eukaryotes. One striking difference between yeast Saccharomyces cerevisiae and metazoans, however, is the distance over which transcriptional activation occurs. In S. cerevisiae, upstream activation sequences (UASs) are generally located within a few hundred base pairs of a target gene, while in Drosophila and mammals, enhancers are often several kilobases away. To study the potential for long-distance activation in S. cerevisiae, we constructed and analyzed reporters in which the UAS-TATA distance varied. Our results show that UASs lose the ability to activate normal transcription as the UAS-TATA distance increases. Surprisingly, transcription does initiate, but proximally to the UAS, regardless of its location. To identify factors affecting long-distance activation, we screened for mutants allowing activation of a reporter when the UAS-TATA distance is 799 bp. These screens identified four loci, SIN4, SPT2, SPT10, and HTA1-HTB1, with sin4 mutations being the strongest. Our results strongly suggest that long-distance activation in S. cerevisiae is normally limited by Sin4 and other factors and that this constraint plays a role in ensuring UAS-core promoter specificity in the compact S. cerevisiae genome. [Abstract/Link to Full Text]

Alper S, McBride SJ, Lackford B, Freedman JH, Schwartz DA
Specificity and complexity of the Caenorhabditis elegans innate immune response.
Mol Cell Biol. 2007 Aug;27(15):5544-53.
In response to infection, Caenorhabditis elegans produces an array of antimicrobial proteins. To understand the C. elegans immune response, we have investigated the regulation of a large, representative sample of candidate antimicrobial genes. We found that all these putative antimicrobial genes are expressed in tissues exposed to the environment, a position from which they can ward off infection. Using RNA interference to inhibit the function of immune signaling pathways in C. elegans, we found that different immune response pathways regulate expression of distinct but overlapping sets of antimicrobial genes. We also show that different bacterial pathogens regulate distinct but overlapping sets of antimicrobial genes. The patterns of genes induced by pathogens do not coincide with any single immune signaling pathway. Thus, even in this simple model system for innate immunity, striking specificity and complexity exist in the immune response. The unique patterns of antimicrobial gene expression observed when C. elegans is exposed to different pathogens or when different immune signaling pathways are perturbed suggest that a large set of yet to be identified pathogen recognition receptors (PRRs) exist in the nematode. These PRRs must interact in a complicated fashion to induce a unique set of antimicrobial genes. We also propose the existence of an "antimicrobial fingerprint," which will aid in assigning newly identified C. elegans innate immunity genes to known immune signaling pathways. [Abstract/Link to Full Text]

Zhao H, Friedman RD, Fournier RE
The locus control region activates serpin gene expression through recruitment of liver-specific transcription factors and RNA polymerase II.
Mol Cell Biol. 2007 Aug;27(15):5286-95.
The human serine protease inhibitor (serpin) gene cluster at 14q32.1 comprises 11 serpin genes, many of which are expressed specifically in hepatic cells. Previous studies identified a locus control region (LCR) upstream of the human alpha1-antitrypsin (alpha1AT) gene that is required for gene activation, chromatin remodeling, and histone acetylation throughout the proximal serpin subcluster. Here we show that the LCR interacts with multiple liver-specific transcription factors, including hepatocyte nuclear factor 3beta (HNF-3beta), HNF-6alpha, CCAAT/enhancer binding protein alpha (C/EBPalpha), and C/EBPbeta. RNA polymerase II is also recruited to the locus through the LCR. Nongenic transcription at both the LCR and an upstream regulatory region was detected, but the deletion of the LCR abolished transcription at both sites. The deletion of HNF-3 and HNF-6 binding sites within the LCR reduced histone acetylation at both the LCR and the upstream regulatory region and decreased the transcription of the alpha1AT, corticosteroid binding globulin, and protein Z-dependent protease inhibitor genes. These results suggest that the LCR activates genes in the proximal serpin subcluster by recruiting liver-specific transcription factors and components of the general transcription machinery to regulatory regions upstream of the alpha1AT gene. [Abstract/Link to Full Text]

Broxmeyer HE, Sehra S, Cooper S, Toney LM, Kusam S, Aloor JJ, Marchal CC, Dinauer MC, Dent AL
Aberrant regulation of hematopoiesis by T cells in BAZF-deficient mice.
Mol Cell Biol. 2007 Aug;27(15):5275-85.
The BAZF (BCL-6b) protein is highly similar to the BCL-6 transcriptional repressor. While BCL-6 has been characterized extensively, relatively little is known about the normal function of BAZF. In order to understand the physiological role of BAZF, we created BAZF-deficient mice. Unlike BCL-6-deficient mice, BAZF-deficient mice are healthy and normal in size. However, BAZF-deficient mice have a hematopoietic progenitor phenotype that is almost identical to that of BCL-6-deficient mice. Compared to wild-type mice, both BAZF-deficient and BCL-6-deficient mice have greatly reduced numbers of cycling hematopoietic progenitor cells (HPC) in the BM and greatly increased numbers of cycling HPC in the spleen. In contrast to HPC from wild-type mice, HPC from BAZF-deficient and BCL-6-deficient mice are resistant to chemokine-induced myelosuppression and do not show a synergistic growth response to granulocyte-macrophage colony-stimulating factor plus stem cell factor. Depletion of CD8 T cells in BAZF-deficient mice reverses several of the hematopoietic defects in these mice. Since both BAZF- and BCL-6-deficient mice have defects in CD8 T-cell differentiation, we hypothesize that both BCL-6 and BAZF regulate HPC homeostasis by an indirect pathway involving CD8 T cells. [Abstract/Link to Full Text]

Bolland DJ, Wood AL, Afshar R, Featherstone K, Oltz EM, Corcoran AE
Antisense intergenic transcription precedes Igh D-to-J recombination and is controlled by the intronic enhancer Emu.
Mol Cell Biol. 2007 Aug;27(15):5523-33.
V(D)J recombination is believed to be regulated by alterations in chromatin accessibility to the recombinase machinery, but the mechanisms responsible remain unclear. We previously proposed that antisense intergenic transcription, activated throughout the mouse Igh VH region in pro-B cells, remodels chromatin for VH-to-DJH recombination. Using RNA fluorescence in situ hybridization, we now show that antisense intergenic transcription occurs throughout the Igh DHJH region before D-to-J recombination, indicating that this is a widespread process in V(D)J recombination. Transcription initiates near the Igh intronic enhancer Emu and is abrogated in mice lacking this enhancer, indicating that Emu regulates DH antisense transcription. Emu was recently demonstrated to regulate DH-to-JH recombination of the Igh locus. Together, these data suggest that Emu controls DH-to-JH recombination by activating this form of germ line Igh transcription, thus providing a long-range, processive mechanism by which Emu can regulate chromatin accessibility throughout the DH region. In contrast, Emu deletion has no effect on VH antisense intergenic transcription, which is rarely associated with DH antisense transcription, suggesting differential regulation and separate roles for these processes at sequential stages of V(D)J recombination. These results support a directive role for antisense intergenic transcription in enabling access to the recombination machinery. [Abstract/Link to Full Text]

Yoshizaki T, Imamura T, Babendure JL, Lu JC, Sonoda N, Olefsky JM
Myosin 5a is an insulin-stimulated Akt2 (protein kinase Bbeta) substrate modulating GLUT4 vesicle translocation.
Mol Cell Biol. 2007 Jul;27(14):5172-83.
Phosphatidylinositol 3-kinase activation of Akt signaling is critical to insulin-stimulated glucose transport and GLUT4 translocation. However, the downstream signaling events following Akt activation which mediate glucose transport stimulation remain relatively unknown. Here we identify an Akt consensus phosphorylation motif in the actin-based motor protein myosin 5a and show that insulin stimulation leads to phosphorylation of myosin 5a at serine 1650. This Akt-mediated phosphorylation event enhances the ability of myosin 5a to interact with the actin cytoskeleton. Small interfering RNA-induced inhibition of myosin 5a and expression of dominant-negative myosin 5a attenuate insulin-stimulated glucose transport and GLUT4 translocation. Furthermore, knockdown of Akt2 or expression of dominant-negative Akt (DN-Akt) abolished insulin-stimulated phosphorylation of myosin 5a, inhibited myosin 5a binding to actin, and blocked insulin-stimulated glucose transport. Taken together, these data indicate that myosin 5a is a newly identified direct substrate of Akt2 and, upon insulin stimulation, phosphorylated myosin 5a facilitates anterograde movement of GLUT4 vesicles along actin to the cell surface. [Abstract/Link to Full Text]

Kininis M, Chen BS, Diehl AG, Isaacs GD, Zhang T, Siepel AC, Clark AG, Kraus WL
Genomic analyses of transcription factor binding, histone acetylation, and gene expression reveal mechanistically distinct classes of estrogen-regulated promoters.
Mol Cell Biol. 2007 Jul;27(14):5090-104.
To explore the global mechanisms of estrogen-regulated transcription, we used chromatin immunoprecipitation coupled with DNA microarrays to determine the localization of RNA polymerase II (Pol II), estrogen receptor alpha (ERalpha), steroid receptor coactivator proteins (SRC), and acetylated histones H3/H4 (AcH) at estrogen-regulated promoters in MCF-7 cells with or without estradiol (E2) treatment. In addition, we correlated factor occupancy with gene expression and the presence of transcription factor binding elements. Using this integrative approach, we defined a set of 58 direct E2 target genes based on E2-regulated Pol II occupancy and classified their promoters based on factor binding, histone modification, and transcriptional output. Many of these direct E2 target genes exhibit interesting modes of regulation and biological activities, some of which may be relevant to the onset and proliferation of breast cancers. Our studies indicate that about one-third of these direct E2 target genes contain promoter-proximal ERalpha-binding sites, which is considerably more than previous estimates. Some of these genes represent possible novel targets for regulation through the ERalpha/AP-1 tethering pathway. Our studies have also revealed several previously uncharacterized global features of E2-regulated gene expression, including strong positive correlations between Pol II occupancy and AcH levels, as well as between the E2-dependent recruitment of ERalpha and SRC at the promoters of E2-stimulated genes. Furthermore, our studies have revealed new mechanistic insights into E2-regulated gene expression, including the absence of SRC binding at E2-repressed genes and the presence of constitutively bound, promoter-proximally paused Pol IIs at some E2-regulated promoters. These mechanistic insights are likely to be relevant for understanding gene regulation by a wide variety of nuclear receptors. [Abstract/Link to Full Text]

Sullivan KE, Reddy AB, Dietzmann K, Suriano AR, Kocieda VP, Stewart M, Bhatia M
Epigenetic regulation of tumor necrosis factor alpha.
Mol Cell Biol. 2007 Jul;27(14):5147-60.
Tumor necrosis factor alpha (TNF-alpha) is a potent cytokine which regulates inflammation via the induction of adhesion molecules and chemokine expression. Its expression is known to be regulated in a complex manner with transcription, message turnover, message splicing, translation, and protein cleavage from the cell surface all being independently regulated. This study examined both cell lines and primary cells to understand the developmental regulation of epigenetic changes at the TNF-alpha locus. We demonstrate that epigenetic modifications of the TNF-alpha locus occur both developmentally and in response to acute stimulation and, importantly, that they actively regulate expression. DNA demethylates early in development, beginning with the hematopoietic stem cell. The TNF-alpha locus migrates from heterochromatin to euchromatin in a progressive fashion, reaching euchromatin slightly later in differentiation. Finally, histone modifications characteristic of a transcriptionally competent gene occur with myeloid differentiation and progress with differentiation. Additional histone modifications characteristic of active gene expression are acquired with stimulation. In each case, manipulation of these epigenetic variables altered the ability of the cell to express TNF-alpha. These studies demonstrate the importance of epigenetic regulation in the control of TNF-alpha expression. These findings may have relevance for inflammatory disorders in which TNF-alpha is overproduced. [Abstract/Link to Full Text]

Marusyk A, Wheeler LJ, Mathews CK, DeGregori J
p53 mediates senescence-like arrest induced by chronic replicational stress.
Mol Cell Biol. 2007 Aug;27(15):5336-51.
Previous studies have shown that exposure of cells to high levels of replicational stress leads to permanent proliferation arrest that does not require p53. We have examined cellular responses to therapeutically relevant low levels of replicational stress that allow limited proliferation. Chronic exposure to low concentrations of hydroxyurea, aphidicolin, or etoposide induced irreversible cell cycle arrest after several population doublings. Inhibition of p53 activity antagonized this arrest and enhanced the long-term proliferation of p53 mutant cells. p21CIP1 was found to be a critical p53 target for arrest induced by hydroxyurea or aphidicolin, but not etoposide, as judged by the ability of p21CIP1 suppression to mimic the effects of p53 disruption. Suppression of Rad51 expression, required for homologous recombination repair, blocked the ability of mutant p53 to antagonize arrest induced by etoposide, but not aphidicolin. Thus, the ability of mutant p53 to prevent arrest induced by replicational stress per se is primarily dependent on preventing p21CIP1 up-regulation. However, when replication stress is associated with DNA strand breaks (such as with etoposide), up-regulation of homologous recombination repair in response to p53 disruption becomes important. Since replicational stress leads to clonal selection of cells with p53 mutations, our results highlight the potential importance of chronic replicational stress in promoting cancer development. [Abstract/Link to Full Text]

Potzner MR, Griffel C, Lütjen-Drecoll E, Bösl MR, Wegner M, Sock E
Prolonged Sox4 expression in oligodendrocytes interferes with normal myelination in the central nervous system.
Mol Cell Biol. 2007 Aug;27(15):5316-26.
The highly related transcription factors Sox4 and Sox11 are both expressed in oligodendrocyte precursors. Yet whether they have a function in oligodendrocyte development is unknown. By overexpressing Sox4 under the control of 3.1 kb of 5' flanking sequences of the myelin basic protein gene in transgenic mice, we extended Sox4 expression in the oligodendrocyte lineage from oligodendrocyte precursors to cells undergoing terminal differentiation. As a consequence of transgene expression, mice develop the full spectrum of phenotypic traits associated with a severe hypomyelination during the first postnatal weeks. Myelin gene expression was severely reduced, and myelin dramatically thinned in several central nervous system (CNS) regions. Despite these disturbances in CNS myelination, the number of oligodendrocytic cells remained unaltered. Considering that apoptosis rates were normal and proliferation only slightly increased, oligodendrocytes likely persist in a premyelinating to early myelinating state. This shows that prolonged Sox4 expression in cells of the oligodendrocyte lineage is incompatible with the acquisition of a fully mature phenotype and argues that the presence of Sox4, and possibly Sox11, in oligodendrocyte precursors may normally prevent premature differentiation. [Abstract/Link to Full Text]

Neuwirth EA, Honma M, Grosovsky AJ
Interchromosomal crossover in human cells is associated with long gene conversion tracts.
Mol Cell Biol. 2007 Aug;27(15):5261-74.
Crossovers have rarely been observed in specific association with interchromosomal gene conversion in mammalian cells. In this investigation two isogenic human B-lymphoblastoid cell lines, TI-112 and TSCER2, were used to select for I-SceI-induced gene conversions that restored function at the selectable thymidine kinase locus. Additionally, a haplotype linkage analysis methodology enabled the rigorous detection of all crossover-associated convertants, whether or not they exhibited loss of heterozygosity. This methodology also permitted characterization of conversion tract length and structure. In TI-112, gene conversion tracts were required to be complex in tract structure and at least 7.0 kb in order to be selectable. The results demonstrated that 85% (39/46) of TI-112 convertants extended more than 11.2 kb and 48% also exhibited a crossover, suggesting a mechanistic link between long tracts and crossover. In contrast, continuous tracts as short as 98 bp are selectable in TSCER2, although selectable gene conversion tracts could include a wide range of lengths. Indeed, only 16% (14/95) of TSCER2 convertants were crossover associated, further suggesting a link between long tracts and crossover. Overall, these results demonstrate that gene conversion tracts can be long in human cells and that crossovers are observable when long tracts are recoverable. [Abstract/Link to Full Text]

Nakaya N, Hemish J, Krasnov P, Kim SY, Stasiv Y, Michurina T, Herman D, Davidoff MS, Middendorff R, Enikolopov G
noxin, a novel stress-induced gene involved in cell cycle and apoptosis.
Mol Cell Biol. 2007 Aug;27(15):5430-44.
We describe a novel stress-induced gene, noxin, and a knockout mouse line with an inactivated noxin gene. The noxin gene does not have sequelogs in the genome and encodes a highly serine-rich protein with predicted phosphorylation sites for ATM, Akt, and DNA-dependent protein kinase kinases; nuclear localization signals; and a Zn finger domain. noxin mRNA and protein levels are under tight control by the cell cycle. noxin, identified as a nitric oxide-inducible gene, is strongly induced by a wide range of stress signals: gamma- and UV irradiation, hydrogen peroxide, adriamycin, and cytokines. This induction is dependent on p53. Noxin accumulates in the nucleus in response to stress and, when ectopically expressed, Noxin arrests the cell cycle at G1; although it also induces p53, the cell cycle arrest function of Noxin is independent of p53 activity. noxin knockout mice are viable and fertile; however, they have an enlarged heart, several altered hematopoietic parameters, and a decreased number of spermatids. Importantly, loss or downregulation of Noxin leads to increased cell death. Our results suggest that Noxin may be a component of the cell defense system: it is activated by various stress stimuli, helps cells to withdraw from cycling, and opposes apoptosis. [Abstract/Link to Full Text]

Lee KY, Jeong JW, Wang J, Ma L, Martin JF, Tsai SY, Lydon JP, DeMayo FJ
Bmp2 is critical for the murine uterine decidual response.
Mol Cell Biol. 2007 Aug;27(15):5468-78.
The process of implantation, necessary for all viviparous birth, consists of tightly regulated events, including apposition of the blastocyst, attachment to the uterine lumen, and differentiation of the uterine stroma. In rodents and primates the uterine stroma undergoes a process called decidualization. Decidualization, the process by which the uterine endometrial stroma proliferates and differentiates into large epithelioid decidual cells, is critical to the establishment of fetal-maternal communication and the progression of implantation. The role of bone morphogenetic protein 2 (Bmp2) in regulating the transformation of the uterine stroma during embryo implantation in the mouse was investigated by the conditional ablation of Bmp2 in the uterus using the (PR-cre) mouse. Bmp2 gene ablation was confirmed by real-time PCR analysis in the PR-cre; Bmp2fl/fl (termed Bmp2d/d) uterus. While littermate controls average 0.9 litter of 6.2+/-0.7 pups per month, Bmp2d/d females are completely infertile. Analysis of the infertility indicates that whereas embryo attachment is normal in the Bmp2d/d as in control mice, the uterine stroma is incapable of undergoing the decidual reaction to support further embryonic development. Recombinant human BMP2 can partially rescue the decidual response, suggesting that the observed phenotypes are not due to a developmental consequence of Bmp2 ablation. Microarray analysis demonstrates that ablation of Bmp2 leads to specific gene changes, including disruption of the Wnt signaling pathway, Progesterone receptor (PR) signaling, and the induction of prostaglandin synthase 2 (Ptgs2). Taken together, these data demonstrate that Bmp2 is a critical regulator of gene expression and function in the murine uterus. [Abstract/Link to Full Text]

Pérez-Fernández J, Román A, De Las Rivas J, Bustelo XR, Dosil M
The 90S preribosome is a multimodular structure that is assembled through a hierarchical mechanism.
Mol Cell Biol. 2007 Aug;27(15):5414-29.
The 90S preribosomal particle is required for the production of the 18S rRNA from a pre-rRNA precursor. Despite the identification of the protein components of this particle, its mechanism of assembly and structural design remain unknown. In this work, we have combined biochemical studies, proteomic techniques, and bioinformatic analyses to shed light into the rules of assembly of the yeast 90S preribosome. Our results indicate that several protein subcomplexes work as discrete assembly subunits that bind in defined steps to the 35S pre-rRNA. The assembly of the t-UTP subunit is an essential step for the engagement of at least five additional subunits in two separate, and mutually independent, assembling routes. One of these routes leads to the formation of an assembly intermediate composed of the U3 snoRNP, the Pwp2p/UTP-B, subunit and the Mpp10p complex. The other assembly route involves the stepwise binding of Rrp5p and the UTP-C subunit. We also report the use of a bioinformatic approach that provides a model for the topological arrangement of protein components within the fully assembled particle. Together, our data identify the mechanism of assembly of the 90S preribosome and offer novel information about its internal architecture. [Abstract/Link to Full Text]

Liu YC, Chen HC, Wu NY, Cheng SC
A novel splicing factor, Yju2, is associated with NTC and acts after Prp2 in promoting the first catalytic reaction of pre-mRNA splicing.
Mol Cell Biol. 2007 Aug;27(15):5403-13.
The Prp19-associated complex (NTC) is essential for pre-mRNA splicing and is associated with the spliceosome during spliceosome activation. NTC is required for specifying interactions of U5 and U6 with pre-mRNA to stabilize their association with the spliceosome after dissociation of U4. Here, we show that a novel splicing factor, Yju2, is associated with components of NTC, and that it is required for pre-mRNA splicing both in vivo and in vitro. During spliceosome assembly, Yju2 is associated with the spliceosome at nearly the same time as NTC but is destabilized after the first catalytic reaction, whereas other NTC components remain associated until the reaction is complete. Extracts depleted of Yju2 could be complemented by recombinant Yju2, suggesting that Yju2 and NTC are not entirely in association with each other. Yju2 is not required for the binding of NTC to the spliceosome or for NTC-mediated spliceosome activation. Complementation analysis of the affinity-isolated spliceosome formed in Yju2-depleted extracts demonstrated that Yju2 acts in concert with an unidentified heat-resistant factor(s) in an ATP-independent manner to promote the first catalytic reaction of pre-mRNA splicing after Prp2-mediated structural rearrangement of the spliceosome. [Abstract/Link to Full Text]

Seiler JA, Conti C, Syed A, Aladjem MI, Pommier Y
The intra-S-phase checkpoint affects both DNA replication initiation and elongation: single-cell and -DNA fiber analyses.
Mol Cell Biol. 2007 Aug;27(16):5806-18.
To investigate the contribution of DNA replication initiation and elongation to the intra-S-phase checkpoint, we examined cells treated with the specific topoisomerase I inhibitor camptothecin. Camptothecin is a potent anticancer agent producing well-characterized replication-mediated DNA double-strand breaks through the collision of replication forks with topoisomerase I cleavage complexes. After a short dose of camptothecin in human colon carcinoma HT29 cells, DNA replication was inhibited rapidly and did not recover for several hours following drug removal. That inhibition occurred preferentially in late-S-phase, compared to early-S-phase, cells and was due to both an inhibition of initiation and elongation, as determined by pulse-labeling nucleotide incorporation in replication foci and DNA fibers. DNA replication was actively inhibited by checkpoint activation since 7-hydroxystaurosporine (UCN-01), the specific Chk1 inhibitor CHIR-124, or transfection with small interfering RNA targeting Chk1 restored both initiation and elongation. Abrogation of the checkpoint markedly enhanced camptothecin-induced DNA damage at replication sites where histone gamma-H2AX colocalized with replication foci. Together, our study demonstrates that the intra-S-phase checkpoint is exerted by Chk1 not only upon replication initiation but also upon DNA elongation. [Abstract/Link to Full Text]

---

Recent Articles in Journal of Cell Science

---

Zenner HL, Collinson LM, Michaux G, Cutler DF
High-pressure freezing provides insights into Weibel-Palade body biogenesis.
J Cell Sci. 2007 Jun 15;120(Pt 12):2117-25.
The Weibel-Palade bodies (WPBs) of endothelial cells play an important role in haemostasis and the initiation of inflammation, yet their biogenesis is poorly understood. Tubulation of their major content protein, von Willebrand factor (VWF), is crucial to WPB function, and so we investigated further the relationship between VWF tubule formation and WPB formation in human umbilical vein endothelial cells (HUVECs). By using high-pressure freezing and freeze substitution before electron microscopy, we visualised VWF tubules in the trans-Golgi network (TGN), as well as VWF subunits in vesicular structures. Tubules were also seen in WPBs that were connected to the TGN by membranous stalks. Tubules are disorganised in the immature WPBs but during maturation we found a dramatic increase in the spatial organisation of the tubules and in organelle electron density. We also found coated budding profiles suggestive of the removal of missorted material after initial formation of these granules. Finally, we discovered that these large, seemingly rigid, organelles flex at hinge points and that the VWF tubules are interrupted at these hinges, facilitating organelle movement around the cell. The use of high-pressure freezing was vital in this study and it suggests that this technique might prove essential to any detailed characterisation of organelle biogenesis. [Abstract/Link to Full Text]

Ma N, Matsunaga S, Takata H, Ono-Maniwa R, Uchiyama S, Fukui K
Nucleolin functions in nucleolus formation and chromosome congression.
J Cell Sci. 2007 Jun 15;120(Pt 12):2091-105.
A complex structure, designated the chromosome periphery, surrounds each chromosome during mitosis. Although several proteins have been shown to localize to the chromosome periphery, their functions during mitosis remain unclear. Here, we used a combination of high-resolution microscopy and RNA-interference-mediated depletion to study the functions of nucleolin, a nucleolar protein localized at the chromosome periphery, in interphase and mitosis. During mitosis, nucleolin was localized in the peripheral region including the vicinity of the outer kinetochore of chromosomes. Staining with an antibody specific for nucleolin phosphorylated by CDC2 revealed that nucleolin was also associated with the spindle poles from prometaphase to anaphase. Nucleolin depletion resulted in disorganization of the nucleoli at interphase. Furthermore, nucleolin-depleted cells showed a prolonged cell cycle with misaligned chromosomes and defects in spindle organization. The misaligned chromosomes showed syntelic kinetochore-microtubule attachments with reduced centromere stretching. Taken together, our results indicate that nucleolin is required for nucleolus formation, and is also involved in chromosome congression and spindle formation. [Abstract/Link to Full Text]

Haraguchi T, Koujin T, Osakada H, Kojidani T, Mori C, Masuda H, Hiraoka Y
Nuclear localization of barrier-to-autointegration factor is correlated with progression of S phase in human cells.
J Cell Sci. 2007 Jun 15;120(Pt 12):1967-77.
Barrier-to-autointegration factor (BAF) is a conserved metazoan protein that plays a critical role in retrovirus infection. To elucidate its role in uninfected cells, we first examined the localization of BAF in both mortal and immortal or cancerous human cell lines. In mortal cell lines (e.g. TIG-1, WI-38 and IMR-90 cells) BAF localization depended on the age of the cell, localizing primarily in the nucleus of >90% of young proliferating cells but only 20-25% of aged senescent cells. In immortal cell lines (e.g. HeLa, SiHa and HT1080 cells) BAF showed heterogeneous localization between the nucleus and cytoplasm. This heterogeneity was lost when the cells were synchronized in S phase. In S-phase-synchronized populations, the percentage of cells with predominantly nuclear BAF increased from 30% (asynchronous controls) to approximately 80%. In HeLa cells, RNAi-induced downregulation of BAF significantly increased the proportion of early S-phase cells that retained high levels of cyclin D3 and cyclin E expression and slowed progression through early S phase. BAF downregulation also caused lamin A to mislocalize away from the nuclear envelope. These results indicate that BAF is required for the integrity of the nuclear lamina and normal progression of S phase in human cells. [Abstract/Link to Full Text]

Reyes A, Sanz M, Duran A, Roncero C
Chitin synthase III requires Chs4p-dependent translocation of Chs3p into the plasma membrane.
J Cell Sci. 2007 Jun 15;120(Pt 12):1998-2009.
In Saccharomyces cerevisiae, Chs4p is required for chitin synthase III (CSIII) activity and hence for chitin synthesis. This protein is transported in vesicles in a polarized fashion independently of the other Chs proteins. Its association with membranes depends not only on prenylation, but also on its interaction with other proteins, mainly Chs3p, which is the catalytic subunit of CSIII and is able to properly direct Chs4p to the bud neck in the absence of prenylation. Chs4p is present in functionally limiting amounts and its overexpression increases Chs3p accumulation at the plasma membrane with a concomitant increase in chitin synthesis. In the absence of Chs4p, Chs3p is delivered to the plasma membrane but fails to accumulate there because it is rapidly endocytosed and accumulates in intracellular vesicles. A blockade of endocytosis stops Chs3p internalization, triggering a significant increase in chitin synthesis. This blockade is independent of Chs4p function, allowing the accumulation of Chs3p at the plasma membrane even in the chs4Delta mutant. However, the absence of Chs4p renders CSIII functionally inactive, independently of Chs3p accumulation at the plasma membrane. Chs4p thus promotes Chs3p translocation into the plasma membrane in a stable and active form. Proper CSIII turnover is maintained through the endocytic internalization of Chs3p. [Abstract/Link to Full Text]

Yao MC, Yao CH, Halasz LM, Fuller P, Rexer CH, Wang SH, Jain R, Coyne RS, Chalker DL
Identification of novel chromatin-associated proteins involved in programmed genome rearrangements in Tetrahymena.
J Cell Sci. 2007 Jun 15;120(Pt 12):1978-89.
Extensive DNA rearrangements occur during the differentiation of the developing somatic macronuclear genome from the germ line micronuclear genome of Tetrahymena thermophila. To identify genes encoding proteins likely to be involved in this process, we devised a cytological screen to find proteins that specifically localize in macronuclear anlagen (Lia proteins) at the stage when rearrangements occur. We compared the localization of these with that of the chromodomain protein, Pdd1p, which is the most abundant known participant in this genome reorganization. We show that in live cells, Pdd1p exhibits dynamic localization, apparently shuttling from the parental to the developing nuclei through cytoplasmic bodies called conjusomes. Visualization of GFP-tagged Pdd1p also highlights the substantial three-dimensional nuclear reorganization in the formation of nuclear foci that occur coincident with DNA rearrangements. We found that late in macronuclear differentiation, four of the newly identified proteins are organized into nuclear foci that also contain Pdd1p. These Lia proteins are encoded by primarily novel genes expressed at the beginning of macronuclear differentiation and have properties or recognizable domains that implicate them in chromatin or nucleic acid binding. Three of the Lia proteins also localize to conjusomes, a result that further implicates this structure in the regulation of DNA rearrangement. [Abstract/Link to Full Text]

Huang JY, Morley G, Li D, Whitaker M
Cdk1 phosphorylation sites on Cdc27 are required for correct chromosomal localisation and APC/C function in syncytial Drosophila embryos.
J Cell Sci. 2007 Jun 15;120(Pt 12):1990-7.
Anaphase-promoting complex or cyclosome (APC/C) controls the metaphase-to-anaphase transition and mitosis exit by triggering the degradation of key cell cycle regulators such as securin and B-type cyclins. However, little is known about the functions of individual APC/C subunits and how they might regulate APC/C activity in space and time. Here, we report that two potential Cdk1 kinase phosphorylation sites are required for the chromosomal localisation of GFP::Cdc27 during mitosis. Either or both of the highly conserved proline residues in the Cdk1 phosphorylation consensus sequence motifs were mutated to alanine (Cdc27 P304A or P456A). The singly mutated fusion proteins, GFP::Cdc27P304A and GFP::Cdc27P456A, can still localise to mitotic chromosomes in a manner identical to wild-type GFP::Cdc27 and are functional in that they can rescue the phenotype of the cdc27L7123 mutant in vivo. However, when both of the Cdk1 phosphorylation sequence motifs were mutated, the resulting GFP::Cdc27P304A,P456A construct was not localised to the chromosomes during mitosis and was no longer functional, as it failed to rescue mutant phenotypes of the cdc27L7123 gene. High levels of cyclin B and cyclin A were detected in mutant third instar larvae brain samples compared with its wild-type control. These results show for the first time that the two potential Cdk1 phosphorylation sites on Drosophila Cdc27 are required for its chromosomal localisation during mitosis and imply that these localisations specific to Cdc27 are crucial for APC/C functions. [Abstract/Link to Full Text]

Negrini M, Ferracin M, Sabbioni S, Croce CM
MicroRNAs in human cancer: from research to therapy.
J Cell Sci. 2007 Jun 1;120(Pt 11):1833-40.
Numerous miRNAs are deregulated in human cancers, and experimental evidence indicates that they can play roles as oncogenes or tumor suppressor genes. Similarly to cancer genes that encode proteins, deregulation of miRNA-encoding genes is associated with genetic or epigenetic alterations, such as deletions, amplifications, point mutations and aberrant DNA methylation. The discovery that miRNAs interact with known oncogenes has established further links with molecular pathways implicated in malignant transformation. Finally, miRNAs can be used as diagnostic markers, and their potential as therapeutic molecules has moved miRNAs from the area of basic research to the field of cancer biotechnology. [Abstract/Link to Full Text]

Kirkpatrick CA, Selleck SB
Heparan sulfate proteoglycans at a glance.
J Cell Sci. 2007 Jun 1;120(Pt 11):1829-32. [Abstract/Link to Full Text]

Li CR, Lee RT, Wang YM, Zheng XD, Wang Y
Candida albicans hyphal morphogenesis occurs in Sec3p-independent and Sec3p-dependent phases separated by septin ring formation.
J Cell Sci. 2007 Jun 1;120(Pt 11):1898-907.
The growing tips of Candida albicans hyphae are sites of polarized exocytosis. Mammalian septins have been implicated in regulating exocytosis and C. albicans septins are known to localize at hyphal tips, although their function here is unknown. Here, we report that C. albicans cells deleted of the exocyst subunit gene SEC3 can grow normal germ tubes, but are unable to maintain tip growth after assembly of the first septin ring, resulting in isotropic expansion of the tip. Deleting either of the septin genes CDC10 or CDC11 caused Sec3p mislocalization and surprisingly, also restored hyphal development in the sec3Delta mutant without rescuing the temperature sensitivity. Co-immunoprecipitation experiments detected association of the septin Cdc3p with the exocyst subunits Sec3p and Sec5p. Our results reveal that C. albicans hyphal development occurs through Sec3p-independent and dependent phases, and provide strong genetic and biochemical evidence for a role of septins in polarized exocytosis. [Abstract/Link to Full Text]

Guidarelli A, Cerioni L, Cantoni O
Inhibition of complex III promotes loss of Ca2+ dependence for mitochondrial superoxide formation and permeability transition evoked by peroxynitrite.
J Cell Sci. 2007 Jun 1;120(Pt 11):1908-14.
In intact U937 cells, peroxynitrite promotes the mitochondrial formation of superoxide via a Ca2+-dependent mechanism involving inhibition of complex III. Superoxide then readily dismutates to H2O2 causing lesions on different biomolecules, including DNA. Here we show that formation of H2O2 and DNA damage are suppressed by inhibition of complex I (by rotenone) or ubisemiquinone formation (by myxothiazol), as well as by a variety of manipulations preventing either the mobilization of Ca2+ or its mitochondrial accumulation. In addition, complex III inhibitors promoted rotenone- or myxothiazol-sensitive formation of H2O2 and DNA strand scission in cells exposed to otherwise inactive concentrations of peroxynitrite. However, under these conditions, the intra-mitochondrial concentration of Ca2+ remained unchanged and the effects of peroxynitrite therefore take place via Ca2+-independent mechanisms. H2O2 formation was paralleled by, and causally linked to, the loss of mitochondrial membrane potential associated with the mitochondrial release of cytochrome c and AIF, and with the mitochondrial accumulation of Bax. These events, although Ca2+ independent, were rapidly followed by death mediated by mitochondrial permeability transition, generally considered a typical Ca2+-dependent event. Thus, enforced inhibition of complex III promotes the loss of Ca2+ dependence of those mitochondrial mechanisms regulating superoxide formation and mitochondrial permeability transition evoked by peroxynitrite. [Abstract/Link to Full Text]

Wu S, Rhee KJ, Zhang M, Franco A, Sears CL
Bacteroides fragilis toxin stimulates intestinal epithelial cell shedding and gamma-secretase-dependent E-cadherin cleavage.
J Cell Sci. 2007 Jun 1;120(Pt 11):1944-52.
Enterotoxigenic Bacteroides fragilis - organisms that live in the colon - secrete a metalloprotease toxin, B. fragilis toxin. This toxin binds to a specific intestinal epithelial cell receptor and stimulates cell proliferation, which is dependent, in part, on E-cadherin degradation and beta-catenin-T-cell-factor nuclear signaling. Gamma-secretase (or presenilin-1) is an intramembrane cleaving protease and is a positive regulator of E-cadherin cleavage and a negative regulator of beta-catenin signaling. Here we examine the mechanistic details of toxin-initiated E-cadherin cleavage. B. fragilis toxin stimulated shedding of cell membrane proteins, including the 80 kDa E-cadherin ectodomain. Shedding of this domain required biologically active toxin and was not mediated by MMP-7, ADAM10 or ADAM17. Inhibition of gamma-secretase blocked toxin-induced proteolysis of the 33 kDa intracellular E-cadherin domain causing cell membrane retention of a distinct beta-catenin pool without diminishing toxin-induced cell proliferation. Unexpectedly, gamma-secretase positively regulated basal cell proliferation dependent on the beta-catenin-T-cell-factor complex. We conclude that toxin induces step-wise cleavage of E-cadherin, which is dependent on toxin metalloprotease and gamma-secretase. Our results suggest that differentially regulated beta-catenin pools associate with the E-cadherin-gamma-secretase adherens junction complex; one pool regulated by gamma-secretase is important to intestinal epithelial cell homeostasis. [Abstract/Link to Full Text]

Chuang YY, Valster A, Coniglio SJ, Backer JM, Symons M
The atypical Rho family GTPase Wrch-1 regulates focal adhesion formation and cell migration.
J Cell Sci. 2007 Jun 1;120(Pt 11):1927-34.
Wrch-1 (Wnt-regulated Cdc42 homolog) is a new member of the Rho family that was identified as a gene transcriptionally upregulated by Wnt-1. Wrch-1 has no detectable GTPase activity and displays very high intrinsic guanine nucleotide exchange, implying that it is constitutively GTP-bound. The biological functions of Wrch-1 largely remain to be characterized. Here, we report that Wrch-1 prominently localizes to focal adhesions. Depletion of Wrch-1 by small interfering RNA increases focal adhesion formation, whereas Wrch-1 overexpression disassembles focal adhesions. Wrch-1 depletion inhibits myosin-light-chain phosphorylation, which in turn leads to an increase in the number of focal adhesions and inhibits cell migration in response to wound healing. Depletion of Wrch-1 also inhibits Akt and JNK activation. Although pharmacological inhibitors of Akt and JNK inhibit cell migration, they do not affect focal adhesions. Thus, our data suggest that Wrch-1 regulates cell migration by multiple mechanisms: on the one hand Wrch-1 controls focal adhesions by regulating myosin light chain and on the other hand Wrch-1 stimulates the activation of Akt and JNK. [Abstract/Link to Full Text]

Alfredsson-Timmins J, Henningson F, Bjerling P
The Clr4 methyltransferase determines the subnuclear localization of the mating-type region in fission yeast.
J Cell Sci. 2007 Jun 1;120(Pt 11):1935-43.
The genome has a non-random spatial distribution in the cell nucleus. In Schizosaccharomyces pombe, it has been shown that the centromeres, telomeres and the mating-type region localize to the nuclear membrane (NM), the former by attaching to the spindle pole body (SPB). In addition, reporter genes inserted into these areas are transcriptionally repressed because of the formation of specialized chromatin structures. Performing live cell analysis we found that in a wild-type strain the mating-type region was positioned in the proximity of the SPB, the location where the pericentromeric heterochromatin is also found. In a strain lacking the histone methyltransferase Clr4, crucial for the formation of heterochromatin, the mating-type region had a random localization in the nucleus. Moreover, in a strain in which the two boundary elements IR-L and IR-R had been deleted, the mating-type region was displaced from its position at the proximity of the SPB, but remained in the vicinity of the NM. Moreover, in all investigated strains with silencing deficiencies the distance between the mating-type region and the SPB increased. This result indicates a correlation between transcriptional derepression and displacement of the region. Two different models of how the mating-type chromatin is organized in the nucleus are discussed. [Abstract/Link to Full Text]

Hajj GN, Lopes MH, Mercadante AF, Veiga SS, da Silveira RB, Santos TG, Ribeiro KC, Juliano MA, Jacchieri SG, Zanata SM, Martins VR
Cellular prion protein interaction with vitronectin supports axonal growth and is compensated by integrins.
J Cell Sci. 2007 Jun 1;120(Pt 11):1915-26.
The physiological functions of the cellular prion protein, PrP(C), as a cell surface pleiotropic receptor are under debate. We report that PrP(C) interacts with vitronectin but not with fibronectin or collagen. The binding sites mediating this PrP(C)-vitronectin interaction were mapped to residues 105-119 of PrP(C) and the residues 307-320 of vitronectin. The two proteins were co-localized in embryonic dorsal root ganglia from wild-type mice. Vitronectin addition to cultured dorsal root ganglia induced axonal growth, which could be mimicked by vitronectin peptide 307-320 and abrogated by anti-PrP(C) antibodies. Full-length vitronectin, but not the vitronectin peptide 307-320, induced axonal growth of dorsal root neurons from two strains of PrP(C)-null mice. Functional assays demonstrated that relative to wild-type cells, PrP(C)-null dorsal root neurons were more responsive to the Arg-Gly-Asp peptide (an integrin-binding site), and exhibited greater alphavbeta3 activity. Our findings indicate that PrP(C) plays an important role in axonal growth, and this function may be rescued in PrP(C)-knockout animals by integrin compensatory mechanisms. [Abstract/Link to Full Text]

Jovceva E, Larsen MR, Waterfield MD, Baum B, Timms JF
Dynamic cofilin phosphorylation in the control of lamellipodial actin homeostasis.
J Cell Sci. 2007 Jun 1;120(Pt 11):1888-97.
During animal cell chemotaxis, signalling at the plasma membrane induces actin polymerisation to drive forward cell movement. Since the cellular pool of actin is limited, efficient protrusion formation also requires the coordinated disassembly of pre-existing actin filaments. To search for proteins that can monitor filamentous and globular actin levels to maintain the balance of polymerisation and disassembly, we followed changes in the proteome induced by RNA interference (RNAi)-mediated alterations in actin signalling. This unbiased approach revealed an increase in the levels of an inactive, phosphorylated form of the actin-severing protein cofilin in cells unable to generate actin-based lamellipodia. Conversely, an increase in F-actin levels induced the dephosphorylation and activation of cofilin via activation of the Ssh phosphatase. Similarly, in the context of acute phosphoinositide 3-kinase (PI3K) signalling, dynamic changes in cofilin phosphorylation were found to depend on the Ssh phosphatase and on changes in lamellipodial F-actin. These results indicate that changes in the extent of cofilin phosphorylation are regulated by Ssh in response to changes in the levels and/or organisation of F-actin. Together with the recent finding that Ssh phosphatase activity is augmented by F-actin binding, these results identify Ssh-dependent regulation of phosphorylated cofilin levels as an important feedback control mechanism that maintains actin filament homeostasis during actin signalling. [Abstract/Link to Full Text]

Bryant DM, Kerr MC, Hammond LA, Joseph SR, Mostov KE, Teasdale RD, Stow JL
EGF induces macropinocytosis and SNX1-modulated recycling of E-cadherin.
J Cell Sci. 2007 May 15;120(Pt 10):1818-28.
In epithelia, junction proteins are endocytosed for modulation of cell-cell adhesion and cell polarity. In response to growth factors, the cell-cell adhesion protein E-cadherin is internalized from the cell surface with degradation or recycling as potential fates. However, the cellular machinery involved in cadherin internalization and recycling remains controversial. Here we investigated EGF-induced E-cadherin internalization. EGF stimulation of MCF-7 cells resulted in Rac1-modulated macropinocytosis of the E-cadherin-catenin complex into endosomal compartments that colocalized with EEA1 and the sorting nexin, SNX1. Depletion of cellular SNX1 levels by siRNA resulted in increased intracellular accumulation and turnover of E-cadherin internalized from the cell surface in response to EGF. Moreover, SNX1 was also required for efficient recycling of internalized E-cadherin and re-establishment of epithelial adhesion. Together, these findings demonstrate a role for SNX1 in retrieval of E-cadherin from a degradative endosomal pathway and in membrane trafficking pathways that regulate E-cadherin recycling. [Abstract/Link to Full Text]

Ramsauer M, D'Amore PA
Contextual role for angiopoietins and TGFbeta1 in blood vessel stabilization.
J Cell Sci. 2007 May 15;120(Pt 10):1810-7.
We used a 3D in-vitro model of angiogenesis to investigate the effects of different growth factors on vessel formation and stabilization in vitro. Vascular endothelial growth factor (VEGF) was the only factor that induced the formation, elongation and sprouting of capillary-like structures (CLS) by bovine retinal capillary endothelial cells (BREC), an effect that was dose-dependent and saturable. Basic fibroblast growth factor 2 (FGF2) enhanced capillary formation in the presence of VEGF, leading to a more complex network of CLS and a higher rate of BrdU incorporation than VEGF alone, indicating that whereas VEGF acts as a morphogen, FGF2 is primarily a mitogen. Addition of transforming growth factor beta1 (TGFbeta1) to the 3D assay along with VEGF and FGF2, reduced tube formation in a dose-dependent manner. When added at the time of cell plating TGFbeta1 completely suppressed formation of VEGF/FGF2-stimulated CLS. Angiopoietin 1 (Ang1) prevented regression of the TGFbeta1-induced CLS, an effect that was blocked by angiopoietin 2 (Ang2), but required the continuous presence of VEGF. [Abstract/Link to Full Text]

Tateishi K, Ashihara E, Takehara N, Nomura T, Honsho S, Nakagami T, Morikawa S, Takahashi T, Ueyama T, Matsubara H, Oh H
Clonally amplified cardiac stem cells are regulated by Sca-1 signaling for efficient cardiovascular regeneration.
J Cell Sci. 2007 May 15;120(Pt 10):1791-800.
Recent studies have shown that cardiac stem cells (CSCs) from the adult mammalian heart can give rise to functional cardiomyocytes; however, the definite surface markers to identify a definitive single entity of CSCs and the molecular mechanisms regulating their growth are so far unknown. Here, we demonstrate a single-cell deposition analysis to isolate individually selected CSCs from adult murine hearts and investigate the signals required for their proliferation and survival. Clonally proliferated CSCs express stem cell antigen-1 (Sca-1) with embryonic stem (ES) cell-like and mesenchymal cell-like characteristics and are associated with telomerase reverse transcriptase (TERT). Using a transgene that expresses a GFP reporter under the control of the TERT promoter, we demonstrated that TERT(GFP)-positive fractions from the heart were enriched for cells expressing Sca-1. Knockdown of Sca-1 transcripts in CSCs led to retarded ex vivo expansion and apoptosis through Akt inactivation. We also show that ongoing CSC proliferation and survival after direct cell-grafting into ischemic myocardium require Sca-1 to upregulate the secreted paracrine effectors that augment neoangiogenesis and limit cardiac apoptosis. Thus, Sca-1 might be an essential component to promote CSC proliferation and survival to directly facilitate early engraftment, and might indirectly exert the effects on late cardiovascular differentiation after CSC transplantation. [Abstract/Link to Full Text]

Levasseur M, Carroll M, Jones KT, McDougall A
A novel mechanism controls the Ca2+ oscillations triggered by activation of ascidian eggs and has an absolute requirement for Cdk1 activity.
J Cell Sci. 2007 May 15;120(Pt 10):1763-71.
Fertilisation in ascidians triggers a series of periodic rises in cytosolic Ca(2+) that are essential for release from metaphase I arrest and progression through meiosis II. These sperm-triggered Ca(2+) oscillations are switched off at exit from meiosis II. Ascidian zygotes provided the first demonstration of the positive feedback loop whereby elevated Cdk1 activity maintained these Ca(2+) oscillations. Since then it has been reported that Cdk1 sensitises the type I inositol trisphosphate [Ins(1,4,5)P(3)] receptor in somatic cells, and that sperm-triggered Ca(2+) oscillations in mouse zygotes stop because the forming pronuclei sequester phospholipase C zeta that was delivered to the egg by the fertilising sperm. Here, using enucleation, we demonstrate in ascidian eggs that Ca(2+) spiking stops at the correct time in the absence of pronuclei. Sequestration of sperm factor is therefore not involved in terminating Ca(2+) spiking for these eggs. Instead we found that microinjection of the Cdk1 inhibitor p21 blocked Ca(2+) spiking induced by ascidian sperm extract (ASE). However, such eggs were still capable of releasing Ca(2+) in response to Ins(1,4,5)P(3) receptor agonists, indicating that ASE-triggered Ca(2+) oscillations can stop even though the response to Ins(1,4,5)P(3) remained elevated. These data suggest that Cdk1 activity promotes Ins(1,4,5)P(3) production in the presence of the sperm factor, rather than sensitising the Ca(2+) releasing machinery to Ins(1,4,5)P(3). These findings suggest a new link between this cell cycle kinase and the Ins(1,4,5)P(3) pathway. [Abstract/Link to Full Text]

O'Connell CB, Khodjakov AL
Cooperative mechanisms of mitotic spindle formation.
J Cell Sci. 2007 May 15;120(Pt 10):1717-22.
Cooperativity is well known to promote the speed of some biochemical reactions by accelerating the activity of enzymes. Recent studies have shown that cooperative interactions also function during the formation of a complex cellular structure, the mitotic spindle. Capture of kinetochores by dynamic astral microtubules was originally proposed as the basis of spindle formation. However, mounting evidence indicates that a more complex series of events occurs. It is now clear that there are multiple microtubule nucleation and capture sites throughout the spindle. Kinetochores, centrosomes and microtubules play multiple roles in establishing connections between spindle components and integrating them into a common structure. These data support a modified search-and-capture model that incorporates additional assembly pathways coordinated by a RanGTP gradient. [Abstract/Link to Full Text]

Mandemakers W, Morais VA, De Strooper B
A cell biological perspective on mitochondrial dysfunction in Parkinson disease and other neurodegenerative diseases.
J Cell Sci. 2007 May 15;120(Pt 10):1707-16.
Dysfunction of mitochondria is frequently proposed to be involved in neurodegenerative disease. Deficiencies in energy supply, free radical generation, Ca(2+) buffering or control of apoptosis, could all theoretically contribute to progressive decline of the central nervous system. Parkinson disease illustrates how mutations in very different genes finally impinge directly or indirectly on mitochondrial function, causing subtle but finally fatal dysfunction of dopaminergic neurons. Neurons in general appear more sensitive than other cells to mutations in genes encoding mitochondrial proteins. Particularly interesting are mutations in genes such as Opa1, Mfn1 and Dnm1l, whose products are involved in the dynamic morphological alterations and subcellular trafficking of mitochondria. These indicate that mitochondrial dynamics are especially important for the long-term maintenance of the nervous system. The emerging evidence clearly demonstrates the crucial role of specific mitochondrial functions in maintaining neuronal circuit integrity. [Abstract/Link to Full Text]

Brown CM
Fluorescence microscopy--avoiding the pitfalls.
J Cell Sci. 2007 May 15;120(Pt 10):1703-5. [Abstract/Link to Full Text]

Berto G, Camera P, Fusco C, Imarisio S, Ambrogio C, Chiarle R, Silengo L, Di Cunto F
The Down syndrome critical region protein TTC3 inhibits neuronal differentiation via RhoA and Citron kinase.
J Cell Sci. 2007 Jun 1;120(Pt 11):1859-67.
The Down syndrome critical region (DSCR) on Chromosome 21 contains many genes whose duplication may lead to the major phenotypic features of Down syndrome and especially the associated mental retardation. However, the functions of DSCR genes are mostly unknown and their possible involvement in key brain developmental events still largely unexplored. In this report we show that the protein TTC3, encoded by one of the main DSCR candidate genes, physically interacts with Citron kinase (CIT-K) and Citron N (CIT-N), two effectors of the RhoA small GTPase that have previously been involved in neuronal proliferation and differentiation. More importantly, we found that TTC3 levels can strongly affect the NGF-induced differentiation of PC12 cells, by a CIT-K-dependent mechanism. Indeed, TTC3 overexpression leads to strong inhibition of neurite extension, which can be reverted by CIT-K RNAi. Conversely, TTC3 knockdown stimulates neurite extension in the same cells. Finally, we find that Rho, but not Rho kinase, is required for TTC3 differentiation-inhibiting activity. Our results suggest that the TTC3-RhoA-CIT-K pathway could be a crucial determinant of in vivo neuronal development, whose hyperactivity may result in detrimental effects on the normal differentiation program. [Abstract/Link to Full Text]

Su Z, Cao L, Zhu Y, Liu X, Huang Z, Huang A, He C
Nogo enhances the adhesion of olfactory ensheathing cells and inhibits their migration.
J Cell Sci. 2007 Jun 1;120(Pt 11):1877-87.
The migration of olfactory ensheathing cells (OECs) is essential for pioneering the olfactory nerve pathway during development and for promoting axonal regeneration when implanted into the injured central nervous system (CNS). In the present study, recombinant Nogo-66 enhanced the adhesion of OECs and inhibited their migration. Using immunocytochemistry and western blot, we showed that the Nogo-66 receptor (NgR) was expressed on OECs. When NgR was released from the cell surface with phosphatidylinositol-specific phospholipase C or neutralized by NgR antibody, the effect of Nogo-66 on OEC adhesion and migration was markedly attenuated. Nogo-66 was found to promote the formation of focal adhesion in OECs and inhibited their membrane protrusion through the activation of RhoA. Furthermore, the co-culture migration assay demonstrated that OEC motility was significantly restricted by Nogo-A expressed on Cos7 cell membranes or oligodendrocytes. Moreover, treatment with anti-NgR antibody facilitated migration of implanted OECs in a spinal cord hemisection injury model. Taken together, we demonstrate, for the first time, that Nogo, a myelin-associated inhibitor of axon regeneration in the CNS, enhances the adhesion and inhibits the migration of OECs via NgR regulation of RhoA. [Abstract/Link to Full Text]

Baarends WM, Wassenaar E, Hoogerbrugge JW, Schoenmakers S, Sun ZW, Grootegoed JA
Increased phosphorylation and dimethylation of XY body histones in the Hr6b-knockout mouse is associated with derepression of the X chromosome.
J Cell Sci. 2007 Jun 1;120(Pt 11):1841-51.
Mono-ubiquitylated H2A marks the transcriptionally silenced XY body during male meiotic prophase. Concomitant with H2A(K119ub1), the ubiquitin-conjugating enzyme HR6B is also enriched on the XY body. We analyzed H2A and H2B ubiquitylation in Hr6b-knockout mouse spermatocytes, but no global changes were detected. Next, we analyzed phosphorylation of the threonine residues T120 and T119 that are adjacent to the K119 and K120 target sites for ubiquitylation in H2A and H2B, respectively. In wild-type cells, H2A(T120ph) and H2B(T119ph) mark meiotically unpaired and silenced chromatin, including the XY body. In Hr6b-knockout spermatocytes, the H2B(T119ph) signal was unchanged, but H2A(T120ph) was enhanced from late pachytene until metaphase I. Furthermore, we found increased H3(K4) dimethylation on the X and Y chromosomes of diplotene Hr6b-knockout spermatocytes, persisting into postmeiotic round spermatids. In these cells, the X and Y chromosomes maintained an unchanged H3(K9m2) level, even when this modification was lost from centromeric heterochromatin. Analysis of gene expression showed derepression of X chromosome genes in postmeiotic Hr6b-knockout spermatids. We conclude that HR6B exerts control over different histone modifications in spermatocytes and spermatids, and that this function contributes to the postmeiotic maintenance of X chromosome silencing. [Abstract/Link to Full Text]

Di Certo MG, Corbi N, Bruno T, Iezzi S, De Nicola F, Desantis A, Ciotti MT, Mattei E, Floridi A, Fanciulli M, Passananti C
NRAGE associates with the anti-apoptotic factor Che-1 and regulates its degradation to induce cell death.
J Cell Sci. 2007 Jun 1;120(Pt 11):1852-8.
Neurotrophin receptor-interacting MAGE homolog (NRAGE) has been recently identified as a cell-death inducer, involved in molecular events driving cells through apoptotic networks during neuronal development. Recently, we have focused on the functional role of Che-1, also known as apoptosis-antagonizing transcription factor (AATF), a protein involved in cell cycle control and gene transcription. Increasing evidence suggests that Che-1 is involved in apoptotic signalling in neural tissues. In cortical neurons Che-1 exhibits an anti-apoptotic activity, protecting cells from neuronal damage induced by amyloid beta-peptide. Here, we report that Che-1 interacts with NRAGE and that an EGFP-NRAGE fusion protein inhibits nuclear localization of Che-1, by sequestering it within the cytoplasmic compartment. Furthermore, NRAGE overexpression downregulates endogenous Che-1 by targeting it for proteasome-dependent degradation. Finally, we propose that Che-1 is a functional antagonist of NRAGE, because its overexpression completely reverts NRAGE-induced cell-death. [Abstract/Link to Full Text]

Pan J, You Y, Huang T, Brody SL
RhoA-mediated apical actin enrichment is required for ciliogenesis and promoted by Foxj1.
J Cell Sci. 2007 Jun 1;120(Pt 11):1868-76.
Programs that direct cellular differentiation are dependent on the strict temporal expression of regulatory factors that can be provided by Rho GTPases. Ciliogenesis is a complex sequence of events involving the generation and docking of basal bodies at the apical membrane, followed by ciliary axoneme generation. Although a cilia proteome has been assembled, programs that direct ciliated cell differentiation are not well established, particularly in mammalian systems. Using mouse primary culture airway epithelial cells, we identified a critical stage of ciliogenesis requiring the temporal establishment of an apical web-like structure of actin for basal body docking and subsequent axoneme growth. Apical web formation and basal body docking were prevented by interruption of actin remodeling and were dependent on RhoA activation. Additional evidence for this program was provided by analysis of Foxj1-null mice that failed to dock basal bodies and lacked apical actin. Foxj1 expression coincided with actin web formation, activated RhoA and RhoB, and persisted despite RhoA inhibition, suggesting that Foxj1 promoted RhoA during ciliogenesis. Apical ezrin localization was also dependent on Foxj1, actin remodeling, and RhoA, but was not critical for ciliogenesis. Thus, temporal Foxj1 and RhoA activity are essential regulatory events for cytoskeletal remodeling during mammalian ciliogenesis. [Abstract/Link to Full Text]

Putney JW
New molecular players in capacitative Ca2+ entry.
J Cell Sci. 2007 Jun 15;120(Pt 12):1959-65.
Capacitative Ca2+ entry links the emptying of intracellular Ca2+ stores to the activation of store-operated Ca2+ channels in the plasma membrane. In the twenty years since the inception of the concept of capacitative Ca2+ entry, a number of activation mechanisms have been proposed, and there has been considerable interest in the possibility that TRP channels function as store-operated channels. However, in the past two years, two major players in both the signaling and permeation mechanisms for store-operated channels have been discovered: Stim1 and the Orai proteins. Stim1 is an endoplasmic reticulum Ca2+ sensor. It appears to act by redistributing within a small component of the endoplasmic reticulum, approaching the plasma membrane, but does not seem to translocate into the plasma membrane. Stim1 signals to plasma membrane Orai proteins, which constitute pore-forming subunits of store-operated channels. [Abstract/Link to Full Text]

Zhao XH, Laschinger C, Arora P, Szászi K, Kapus A, McCulloch CA
Force activates smooth muscle alpha-actin promoter activity through the Rho signaling pathway.
J Cell Sci. 2007 May 15;120(Pt 10):1801-9.
In pressure or volume overload, hypertrophic growth of the myocardium is associated with myofibroblast differentiation, a process in which cardiac fibroblasts express smooth muscle alpha-actin (SMA). The signaling mechanisms that mediate force-induced myofibroblast differentiation and SMA expression are not defined. We examined the role of the Rho-Rho-kinase pathway in force-induced SMA expression in fibroblasts using an in vitro model system that applies static tensile forces (0.65 pN/microm(2)) to integrins via collagen-coated magnetite beads. Force maximally induced RhoA activation at 10 minutes that was localized to force application sites and required intact actin filaments. Force application induced phosphorylation of LIM kinase (5-10 minutes) and an early dephosphorylation of cofilin (5 minutes) that was followed by prolonged cofilin phosphorylation. These responses were blocked by Y27632, an inhibitor of Rho kinase. Force promoted actin filament assembly at force application sites (10-20 minutes), a process that required Rho kinase and cofilin. Force application induced nuclear translocation of the transcriptional co-activator MRTF-A but not MRTF-B. Nuclear translocation of MRTF-A required Rho kinase and intact actin filaments. Force caused 3.5-fold increases of SMA promoter activity that were completely blocked by transfection of cells with dominant-negative MRTF-A or by inhibition of Rho kinase or by actin filament disassembly. These data indicate that mechanical forces mediate actin assembly through the Rho-Rho-kinase-LIMK cofilin pathway. Force-mediated actin filament assembly promotes nuclear translocation of MRTF and subsequent activation of the SMA promoter to enhance SMA expression. [Abstract/Link to Full Text]

Abell BM, Rabu C, Leznicki P, Young JC, High S
Post-translational integration of tail-anchored proteins is facilitated by defined molecular chaperones.
J Cell Sci. 2007 May 15;120(Pt 10):1743-51.
Tail-anchored (TA) proteins provide an ideal model for studying post-translational integration at the endoplasmic reticulum (ER) of eukaryotes. There are multiple pathways for delivering TA proteins from the cytosol to the ER membrane yet, whereas an ATP-dependent route predominates, none of the cytosolic components involved had been identified. In this study we have directly addressed this issue and identify novel interactions between a model TA protein and the two cytosolic chaperones Hsp40 and Hsc70. To investigate their function, we have reconstituted the membrane integration of TA proteins using purified components. Remarkably, we find that a combination of Hsc70 and Hsp40 can completely substitute for the ATP-dependent factors present in cytosol. On the basis of this in vitro analysis, we conclude that this chaperone pair can efficiently facilitate the ATP-dependent integration of TA proteins. [Abstract/Link to Full Text]

---

Recent Articles in Journal of Cellular and Molecular Medicine

---

Esposito G, Scuderi C, Lu J, Savani C, De Filippis D, Iuvone T, Steardo L, Sheen V, Steardo L
S100B induces tau protein hyperphosphorylation via Dickopff-1 up-regulation and disrupts the Wnt pathway in human neural stem cells.
J Cell Mol Med. 2007 Nov 16;
Previous studies suggest that levels of the astrocyte-derived S100B protein, such as those occurring in brain extracellular spaces consequent to persistent astroglial activation, may have a pathogenetic role in Alzheimer's disease (AD). Although S100B was reported to promote beta amyloid precursor protein (APP) overexpression, no clear mechanistic relationship between S100B and formation of neurofibrillary tangles (NFTs) is established. This in vitro study has been aimed at investigating whether S100B is able to disrupt Wnt pathway and lead to tau protein hyperphosphorylation. Utilizing Western blot, EMSA, supershift and RT-PCR techniques, it has been demonstrated that micromolar S100B concentrations stimulate c-Jun N-terminal kinase (JNK) phosphorylation through the receptor for advanced glycation ending products (RAGE), and subsequently activate nuclear AP-1/cJun transcription, in cultured human neural stem cells (NSCs). In addition, as revealed by Western blot, siRNA and immunofluorescence analysis, S100B-induced JNK activation increased expression of Dickopff-1 (DKK-1) that, in turn, promoted Glycogen Synthase Kinase 3beta (GSK-3beta) phosphorylation and beta-catenin degradation, causing canonical Wnt pathway disruption and tau protein hyperphosphorylation. These findings propose a previously unrecognized link between S100B and tau hyperphosphorylation, suggesting S100B can contribute to NFT formation in AD and in all other conditions in which neuroinflammation may have a crucial role. [Abstract/Link to Full Text]

Coste O, Pierre S, Marian C, Brenneis C, Angioni C, Schmidt H, Popp L, Geisslinger G, Scholich K
Antinociceptive activity of the S1P-receptor agonist FTY720.
J Cell Mol Med. 2007 Nov 16;
FTY720 is a novel immunosuppressive drug that inhibits the egress of lymphocytes from secondary lymphoid tissues and thymus. In its phosphorylated form FTY720 is a potent S1P receptor agonist. Recently it was also shown that FTY720 can reduce prostaglandin synthesis through the direct inhibition of the cytosolic phospholipase A2 (cPLA2). Since prostaglandins are important mediators of nociception, we studied the effects of FTY720 in different models of nociception. We found that intraperitoneal administration of FTY720 reduced dose-dependently the nociceptive behaviour of rats in the formalin assay. Although the antinociceptive doses of FTY720 were too low to alter the lymphocyte count, prostanoid concentrations in the plasma were dramatically reduced. Surprisingly, intrathecally administered FTY720 reduced the nociceptive behaviour in the formalin assay without altering spinal prostaglandin synthesis, indicating that additional antinociceptive mechanisms beside the inhibition of prostaglandin synthesis are involved. Accordingly, FTY720 reduced also the nociceptive behaviour in the spared nerve injury model for neuropathic pain which does not depend on prostaglandin synthesis. In this model the antinociceptive effect of FTY720 was similar to gabapentin, a commonly used drug to treat neuropathic pain. Taken together we show for the first time that FTY720 possesses antinociceptive properties and that FTY720 reduces nociceptive behaviour during neuropathic pain. [Abstract/Link to Full Text]

Smadja DM, Basire A, Amelot A, Conte A, Bičche I, Le Bonniec BF, Aiach M, Gaussem P
Thrombin bound to a fibrin clot confers angiogenic and hemostatic properties on endothelial progenitor cells.
J Cell Mol Med. 2007 Nov 16;
Recent data suggest that endothelial progenitor cells (EPCs) are involved in recanalizing venous thrombi. We examined the impact of a fibrin network, and particularly of adsorbed thrombin, on EPCs derived from cord blood CD34+ cells. Fibrin networks generated in microplates by adding CaCl(2) to platelet-depleted plasma retained adsorbed thrombin at the average concentration of 4.2 nM per well. EPCs expressed high levels of endothelial cell protein C receptor (EPCR) and thrombomodulin, allowing the generation of activated protein C on the fibrin matrix in the presence of exogenous human protein C. The fibrin matrix induced significant EPC proliferation and, when placed in the lower chamber of a Boyden device, strongly enhanced EPC migration. These effects were partly inhibited by hirudin (by 41% and 66%, respectively), which suggests that fibrin adsorbed thrombin interacts with EPCs via the thrombin receptor PAR-1. Finally, spontaneous lysis of the fibrin network, studied by measuring D-dimer release into the supernatant, was inhibited by EPCs but not by control mononuclear cells. Such an effect was associated with a ten fold increase in plasminogen activator inhibitor-1 (PAI-1) secretion by EPCs cultivated in fibrin matrix. Overall, our data show that EPCs, in addition to their angiogenic potential, have both anticoagulant and antifibrinolytic properties. Thrombin may modulate these properties and contribute to thrombus recanalization by EPCs. [Abstract/Link to Full Text]

Fiegel HC, Kaufmann PM, Bruns H, Kluth D, Horch RE, Vacanti JP, Kneser U
Review: Hepatic Tissue Engineering.
J Cell Mol Med. 2007 Nov 16;
Today, liver transplantation is still the only curative treatment for liver failure due to end-stages liver diseases. Donor organ shortage, high cost and the need of immunosuppressive medications are still the major limitations in the field of liver transplantation. Thus, alternative innovative cell-based liver directed therapies, e.g. liver tissue engineering, are under investigation with the aim, that in future an artificial liver tissue could be created and be used for the replacement of the liver function in patients. Using cells instead of organs in this setting should permit (i) expansion of cells in an in vitro phase, (ii) genetic or immunological manipulation of cells for transplantation, (iii) tissue typing and cryopreservation in a cell bank, and (iv) the ex vivo genetic modification of patient's own cells prior re-implantation. Function and differentiation of liver cells are influenced by the three-dimensional organ architecture. The use of polymeric matrices permits the three dimensional formation of a neo-tissue and specific stimulation by adequate modification of the matrix-surface which might be essential for appropriate differentiation of transplanted cells. Additionally, culturing hepatocytes on three dimensional matrices permits culture in a flow bioreactor system with increased function and survival of the cultured cells. Based on bioreactor technology, bioartificial liver devices (BAL) are developed for extracorporeal liver support. Although BALs improved clinical and metabolic conditions, increased patient survival rates have not been proven yet. For intra-corporeal liver replacement, a concept which combines Tissue Engineering using three-dimensional, highly porous matrices with cell transplantation could be useful. In such a concept, whole liver mass transplantation, long term engraftment and function as well as correction of a metabolic defect in animal models could be achieved with a principally reversible procedure. Future studies have to investigate, which environmental conditions and transplantation system would be most suitable for the development of artificial functional liver tissue including blood supply for a potential use in a clinical setting. [Abstract/Link to Full Text]

Cenini G, Sultana R, Memo M, Butterfield DA
Elevated Levels of Pro-apoptotic p53 and Its Oxidative Modification by the Lipid Peroxidation Product, HNE, in Brain from Subjects with Amnestic Mild Cognitive Impairment and Alzheimer's Disease.
J Cell Mol Med. 2007 Nov 16;
Oxidative stress has been implicated in the pathogenesis of Alzheimer's disease (AD). Both AD and arguably its earlier form, mild cognitive impairment (MCI), have elevated membrane oxidative damage in brain. The tumor suppressor and transcription factor p53 plays a pivotal function in neuronal apoptosis triggered by oxidative stress. Apoptosis contributes to neuronal death in many neurological disorders, including AD. In this study, we investigated p53 expression in a specific region of the cerebral cortex, namely the inferior parietal lobule (IPL), in MCI and AD brain, to test the hypothesis that alterations of this pro-apoptotic protein may be involved in neuronal death in the progression of AD. By immunoprecipitation assay, we also investigated whether 4-hydroxy-2-trans-nonenal (HNE), an aldehydic product of lipid peroxidation, was bound in excess to p53 in IPL from subjects with MCI and AD compared to control. Overall, the data provide evidence that p53 is involved in the neuronal death in both MCI and AD, suggesting that the observed alterations are early events in the progression of AD. In addition, HNE may be a novel non-protein mediator of oxidative stress-induced neuronal apoptosis. [Abstract/Link to Full Text]

Samali A
TRIBUTE TO Professor Richard A. Lockshin.
J Cell Mol Med. 2007 Nov 16; [Abstract/Link to Full Text]

Samali A
"Apoptosis Review Series"
J Cell Mol Med. 2007 Nov 16; [Abstract/Link to Full Text]

Durkin ME, Yuan BZ, Zhou X, Zimonjic DB, Lowy DR, Thorgeirsson SS, Popescu NC
DLC-1:a Rho GTPase-activating protein and tumour suppressor.
J Cell Mol Med. 2007 Sep-Oct;11(5):1185-207.
The deleted in liver cancer 1 (DLC-1) gene encodes a GTPase activating protein that acts as a negative regulator of the Rho family of small GTPases. Rho proteins transduce signals that influence cell morphology and physiology, and their aberrant up-regulation is a key factor in the neoplastic process, including metastasis. Since its discovery, compelling evidence has accumulated that demonstrates a role for DLC-1 as a bona fide tumour suppressor gene in different types of human cancer. Loss of DLC-1 expression mediated by genetic and epigenetic mechanisms has been associated with the development of many human cancers, and restoration of DLC-1 expression inhibited the growth of tumour cells in vivo and in vitro. Two closely related genes, DLC-2 and DLC-3, may also be tumour suppressors. This review presents the current status of progress in understanding the biological functions of DLC-1 and its relatives and their roles in neoplasia. [Abstract/Link to Full Text]

Mandache E, Popescu LM, Gherghiceanu M
Myocardial interstitial Cajal-like cells (ICLC) and their nanostructural relationships with intercalated discs: shed vesicles as intermediates.
J Cell Mol Med. 2007 Sep-Oct;11(5):1175-84.
Intercalated discs (ID) are complex junctional units that connect cardiac myocytes mechanically and electrochemically. However, there is limited information concerning the cardiomyocyte interaction with interstitial non-muscle cells. Our previous studies showed that myocardial interstitial Cajal-like cells (ICLC) are located in between cardiomyocytes, blood capillaries and nerve fibres. Typically, ICLC have several very long, moniliform, cytoplasmic processes which establish closed contacts with nerve fibres, as well as each other. We report here ultrastructural evidence concerning the relationships of ICLC processes with ID. The ICLC cytoplasmic prolongations (tens micrometers length) preferentially pass by or stop nearby the ID. Transmission electron microscopy emphasized three distinct connecting features between the tips of ICLC extensions and myocytes at the 'mouth' of ID: free or budding shed vesicles, exocytotic multi-vesicular bodies and direct contacts. In the last case, electron-dense repetitive nanostructures ('pillars') (35-40 nm high and 100-150 nm wide, similar to adhesion molecules) fasten the ICLC to the myocytes. All these features suggest a juxtacrine and/or paracrine intercellular mutual modulation of ICLC and cardiomyocytes in the microenvironment of ID, possibly monitoring the cardiac functions, particularly the electrical activity. [Abstract/Link to Full Text]

Valkovskaya N, Kayed H, Felix K, Hartmann D, Giese NA, Osinsky SP, Friess H, Kleeff J
ADAM8 expression is associated with increased invasiveness and reduced patient survival in pancreatic cancer.
J Cell Mol Med. 2007 Sep-Oct;11(5):1162-74.
ADAM8 belongs to a family of transmembrane proteins implicated in cell-cell interactions, proteolysis of membrane proteins, and various aspects of carcinogenesis. In the present study, we aimed to evaluate the expression and function of ADAM8 in pancreatic cancer. ADAM8 mRNA levels were analysed by quantitative RT-PCR and correlated to patient survival. Immunohistochemistry was performed to localize ADAM8 in pancreatic tis-sues. Silencing of ADAM8 expression was carried out by transfection with specific siRNA oligonucleotides. Cell growth and invasion assays were used to assess the functional consequences of ADAM8 silencing. SELDI-TOF-MS was performed to detect the proteolytic activity of ADAM8 in pancreatic cancer cells. ADAM8 mRNA was significantly overexpressed in pancreatic ductal adenocarcinoma (PDAC) compared with normal pancreatic tissues (5.3-fold increase; P= 0.0008), and high ADAM8 mRNA and protein expression levels correlated with reduced survival time of PDAC patients (P= 0.048 and P= 0.065, respectively). Silencing of ADAM8 expression did not significantly influence pancreatic cancer cell growth but suppressed invasiveness. In addition, decreased proteolytic activity was measured in cell culture supernatants following silencing of ADAM8. In conclusion, ADAM8 is overexpressed in PDAC, influences cancer cell invasiveness and correlates with reduced survival, suggesting that ADAM8 might be a potential target in pancreatic cancer therapy. [Abstract/Link to Full Text]

Smadja DM, Bičche I, Helley D, Laurendeau I, Simonin G, Muller L, Aiach M, Gaussem P
Increased VEGFR2 expression during human late endothelial progenitor cells expansion enhances in vitro angiogenesis with up-regulation of integrin alpha(6).
J Cell Mol Med. 2007 Sep-Oct;11(5):1149-61.
In vitro expansion of late endothelial progenitor cells (EPCs) might yield a cell therapy product useful for myocardial and leg ischaemia, but the influence of EPC expansion on the angiogenic properties of these cells is unknown. In the present study, we investigated the effect of in vitro EPC expansion on vascular endothelial growth factor (VEGF) receptor expression. EPCs were obtained from CD34(+) cord blood cells and expanded for up to 5 weeks. Real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) showed that VEGFR2 expression, contrary to VEGFR1 and VEGFR3 expression, was significantly higher on expanded EPCs than on freshly isolated CD34(+) cells or on human umbilical vein endothelial cells (HUVECs). Quantitative flow cytometry confirmed that VEGFR2 density on EPCs increased during the expansion process and was significantly higher than on HUVECs. The impact of VEGFR2 increase was studied on the three theoretical steps of angiogenesis, i.e., EPC proliferation, migration and differentiation. VEGFR2 up-regulation had no effect on VEGF-induced cell proliferation, but significantly enhanced EPC migration and pseudotubes formation dependent on integrin alpha(6) subunit overexpression. In vitro expansion of late EPCs increases the expression of VEGFR2, the main VEGF receptor, with possible implications for EPC-based angiogenic therapy. [Abstract/Link to Full Text]

Rossner P, Terry MB, Gammon MD, Agrawal M, Zhang FF, Ferris JS, Teitelbaum SL, Eng SM, Gaudet MM, Neugut AI, Santella RM
Plasma protein carbonyl levels and breast cancer risk.
J Cell Mol Med. 2007 Sep-Oct;11(5):1138-48.
To study the role of oxidative stress in breast cancer risk, we analysed plasma levels of protein carbonyls in 1050 cases and 1107 controls. We found a statistically significant trend in breast cancer risk in relation to increasing quartiles of plasma protein carbonyl levels (OR = 1.2, 95% CI = 0.9-1.5; OR = 1.5, 95% CI = 1.2-2.0; OR = 1.6, 95% CI = 1.2-2.1, for the 2(nd), 3(rd) and 4(th) quartile relative to the lowest quartile, respectively, P for trend = 0.0001). The increase in risk was similar for younger (<50 years) and older women, more pronounced among women with higher physical activity levels (0.7 hrs/week for 4(th) quartile versus lowest quartile OR = 2.0, 95% CI = 1.4-3.0), higher alcohol consumption (> or = 15 grams/day for 4(th) quartile versus lowest quartile OR = 2.3, 95% CI = 1.1-4.7), and hormone replacement therapy use (HRT, OR = 2.6, 95% CI = 1.6-4.4 for 4(th) quartile versus lowest quartile). The multiplicative interaction terms were statistically significant only for physical activity and HRT. The positive association between plasma protein carbonyl levels and breast cancer risk was also observed when the analysis was restricted to women who had not received chemotherapy or radiation therapy prior to blood collection. Among controls, oxidized protein levels significantly increased with cigarette smoking and higher fruit and vegetable consumption, and decreased with alcohol consumption >30 grams per day. Women with higher levels of plasma protein carbonyl and urinary 15F(2t)-isoprostane had an 80% increase in breast cancer risk (OR = 1.8, 95% CI = 1.2-2.6) compared to women with levels below the median for both markers of oxidative stress. In summary, our results suggest that increased plasma protein carbonyl levels may be associated with breast cancer risk. [Abstract/Link to Full Text]

Fahey AJ, Adrian Robins R, Constantinescu CS
Curcumin modulation of IFN-beta and IL-12 signalling and cytokine induction in human T cells.
J Cell Mol Med. 2007 Sep-Oct;11(5):1129-37.
Curcumin is a polyphenol derived from the dietary spice turmeric. It possesses diverse anti-inflammatory and anti-cancer properties. Curcumin has been shown to exhibit an inhibitory effect on the production of inflammatory cytokines by human monocytes and has inhibited the animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE) in association with a decrease in interleukin 12 (IL-12) production and signal transducer and activator of transcription 4 (STAT4) activation. The type I interferon (IFN) IFN-has the ability to suppress IL-12. Both IL-12 and IFN-alpha/beta signal through the activation by phosphorylation of STAT4. Our aim was to investigate the effects of curcumin on the ability of T cells to respond to IL-12 or IFN-alpha/beta. We report that curcumin decreases IL-12-induced STAT4 phosphorylation, IFN-gamma production, and IL-12 Rbeta1 and beta2 expression. IFN-beta-induced STAT4 phosphorylation, IL-10 production and IFN receptor (IFNAR) subunits 1 and 2 expression were enhanced by curcumin. Curcumin increased IFN-alpha-induced IL-10 and IFNAR1 expression. Prior exposure to curcumin decreased IFN-alpha-induced IFNAR2 expression and did not modify the level of IFN-alpha-induced pSTAT4 generation. Thus, the effect of curcumin on STAT4 activation in T cells is dependent upon the stimulus to which the T cells have been exposed. [Abstract/Link to Full Text]

Mihai S, Chiriac MT, Herrero-González JE, Goodall M, Jefferis R, Savage CO, Zillikens D, Sitaru C
IgG4 autoantibodies induce dermal-epidermal separation.
J Cell Mol Med. 2007 Sep-Oct;11(5):1117-28.
Bullous pemphigoid (BP) is a sub-epidermal autoimmune blistering disease associated with autoantibodies to the dermal-epidermal junction (DEJ). Patients' autoantibodies induce dermal-epidermal separation when co-incubated with cryosections of human skin and leucocytes from healthy volunteers. IgG autoantibodies trigger complement and/or leucocyte activation resulting in specific pathology in several autoimmune conditions. In these diseases, IgG1 and IgG3 isotypes, but not the IgG4 subclass, are thought to trigger inflammatory pathways resulting in tissue damage. The capacity of IgG4 autoantibodies to mediate tissue damage has not yet been demonstrated. In this study, we isolated IgG1 and IgG4 autoantibodies from bullous pemhigoid patients'serum and analysed their blister-inducing potential in our cryosection assay. As expected, complement-fixing IgG1 autoantibodies induced sub-epidermal splits in this experimental model. Purified IgG4 did not fix complement, but, interestingly, like IgG1, activated leucocytes and induced dermal-epidermal separation. The potential of IgG4 autoantibodies to induce Fc-dependent dermal-epidermal separation was significantly lower compared to IgG1. Our results demonstrate that IgG4 autoantibodies are able to activate leucocytes and point to a hitherto less recognized function of IgG4. Moreover, for the first time, we clearly demonstrate that BP IgG4 autoantibodies have the capacity to induce leucocyte-dependent tissue damage. [Abstract/Link to Full Text]

Tsujikawa K, Koike K, Kitae K, Shinkawa A, Arima H, Suzuki T, Tsuchiya M, Makino Y, Furukawa T, Konishi N, Yamamoto H
Expression and sub-cellular localization of human ABH family molecules.
J Cell Mol Med. 2007 Sep-Oct;11(5):1105-16.
AlkB is an Escherichia coli protein that catalyses the oxidative demethylation of 1-methyladenine and 3-methylcytosine in DNA and RNA. The enzyme activity of AlkB is dependent on a 2-oxoglutarate- and Fe(II)-dependent (2OG-Fe[II]) oxygenase domain. Human AlkB homologues (hABH), hABH1, hABH2 and hABH3, which also possess the 2OG-Fe(II) oxygenase domain, have previously been identified. Recent bioinformatics analysis suggests the existence of an additional five ABH genes in humans. In this study, we identified the hABH4-hABH7 mRNAs and determined their expression in human tissues. Moreover, an hABH2 splice variant lacking the 2OG-Fe(II) oxygenase domain and a new gene, hABH8, were cloned from testis cDNA. hABH8 possesses not only the 2OG-Fe(II) oxygenase domain but both an RNA-binding motif and a methyl-transferase domain. mRNA of the eight hABH molecules was detected in the 16 normal human tissues examined. The sub-cellular localization of EmGFP-hABH8 was restricted to the cytoplasm. EmGFP-hABH1, 3, 4, 6 and 7 were localized in both the cytoplasm and nuclei. Interestingly, the EmGFP-hABH2 splice variant localized in nucleoplasm with a dot-like pattern. In some HeLa cells transfected with EmGFP-hABH5, dot-like fluorescence was also detected in the cytoplasm. These observations provide important information for the future annotation of the hABH family of molecules. [Abstract/Link to Full Text]

Du XJ
Re-modelling 'hostile' milieu of diseased myocardium via paracrine function of transplanted cells or relaxin.
J Cell Mol Med. 2007 Sep-Oct;11(5):1101-4.
While the approaches of regenerating cardiac muscle remain undetermined, recent evidence indicates that paracrine function of transplanted cells contributes significantly to the beneficial effects of cell therapy. Combination of such paracrine function of grafted cells with extracellular matrix remodelling by relaxin represents a promising complement to cell-based therapy for cardiac repair and muscle regeneration. [Abstract/Link to Full Text]

Formigli L, Perna AM, Meacci E, Cinci L, Margheri M, Nistri S, Tani A, Silvertown J, Orlandini G, Porciani C, Zecchi-Orlandini S, Medin J, Bani D
Paracrine effects of transplanted myoblasts and relaxin on post-infarction heart remodelling.
J Cell Mol Med. 2007 Sep-Oct;11(5):1087-100.
In the post-infarcted heart, grafting of precursor cells may partially restore heart function but the improvement is modest and the mechanisms involved remain to be elucidated. Here, we explored this issue by transplanting C2C12 myoblasts, genetically engineered to express enhanced green fluorescent protein (eGFP) or eGFP and the cardiotropic hormone relaxin (RLX) through coronary venous route to swine with experimental chronic myocardial infarction. The rationale was to deliver constant, biologically effective levels of RLX at the site of cell engraftment. One month after engraftment, histological analysis showed that C2C12 myoblasts selectively settled in the ischaemic scar and were located around blood vessels showing an activated endothelium (ICAM-1-,VCAM-positive). C2C12 myoblasts did not trans-differentiate towards a cardiac phenotype, but did induce extracellular matrix remodelling by the secretion of matrix metalloproteases (MMP) and increase microvessel density through the expression of vascular endothelial growth factor (VEGF). Relaxin-producing C2C12 myoblasts displayed greater efficacy to engraft the post-ischaemic scar and to induce extracellular matrix re-modelling and angiogenesis as compared with the control cells. By echocardiography, C2C12-engrafted swine showed improved heart contractility compared with the ungrafted controls, especially those producing RLX. We suggest that the beneficial effects of myoblast grafting on cardiac function are primarily dependent on the paracrine effects of transplanted cells on extracellular matrix remodelling and vascularization. The combined treatment with myoblast transplantation and local RLX production may be helpful in preventing deleterious cardiac remodelling and may hold therapeutic possibility for post-infarcted patients. [Abstract/Link to Full Text]

Cho WJ, Chow AK, Schulz R, Daniel EE
Matrix metalloproteinase-2, caveolins, focal adhesion kinase and c-Kit in cells of the mouse myocardium.
J Cell Mol Med. 2007 Sep-Oct;11(5):1069-86.
Matrix metalloproteinase-2 (MMP-2) may play roles at intracellular and extracellular sites of the heart in ischaemia/reperfusion injury. Caveolins (Cav-1, -2 and -3) are lipid raft proteins which play roles in cell sig-nalling. This study examined, using immunohistochemistry and two photon confocal microscopy, if MMP-2 and caveolins co-localize at the plasma membrane of cardiac cells: cardiomyocytes (CM), fibroblasts (FB) and capillary endothelial cells (CEC) in the left ventricle (LV) of the Cav-1(+/+) and Cav-1(-/-) mouse heart. In Cav-1(+/+) mouse LV MMP-2 and Cav-1 co-localized at CM plasma membranes, and at multiple locations in FB and CEC. MMP-2 co-localized with Cav-2 only at CEC. MMP-2 co-localized with Cav-3 at CM plasma membranes and Z-lines, and partially at FB and CEC. In Cav-1(-/-) LV Cav-1 and MMP-2 were absent or reduced everywhere. Cav-2 appeared at CEC despite the absence of Cav-1. Cav-3 appeared at CM plasma membranes and Z-lines, FB and CEC. Also, FAK in FB and c-Kit in interstitial Cajal-like cells (ICLC) were completely absent. By transmission electron microscopy in Cav-1(+/+), regular size caveolae (Cav) were at CEC, irregular size Cav were at CM and a few were at FB. In Cav-1(-/-) there were few Cav at CM and FB and some at CEC. To conclude, MMP-2 is closely associated with caveolins at FB and CEC as well as at CM. Also, MMP-2 is closely associated with FAK at FB and c-Kit at ICLC. Thus, Cav-1 expression is not necessary for Cav-2 expression. Cav-3 or Cav-3 with Cav-2 has the capability to make Cav. [Abstract/Link to Full Text]

Palmieri G, Casula M, Sini MC, Ascierto PA, Cossu A
Issues affecting molecular staging in the management of patients with melanoma.
J Cell Mol Med. 2007 Sep-Oct;11(5):1052-68.
Prediction of metastatic potential remains one of the main goals to be pursued in order to better assess the risk subgroups of patients with melanoma. Detection of occult melanoma cells in peripheral blood (circulating metastatic cells [CMC]) or in sentinel lymph nodes (sentinel node metastatic cells [SNMC]), could significantly contribute to better predict survival in melanoma patients. An overview of the numerous published studies indicate the existence of several drawbacks about either the reliability of the approaches for identification of occult melanoma cells or the clinical value of CMC and SNMC as prognostic factors among melanoma patients. In this sense, characterization of the molecular mechanisms involved in development and progression of melanoma (referred to as melanomagenesis) could contribute to better classify the different subsets of melanoma patients. Increasing evidence suggest that melanoma develops as a result of accumulated abnormalities in genetic pathways within the melanocytic lineage. The different molecular mechanisms may have separate roles or cooperate during all evolutionary phases of melanocytic tumourigenesis, generating different subsets of melanoma patients with distinct aggressiveness, clinical behaviour, and response to therapy. All these features associated with either the dissemination of occult metastatic cells or the melanomagenesis might be useful to adequately manage the melanoma patients with different prognosis as well as to better address the different melanoma subsets toward more appropriate therapeutic approaches. [Abstract/Link to Full Text]

Gressner OA, Weiskirchen R, Gressner AM
Biomarkers of hepatic fibrosis, fibrogenesis and genetic pre-disposition pending between fiction and reality.
J Cell Mol Med. 2007 Sep-Oct;11(5):1031-51.
Fibrosis is a frequent, life-threatening complication of most chronic liver diseases. Despite major achievements in the understanding of its pathogenesis, the translation of this knowledge into clinical practice is still limited. In particular, non-invasive and reliable (serum-) biomarkers indicating the activity of fibrogenesis are scarce. Class I biomarkers are defined as serum components having a direct relation to the mechanism of fibrogenesis, either as secreted matrix-related components of activated hepatic stellate cells and fibroblasts or as mediators of extracellular matrix (ECM) synthesis or turnover. They reflect primarily the activity of the fibrogenic process. Many of them, however, proved to be disappointing with regard to sensitivity and specificity. Up to now hyaluronan turned out to be the relative best type I serum marker. Class II biomarkers comprise in general rather simple standard laboratory tests, which are grouped into panels. They fulfil most criteria for detection and staging of fibrosis and to a lesser extent grading of fibrogenic activity. More than 20 scores are currently available, among which Fibrotest is the most popular one. However, the diagnostic use of many of these scores is still limited and standardization of the assays is only partially realized. Combining of panel markers in sequential algorithms might increase their diagnostic validity. The translation of genetic pre-disposition biomarkers into clinical practice has not yet started, but some polymorphisms indicate a link to progression and outcome of fibrogenesis. Parallel to serum markers non-invasive physical techniques, for example, transient elastography, are developed, which can be combined with serum tests and profiling of serum proteins and glycans. [Abstract/Link to Full Text]

Ball SG, Shuttleworth CA, Kielty CM
Mesenchymal stem cells and neovascularization: role of platelet-derived growth factor receptors.
J Cell Mol Med. 2007 Sep-Oct;11(5):1012-30.
There is now accumulating evidence that bone marrow-derived mesenchymal stem cells (MSCs) make an important contribution to postnatal vasculogenesis, especially during tissue ischaemia and tumour vascularization. Identifying mechanisms which regulate the role of MSCs in vasculogenesis is a key therapeutic objective, since while increased neovascularization can be advantageous during tissue ischaemia, it is deleterious during tumourigenesis. The potent angiogenic stimulant vascular endothelial growth factor (VEGF) is known to regulate MSC mobilization and recruitment to sites of neovascularization, as well as directing the differentiation of MSCs to a vascular cell fate. Despite the fact that MSCs did not express VEGF receptors, we have recently identified that VEGF-A can stimulate platelet-derived growth factor (PDGF) receptors, which regulates MSC migration and proliferation. This review focuses on the role of PDGF receptors in regulating the vascular cell fate of MSCs, with emphasis on the function of the novel VEGF-A/PDGF receptor signalling mechanism. [Abstract/Link to Full Text]

Mimeault M, Hauke R, Mehta PP, Batra SK
Recent advances in cancer stem/progenitor cell research: therapeutic implications for overcoming resistance to the most aggressive cancers.
J Cell Mol Med. 2007 Sep-Oct;11(5):981-1011.
Overcoming intrinsic and acquired resistance of cancer stem/progenitor cells to current clinical treatments represents a major challenge in treating and curing the most aggressive and metastatic cancers. This review summarizes recent advances in our understanding of the cellular origin and molecular mechanisms at the basis of cancer initiation and progression as well as the heterogeneity of cancers arising from the malignant transformation of adult stem/progenitor cells. We describe the critical functions provided by several growth factor cascades, including epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), stem cell factor (SCF) receptor (KIT), hedgehog and Wnt/beta-catenin signalling pathways that are frequently activated in cancer progenitor cells and are involved in their sustained growth, survival, invasion and drug resistance. Of therapeutic interest, we also discuss recent progress in the development of new drug combinations to treat the highly aggressive and metastatic cancers including refractory/relapsed leukaemias, melanoma and head and neck, brain, lung, breast, ovary, prostate, pancreas and gastrointestinal cancers which remain incurable in the clinics. The emphasis is on new therapeutic strategies consisting of molecular targeting of distinct oncogenic signalling elements activated in the cancer progenitor cells and their local microenvironment during cancer progression. These new targeted therapies should improve the efficacy of current therapeutic treatments against aggressive cancers, and thereby preventing disease relapse and enhancing patient survival. [Abstract/Link to Full Text]

Salanueva IJ, Cerezo A, Guadamillas MC, del Pozo MA
Integrin regulation of caveolin function.
J Cell Mol Med. 2007 Sep-Oct;11(5):969-80.
Caveolae are unique organelles that are found in the plasma membrane of many cell types. They participate in various processes such as lipid recycling, cellular signalling and endocytosis. A variety of signalling molecules localize to caveolae in response to various stimuli, providing a potential mechanism for the spatial regulation of signal transduction pathways. Caveolin-1, a constitutive protein of caveolae, has been implicated in the regulation of cell growth, lipid trafficking, endocytosis and cell migration. Phosphorylation of caveolin-1 on Tyr 14 is involved in integrin-regulated caveolae trafficking and also in signalling at focal adhesions in migrating cells. In this review, we focus on recent studies that describe the role of caveolin-1 in integrin signal transduction, and how this interplay links extracellular matrix anchorage to cell proliferation, polarity and directional migration. [Abstract/Link to Full Text]

Nakayama S, Kajioka S, Goto K, Takaki M, Liu HN
Calcium-associated mechanisms in gut pacemaker activity.
J Cell Mol Med. 2007 Sep-Oct;11(5):958-68.
A considerable body of evidence has revealed that interstitial cells of Cajal (ICC), identified with c-Kit-immunoreactivity, act as gut pacemaker cells, with spontaneous Ca(2+) activity in ICC as the probable primary mechanism. Namely, intracellular (cytosolic) Ca(2+) oscillations in ICC periodically activate plasmalemmal Ca(2+)-dependent ion channels and thereby generate pacemaker potentials. This review will, thus, focus on Ca(2+)-associated mechanisms in ICC in the gastrointestinal (GI) tract, including auxiliary organs. [Abstract/Link to Full Text]

Zhang WJ, Liu W, Cui L, Cao Y
Tissue engineering of blood vessel.
J Cell Mol Med. 2007 Sep-Oct;11(5):945-57.
Vascular grafts are in large demand for coronary and peripheral bypass surgeries. Although synthetic grafts have been developed, replacement of vessels with purely synthetic polymeric conduits often leads to the failure of such graft, especially in the grafts less than 6 mm in diameter or in the areas of low blood flow, mainly due to the early formation of thrombosis. Moreover, the commonly used materials lack growth potential, and long-term results have revealed several material-related failures, such as stenosis, thromboembolization, calcium deposition and infection. Tissue engineering has become a promising approach for generating a bio-compatible vessel graft with growth potential. Since the first success of constructing blood vessels with collagen and cultured vascular cells by Weinberg and Bell, there has been considerable progress in the area of vessel engineering. To date, tissue- engineered blood vessels (TEBVs) could be successfully constructed in vitro, and be used to repair the vascular defects in animal models. This review describes the major progress in the field, including the seeding cell sources, the biodegradable scaffolds, the construction technologies, as well as the encouraging achievements in clinical applications. The remaining challenges are also discussed. [Abstract/Link to Full Text]

Guillot PV, Cui W, Fisk NM, Polak DJ
Stem cell differentiation and expansion for clinical applications of tissue engineering.
J Cell Mol Med. 2007 Sep-Oct;11(5):935-44.
This invited review discusses the latest advances stem cell biology, tissue engineering and the transition from bench to bedside. An overview is presented as to which the best cell source might be for cell therapy and tissue engineering applications, best biomaterials currently available and the challenges the field faces to translate basic research into therapies for a large number of human diseases. [Abstract/Link to Full Text]

Huang C, Miller RT
The calcium-sensing receptor and its interacting proteins.
J Cell Mol Med. 2007 Sep-Oct;11(5):923-34.
Seven membrane-spanning, or G protein-coupled receptors were originally thought to act through het-erotrimeric G proteins that in turn activate intracellular enzymes or ion channels, creating relatively simple, linear signalling pathways. Although this basic model remains true in that this family does act via a relatively small number of G proteins, these signalling systems are considerably more complex because the receptors interact with or are located near additional proteins that are often unique to a receptor or subset of receptors. These additional proteins give receptors their unique signalling personalities. The extracellular Ca-sensing receptor (CaR) signals via Galpha(i), Galpha(q) and Galpha(12/13), but its effects in vivo demonstrate that the signalling pathways controlled by these subunits are not sufficient to explain all its biologic effects. Additional structural or signalling proteins that interact with the CaR may explain its behaviour more fully. Although the CaR is less well studied in this respect than other receptors, several CaR-interacting proteins such as filamin, a potential scaffolding protein, receptor activity modifying proteins (RAMPs) and potassium channels may contribute to the unique characteristics of the CaR. The CaR also appears to interact with additional proteins common to other G protein-coupled receptors such as arrestins, G protein receptor kinases, protein kinase C, caveolin and proteins in the ubiquitination pathway. These proteins probably represent a few initial members of CaR-based signalling complex. These and other proteins may not all be associated with the CaR in all tissues, but they form the basis for understanding the complete nature of CaR signalling. [Abstract/Link to Full Text]

Hu J, Spiegel AM
Structure and function of the human calcium-sensing receptor: insights from natural and engineered mutations and allosteric modulators.
J Cell Mol Med. 2007 Sep-Oct;11(5):908-22.
The human extracellular Ca(2+)-sensing receptor (CaR), a member of the G protein-coupled receptor family 3, plays a key role in the regulation of extracellular calcium homeostasis. It is one of just a few G protein-coupled receptors with a large number of naturally occurring mutations identified in patients. In contrast to the small sizes of its agonists, this large dimeric receptor consists of domains with topologically distinctive orthosteric and allosteric sites. Information derived from studies of naturally occurring mutations, engineered mutations, allosteric modulators and crystal structures of the agonist-binding domain of homologous type 1 metabotropic glutamate receptor and G protein-coupled rhodopsin offers new insights into the structure and function of the CaR. [Abstract/Link to Full Text]

Hofer AM
Review series on the extracellular Ca(2+)-sensing receptor.
J Cell Mol Med. 2007 Sep-Oct;11(5):906-7. [Abstract/Link to Full Text]

Courtoy P
A tribute to Professor Christian de Duve on his 90th birthday.
J Cell Mol Med. 2007 Sep-Oct;11(5):902-5. [Abstract/Link to Full Text]

---

Recent Articles in Molecules and Cells

---

Park SY, Chae CB
Toxic levels of amyloid beta peptide do not induce VEGF synthesis.
Mol Cells. 2007 Aug 31;24(1):69-75.
Alzheimer's disease is a neurodegenerative disorder associated with progressive loss of cognitive function and memory. Amyloid beta peptide (Abeta) is the major component of senile plaques and is known to exert its cytotoxic effect mainly by producing H2O2. Vascular endothelial growth factor (VEGF) is elevated in the cerebrospinal fluid (CSF) and brain of AD patients, and H2O2 is one of the factors that induce VEGF. Therefore, we tested whether Abeta might be responsible for the increased VEGF synthesis. We found that Abeta induced the production of H2O2 in vitro. Comparison of the amount of H2O2 required to induce VEGF synthesis in HN33 cells and the amount of H2O2 produced by 10 muM Abeta1-42 in vitro suggested that a toxic concentration of Abeta might induce VEGF synthesis in these cells. However, toxic concentrations of Abeta failed to induce VEGF synthesis in several cell systems. They also had no effect on antioxidant enzymes such as glutathione peroxidase, catalase, and peroxiredoxin in HN33 cells. Cu2+, Zn2+ and Fe3+ are known to accumulate in the brains of AD patients and promote aggregation of Abeta, and Cu2+ by itself induces synthesis of VEGF. However, there was no synergistic effect between Cu2+ and Abeta1-42 in the induction of VEGF synthesis and Zn2+ and Fe3+ also had no effect on the synthesis of VEGF, alone or in combination with Abeta. [Abstract/Link to Full Text]

Kim J, Jo BH, Lee KL, Yoon ES, Ryu GH, Chung KW
Identification of new microsatellite markers in Panax ginseng.
Mol Cells. 2007 Aug 31;24(1):60-8.
Microsatellites, also called simple sequence repeats (SSR), are very useful molecular genetic markers commonly used in crop breeding, species identification and linkage analysis. In the present study, we constructed a microsatellite-enriched genomic library of Panax ginseng, and identified 251 novel microsatellite sequences. Tri-nt repeat units were the most abundant (46.6%), followed by di-nt repeats (35.5%). The (AG)n motif was most common (23.1%), followed by the (AAC)n motif (22.3%). From the genotyping of 94 microsatellites using marker-specific primer sets, we identified 11 intraspecific polymorphic markers as well as 14 possible interspecific polymorphic markers differing between P. ginseng and P. quinquefolius. The exact allele structures of the polymorphic markers were determined and the alleles were named. This study represents the first report of the bulk isolation of microsatellites by screening a microsatellite-enriched genomic library in P. ginseng. The microsatellite markers could be useful for linkage analysis, genetic breeding and authentication of Panax species. [Abstract/Link to Full Text]

Kim DW, Shibato J, Agrawal GK, Fujihara S, Iwahashi H, Kim du H, Shim IeS, Rakwal R
Gene transcription in the leaves of rice undergoing salt-induced morphological changes (Oryza sativa L.).
Mol Cells. 2007 Aug 31;24(1):45-59.
We describe the gene expression profile of third leaves of rice (cv. Nipponbare) seedlings subjected to salt stress (130 mM NaCl). Transcripts of Mn-SOD, Cu/Zn-SOD,cytosolic and stromal APX, GR and CatB were regulated, whereas expression of thylakoid-bound APX and CatA were down-regulated. The levels of the compatible solute proline and of transcripts of its biosynthetic gene, Delta1-pyrroline-5-carboxylate synthetase (P5CS), were strongly increased by salt stress. Interestingly, a potential compatible solute, gamma-aminobutyric acid (GABA), was also found to be strongly induced by salt stress along with marked up-regulation of transcripts of GABA-transaminase. A dye-swap rice DNA microarray analysis identified a large number of genes whose expression in third leaves was altered by salt stress. Among 149 genes whose expression was altered at all the times assayed (3, 4 and 6 days) during salt stress, there were 47 annotated novel genes and 76 unknown genes. These results provide new insight into the effect of salt stress on the expression of genes related to antioxidant enzymes, proline and GABA as well as of genes in several functional categories. [Abstract/Link to Full Text]

Kwon SI, Lee H, An CS
Differential expression of three catalase genes in the small radish (Rhaphanus sativus L. var. sativus).
Mol Cells. 2007 Aug 31;24(1):37-44.
Three catalase cDNA clones were isolated from the small radish (Raphanus sativus L.). Their nucleotide and deduced amino acid sequences showed the greatest homology to those of Arabidopsis. Genomic Southern blot analysis, using RsCat1 cDNA as a probe, showed that catalases are encoded by small multigene family in the small radish. Nondenaturing polyacrylamide gels revealed the presence of several catalase isozymes, the levels of which varied among the organs examined. The isozyme activities were assigned the individual catalase genes by Northern analysis using total RNA from different organs. The three catalase genes were differentially expressed in response to treatments such as white light, xenobiotics, osmoticum, and UV. Their expression in seedlings was controlled by the circadian clock under a light/dark cycle and/or in constant light. Interestingly, RsCat1 transcripts peaked in the morning, while those of RsCat2 and RsCat3 peaked in the early evening. Our results suggest that the RsCat enzymes are involved in defense against the oxidative stress induced by environmental changes. [Abstract/Link to Full Text]

Lim H, Kim K, Han D, Oh J, Kim Y
Crystal structure of TTC0263, a thermophilic TPR protein from Thermus thermophilus HB27.
Mol Cells. 2007 Aug 31;24(1):27-36.
The hypothetical protein TTC0263 of Thermus thermophilus HB27 is a thermophilic tetratricopeptide repeat (TPR)-containing protein. In the present study, the TPR region (residues 26-230) was resolved at 2.5 A with R-factors of R/Rfree = 23.6%/28.6%. TTC0263 consists of 11 helices that form five TPR units. Uniquely, it contains one atypical "extended" TPR (eTPR) unit. This comprises extended helical residues near the loop region of TTC0263, such that the helical length of eTPR is longer than that of the canonical TPR sequence. In addition, the hybrid TPR domain of TTC0263 possesses oligomer-forming characteristics. TPR domains are generally involved in forming multi-subunit complexes by interacting with each other or with other subunit proteins. The dynamic structure of TTC0263 described here goes some way to explaining how TPR domains mediate the formation of multi-subunit complexes. [Abstract/Link to Full Text]

Rahman ML, Chu SH, Choi MS, Qiao YL, Jiang W, Piao R, Khanam S, Cho YI, Jeung JU, Jena K, Koh HJ
Identification of QTLs for some agronomic traits in rice using an introgression line from Oryza minuta.
Mol Cells. 2007 Aug 31;24(1):16-26.
Wild progenitor species provide potential gene sources for complex traits such as yield and multiple resistances to biotic and abiotic stresses, and thus are expected to contribute to sustainable food supplies. An introgression line 'IR71033-121-15' was derived from a wild species Oryza minuta (2n = 48, BBCC, Acc No. 101141) at IRRI. Introgression analysis using 530 SSR and STS markers revealed that at least 14 chromosomal segments distributed over 12 chromosomes had been introgressed from O. minuta. An F2:3 population from the cross between IR71033 and Junambyeo (a Korean japonica cultivar) consisting of 146 lines was used for quantitative trait loci (QTL) analysis of 16 agronomic traits. A total of 36 single-locus QTLs (S-QTLs) and 45 digenic epistasis (E-QTLs) were identified. In spite of it's inferiority of O. minuta for most of the traits studied, its alleles contributed positively to 57% of the QTLs. The other QTLs originated from either parent, IR71033 or Junambyeo. QTLs for phenotypically correlated traits were mostly detected on introgressed segments. Fourteen QTLs corresponded to QTLs reported earlier, indicating that these QTLs are stable across genetic backgrounds. Twenty-two QTLs controlling yield and its components had not been detected in previous QTL studies. Of these, thirteen consisted of potentially novel alleles from O. minuta. QTLs from O. minuta introgression could be new sources of natural variation for the genetic improvement of rice. [Abstract/Link to Full Text]

Lee YA, Kang SS, Baek SH, Jung JC, Jin EJ, Tak EN, Sonn JK
Redifferentiation of dedifferentiated chondrocytes on chitosan membranes and involvement of PKCalpha and P38 MAP kinase.
Mol Cells. 2007 Aug 31;24(1):9-15.
To investigate the effects of chitosan on the redifferentiation of dedifferentiated chondrocytes, we used chondrocytes obtained from a micromass culture system. Micromass cultures of chick wing bud mesenchymal cells yielded differentiated chondrocytes, but these dedifferentiated during serial monolayer subculture. When the dedifferentiated chondrocytes were cultured on chitosan membranes they regained the phenotype of differentiated chondrocytes. Expression of protein kinase C (PKC) increased during chondrogenesis, decreased during dedifferentiation, and increased again during redifferentiation. Treatment of the cultures with phorbol 12-myristate 13-acetate (PMA) inhibited redifferentiation and down-regulated PKC. In addition, the expression of p38 mitogen-activated protein (MAP) kinase increased during redifferentiation, and its inhibition suppressed redifferentiation. These findings establish a culture system for producing chondrocytes, point to a new role of chitosan in the redifferentiation of dedifferentiated chondrocytes, and show that PKC and p38 MAP kinase activities are required for chondrocyte redifferentiation in this model system. [Abstract/Link to Full Text]

Yoon SR, Chung JW, Choi I
Development of natural killer cells from hematopoietic stem cells.
Mol Cells. 2007 Aug 31;24(1):1-8.
Natural killer (NK) cells play a crucial role in innate immune system and tumor surveillance. NK cells are derived from CD34+hematopoietic stem cells and undergo differentiation via precursor NK cells in bone marrow (BM) through sequential acquisition of functional surface receptors. During differentiation of NK cells, many factors are involved including cytokines, membrane factors and transcription factors as well as microenvironment of BM. NK cells express their own repertoire of receptors including activating and inhibitory receptors that bind to major histocompatibility complex (MHC) class I or class I-related molecules. The balance between activating and inhibitory receptors determines the function of NK cells to kill targets. Binding of NK cell inhibitory receptors to their MHC class I-ligand renders the target cells to be protected from NK cell-mediated cytotoxicity. Thus, NK cells are able to discriminate self from non-self through MHC class I-binding inhibitory receptor. Using intrinsic properties of NK cells, NK cells are emerging to apply as therapeutic agents against many types of cancers. Recently, NK cell alloactivity has also been exploited in killer cell immunoglobulin-like receptor mismatched haploidentical stem cell transplantation to reduce the rate of relapse and graft versus host disease. In this review, we discuss the basic mechanisms of NK cell differentiation, diversity of NK cell receptors, and clinical applications of NK cells for anti-cancer immunotherapy. [Abstract/Link to Full Text]

Kim HB, Bae JH, Lim JD, Yu CY, An CS
Expression of a functional type-I chalcone isomerase gene is localized to the infected cells of root nodules of Elaeagnus umbellata.
Mol Cells. 2007 Jun 30;23(3):405-9.
A putative type-I chalcone isomerase (CHI) cDNA clone EuNOD-CHI was previously isolated from the root nodule of Elaeagnus umbellata [Kim et al. (2003)]. To see if it encodes a functional CHI, we ectopically overexpressed it in the Arabidopsis (Arabidopsis thaliana) transparent testa 5 (tt5) mutant, which is defective in naringenin production and has yellow seeds due to proanthocyanidin deficiency. Ectopic overexpression of EuNOD-CHI resulted in recovery of normal seed coat color. Naringenin produced by CHI from naringenin chalcone was detected in the transgenic lines like in the wild-type, whereas it was absent from the tt5 mutant. We conclude that EuNOD-CHI encodes a functional type-I CHI. In situ hybridization revealed that EuNOD-CHI expression is localized to the infected cells of the fixation zone in root nodules. [Abstract/Link to Full Text]

Lee TH, Kwak HB, Kim HH, Lee ZH, Chung DK, Baek NI, Kim J
Methanol extracts of Stewartia koreana inhibit cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) gene expression by blocking NF-kappaB transactivation in LPS-activated RAW 264.7 cells.
Mol Cells. 2007 Jun 30;23(3):398-404.
Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are involved in various pathophysiological processes such as inflammation and carcinogenesis. In a search for inhibitors of COX-2 and iNOS production we found that extracts of Stewartia koreana strongly inhibited NO and PGE2 production in LPS-treated macrophage RAW 264.7 cells. We have now shown that the mRNA and protein levels of iNOS and COX-2 are reduced by the Stewartia koreana extract (SKE). SKE inhibited expression of an NF-kappaB reporter gene in response to LPS, and gel mobility shift assays revealed that SKE reduced NF-kappaB DNA-binding activity. The extract also inhibited LPS-induced phosphorylation of IkappaB-alpha and nuclear translocation of p65. Administration of the extract reduced the symptoms of arthritis in a collagen-induced arthritic mouse model. These results indicate that Stewartia extracts contain potentially useful agents for preventing and treating inflammatory diseases. [Abstract/Link to Full Text]

Ruan XZ, Yan F, Zhao XY, Wang CT, Song M, Yang HS, Deng HX, Wei YQ
Identification and characterization of two novel variants of the DUF1208 protein FAM92A1.
Mol Cells. 2007 Jun 30;23(3):391-7.
FAM92A1 (named FAM92A1-271) belongs to the family of proteins with conserved DUF1208 domains. Its function remains elusive. We identified two novel transcript variants (FAM92A1-251, FAM92A1-289) of FAM92A1. The presence of these transcripts in cancerous and normal cells, as well as their influence on cell proliferation and apoptosis, were investigated. The subcellular location of FAM92A1 was determined by fluorescence microscopy. We found that FAM92A1-271 and FAM92A1- 289 were highly expressed in both normal and cancerous cells, but FAM92A1-251 was only expressed at a moderate level in both types of cell. Overexpression of FAM92A1-271, FAM92A1-251 and FAM92A1-289 inhibited cell proliferation, caused S-phase arrest and induced apoptosis. Subcellular localization showed that FAM92A1 localizes to the nucleus. Our results show that FAM92A1 has different splicing variants, and that it may take part in regulating cell proliferation and apoptosis. [Abstract/Link to Full Text]

Kim KH, Jeon HK, Kang S, Sultana T, Kim GJ, Eom K, Park JK
Characterization of the complete mitochondrial genome of Diphyllobothrium nihonkaiense (Diphyllobothriidae: Cestoda), and development of molecular markers for differentiating fish tapeworms.
Mol Cells. 2007 Jun 30;23(3):379-90.
We sequenced and characterized the complete mitochondrial genome of the Japanese fish tapeworm D. nihonkaiense. The genome is a circular-DNA molecule of 13607 bp (one nucleotide shorter than that of D. latum mtDNA) containing 12 protein-coding genes (lacking atp8), 22 tRNA genes and two rRNA genes. Gene order and genome content are identical to those of the other cestodes reported thus far, including its congener D. latum. The only exception is Hymenolepis diminuta in which the positions of trnS2 and trnL1 are switched. We tested a PCR-based molecular assay designed to rapidly and accurately differentiate between D. nihonkaiense and D. latum using species-specific primers based on a comparison of their mtDNA sequences. We found the PCR-based system to be very reliable and specific, and suggest that PCR-based identification methods using mtDNA sequences could contribute to the study of the epidemiology and larval ecology of Diphyllobothrium species. [Abstract/Link to Full Text]

Seo SN, Lee JH, Kim YM
Characterization of an iron- and manganese-containing superoxide dismutase from Methylobacillus sp. strain SK1 DSM 8269.
Mol Cells. 2007 Jun 30;23(3):370-8.
A superoxide dismutase was purified 62-fold in seven steps to homogeneity from Methylobacillus sp. strain SK1, an obligate methanol-oxidizing bacterium, with a yield of 9.6%. The final specific activity was 4,831 units per milligram protein as determined by an assay based on a 50% decrease in the rate of cytochrome c reduction. The molecular weight of the native enzyme was estimated to be 44,000. Sodium dodecyl sulfate gel electrophoresis revealed two identical subunits of molecular weight 23,100. The isoelectric point of the purified enzyme was found to be 4.4. Maximum activity of the enzyme was measured at pH 8. The enzyme was stable at pH range from 6 to 8 and at high temperature. The enzyme showed an absorption peak at 280 nm with a shoulder at 292 nm. Hydrogen peroxide and sodium azide, but not sodium cyanide, was found to inhibit the purified enzyme. The enzyme activity in cell-free extracts prepared from cells grown in manganese-rich medium, however, was not inhibited by hydrogen peroxide but inhibited by sodium azide. The activity in cell extracts from cells grown in iron-rich medium was found to be highly sensitive to hydrogen peroxide and sodium azide. One mol of native enzyme was found to contain 1.1 g-atom of iron and 0.7 g-atom of manganese. The N-terminal amino acid sequence of the purified enzyme was Ala-Tyr-Thr-Leu-Pro-Pro-Leu-Asn-Tyr-Ala-Tyr. The superoxide dismutase of Methylobacillus sp. strain SK1 was found to have antigenic sites identical to those of Methylobacillus glycogenes enzyme. The enzyme, however, shared no antigenic sites with Mycobacterium sp. strain JC1, Methylovorus sp. strain SS1, Methylobacterium sp. strain SY1, and Methylosinus trichosproium enzymes. [Abstract/Link to Full Text]

Kim BJ, Jeon JH, Kim SJ, So I, Kim KW
Regulation of transient receptor potential melastatin 7 (TRPM7) currents by mitochondria.
Mol Cells. 2007 Jun 30;23(3):363-9.
Mitochondria play a central role in energy-generating processes and may be involved in the regulation of channels and receptors. Here we investigated TRPM7, an ion channel and functional kinase, and its regulation by mitochondria. Proton ionophores such as CCCP elicited a rapid decrease in outward TRPM7 whole-cell currents but a slight increase in inward currents with pipette solutions containing no MgATP. With pipette solutions containing 3 mM MgATP, however, CCCP increased both outward and inward TRPM7 currents. This effect was reproducible and fully reversible, and repeated application of CCCP yielded similar decreases in current amplitude. Oligomycin, an inhibitor of F1/FO-ATP synthase, inhibited outward whole-cell currents but did not affect inward currents. The respiratory chain complex I inhibitor, rotenone, and complex III inhibitor, antimycin A, were without effect as were kaempferol, an activator of the mitochondrial Ca2+ uniporter, and ruthenium red, an inhibitor of the mitochondrial Ca2+ uniporter. These results suggest that the inner membrane potential (as regulated by proton ionophores) and the F1/FO-ATP synthase of mitochondria are important in regulating TRPM7 channels. [Abstract/Link to Full Text]

Park JY, Hwang EM, Park N, Kim E, Kim DG, Kang D, Han J, Choi WS, Ryu PD, Hong SG
Gateway RFP-fusion vectors for high throughput functional analysis of genes.
Mol Cells. 2007 Jun 30;23(3):357-62.
There is an increasing demand for high throughput (HTP) methods for gene analysis on a genome-wide scale. However, the current repertoire of HTP detection methodologies allows only a limited range of cellular phenotypes to be studied. We have constructed two HTP-optimized expression vectors generated from the red fluorescent reporter protein (RFP) gene. These vectors produce RFP-tagged target proteins in a multiple expression system using gateway cloning technology (GCT). The RFP tag was fused with the cloned genes, thereby allowing us localize the expressed proteins in mammalian cells. The effectiveness of the vectors was evaluated using an HTP-screening system. Sixty representative human C2 domains were tagged with RFP and overexpressed in HiB5 neuronal progenitor cells, and we studied in detail two C2 domains that promoted the neuronal differentiation of HiB5 cells. Our results show that the two vectors developed in this study are useful for functional gene analysis using an HTP-screening system on a genome-wide scale. [Abstract/Link to Full Text]

Hong CP, Piao ZY, Kang TW, Batley J, Yang TJ, Hur YK, Bhak J, Park BS, Edwards D, Lim YP
Genomic distribution of simple sequence repeats in Brassica rapa.
Mol Cells. 2007 Jun 30;23(3):349-56.
Simple Sequence Repeats (SSRs) represent short tandem duplications found within all eukaryotic organisms. To examine the distribution of SSRs in the genome of Brassica rapa ssp. pekinensis, SSRs from different genomic regions representing 17.7 Mb of genomic sequence were surveyed. SSRs appear more abundant in non-coding regions (86.6%) than in coding regions (13.4%). Comparison of SSR densities in different genomic regions demonstrated that SSR density was greatest within the 5'-flanking regions of the predicted genes. The proportion of different repeat motifs varied between genomic regions, with trinucleotide SSRs more prevalent in predicted coding regions, reflecting the codon structure in these regions. SSRs were also preferentially associated with gene-rich regions, with peri-centromeric heterochromatin SSRs mostly associated with retrotransposons. These results indicate that the distribution of SSRs in the genome is non-random. Comparison of SSR abundance between B. rapa and the closely related species Arabidopsis thaliana suggests a greater abundance of SSRs in B. rapa, which may be due to the proposed genome triplication. Our results provide a comprehensive view of SSR genomic distribution and evolution in Brassica for comparison with the sequenced genomes of A. thaliana and Oryza sativa. [Abstract/Link to Full Text]

Kim S, Choi H, Park ZY
Improved detection of multi-phosphorylated peptides by LC-MS/MS without phosphopeptide enrichment.
Mol Cells. 2007 Jun 30;23(3):340-8.
Although considerable effort has been devoted in the mass spectrometric analysis of phosphorylated peptides, successful identification of multi-phosphorylated peptides in enzymatically digested protein samples still remains challenging. The ionization behavior of multi-phosphorylated peptides appears to be somewhat different from that of mono- or di-phosphorylated peptides. In this study, we demonstrate increased sensitivity of detection of multi-phosphorylated peptides of beta casein without using phosphopeptide enrichment techniques. Proteinase K digestion alone increased the detection limit of beta casein multi-phosphorylated peptides in the LC-MS analysis almost 500 fold, compared to conventional trypsin digestion (~50 pmol). In order to understand this effect, various factors affecting the ionization of phosphopeptides were investigated. Unlike ionizations of phosphopeptides with minor modifications, those of multi-phosphorylated peptides appeared to be subject to effects such as selectively suppressed ionization by more ionizable peptides and decreased ionization efficiency by multi-phosphorylation. The enhanced detection limit of multi- phosphorylated peptides resulting from proteinase K digestion was validated using a complex protein sample, namely a lysate of HEK 293 cells. Compared to trypsin digestion, the numbers of phosphopeptides identified and modification sites per peptide were noticeably increased by proteinase K digestion. Non-specific proteases such as proteinase K and elastase have been used in the past to increase detection of phosphorylation sites but the effectiveness of proteinase K digestion for multi-phosphorylated peptides has not been reported. [Abstract/Link to Full Text]

Park YY, Teyssier C, Vanacker JM, Choi HS
Distinct repressive properties of the mammalian and fish orphan nuclear receptors SHP and DAX-1.
Mol Cells. 2007 Jun 30;23(3):331-9.
It has been suggested that the structure and function of nuclear receptors are evolutionally conserved. Here, we compare the molecular functions of the nile tilapia (Oreochromis niloticus) small heterodimer partner (nSHP/NR0B2) and the Dosage-sensitive sex reversal AHC critical region on X chromosome gene 1 (nDAX-1/NR0B1) with those of human SHP and DAX-1 (hSHP and hDAX-1, respectively). We found that, upon transient cotransfection of human cells, nDAX-1 repressed the activity of tilapia SF-1 (nSF-1) but not that of human SF-1, although the physical interaction with human SF-1 was retained. Similarly, nSHP repressed the activity of nSF-1, whereas hSHP did not, pointing to divergent evolution of SHP/SF-1 in fish and human. We thus propose that the repressive functions of SHP and DAX-1 have been conserved in fish and mammals although with different transcriptional targets and mechanisms. These differences provide new insights into the physiological diversification of atypical orphan nuclear receptors during vertebrate evolution. [Abstract/Link to Full Text]

Jia H, Yi D, Yu J, Xue S, Xiang Y, Zhang C, Zhang Z, Zhang L, Ma Z
Mapping QTLs for tissue culture response of mature wheat embryos.
Mol Cells. 2007 Jun 30;23(3):323-30.
The mature wheat embryo is arguably one of the best explants for genetic transformation because of its unlimited availability and lack of growth season restriction. However, an efficient regeneration system using mature wheat embryos (Triticum aestivum L.) is still not available. To identify genes related to the tissue culture response (TCR) of wheat, QTLs for callus induction from mature embryos and callus regeneration were mapped using an RIL population derived from the cross of "Wangshuibai" with "Nanda2419" which has a good TCR. By whole genome scanning we identified five, four and four chromosome regions conditioning, respectively, percent embryos forming a callus (PEFC), percent calli regenerating plantlets (PCRP), and number of plantlets per regenerating callus (NPRC). The major QTLs QPefc.nau-2A and QPcrp.nau-2A were mapped to the long arm of chromosome 2A, explaining up to 22.8% and 17.6% of the respective phenotypic variance. Moreover, two major QTLs for NPRC were detected on chromosomes 2D and 5D; these together explained 51.6% of the phenotypic variance. We found that chromosomes 2A, 2D, 5A, 5B and 5D were associated via different intervals with at least two of the three TCR indexes used. Based on this study and other reports, the TCRs of different explant types of wheat may be under the control of shared or tightly linked genes, while different genes or gene combinations may govern the stages from callus induction to plantlet regeneration. The importance of group 2 and 5 chromosomes in controlling the TCRs of Triticeae crops and the likely conservation of the corresponding genes in cereals are discussed. [Abstract/Link to Full Text]

Gu X, Zheng M, Fei X, Yang Z, Li F, Ji C, Xie Y, Mao Y
ZNF435, a novel human SCAN-containing zinc finger protein, inhibits AP-1-mediated transcriptional activation.
Mol Cells. 2007 Jun 30;23(3):316-22.
Zinc finger transcription factor genes are a significant fraction of the genes in the vertebrate genome. Here we report the isolation and characterization of a human zinc finger-containing gene, ZNF435, from a fetal brain cDNA library. ZNF435 cDNA is 1290 base pairs in length and contains an open reading frame encoding 349 amino acids with four C2H2-type zinc fingers at its carboxyl terminus and a SCAN motif at its amino terminus. RT-PCR results showed that ZNF435 was expressed in all tested tissues. A ZNF435-GFP fusion protein was located in the nucleus and the four zinc fingers acted as nuclear localization signals (NLSs). ZNF435 was found to be capable of homo-association, and this effect was independent of its zinc fingers. Furthermore, ZNF435 proved to be a transcription repressor as its overexpression in AD293 cells inhibited the transcriptional activities of AP-1. [Abstract/Link to Full Text]

Kwon SJ, Lim WS, Park SH, Park MR, Kim KH
Molecular characterization of a dsRNA mycovirus, Fusarium graminearum virus-DK21, which is phylogenetically related to hypoviruses but has a genome organization and gene expression strategy resembling those of plant potex-like viruses.
Mol Cells. 2007 Jun 30;23(3):304-15.
Fusarium graminearum causes a serious scab disease of small grains in Korea. The nucleotide sequence of the genomic RNA of a double-stranded RNA (dsRNA) virus, Fusarium graminearum virus-DK21 (FgV-DK21), from F. graminearum strain DK21, which is associated with hypovirulence in F. graminearum, was determined and compared to the genome sequences of other mycoviruses, including Cryponectria hypoviruses. The FgV-DK21 dsRNA consists of 6,624 nucleotides, excluding the 3'-terminal poly(A) tail. The viral genome has 53- and 46-nucleotide 5' and 3' untranslated regions (UTRs), respectively, and five putative open reading frames. A phylogenetic analysis of the deduced amino acid sequence of ORF1, which encodes a putative RNA-dependent RNA polymerase, and those of other mycoviruses revealed that this organism forms a distinct virus clade with other hypoviruses, and is more distantly related to other mycoviruses (3.8 to 24.0% identity). However, pairwise sequence comparisons of the nucleotide and deduced amino acid sequences of ORFs 2 through 5 revealed no close relationships to other protein sequences currently available in GenBank. Analyses of RNA accumulation by Northern blot and primer extension indicated that these putative gene products are expressed from at least two different subgenomic RNAs (sgRNAs), in contrast to the cases in other hypoviruses. This study suggests the existence of a new, as yet unassigned, genus of mycoviruses that exhibits a potex-like genome organization and sgRNA accumulation. [Abstract/Link to Full Text]

Lee YM, Rhee JS, Hwang DS, Kim IC, Raisuddin S, Lee JS
Mining of biomarker genes from expressed sequence tags and differential display reverse transcriptase-polymerase chain reaction in the self-fertilizing fish, Kryptolebias marmoratus and their expression patterns in response to exposure to an endocrine-disrupting alkylphenol, bisphenol A.
Mol Cells. 2007 Jun 30;23(3):287-303.
Expressed sequence tags (ESTs) and differentially expressed cDNAs from the self-fertilizing fish, Kryptolebias marmoratus were mined to develop alternative biomarkers for endocrine-disrupting chemicals (EDCs). 1,577 K. marmoratus cDNA clones were randomly sequenced from the 5'-end. These clones corresponded to 1,518 and 1,519 genes in medaka dbEST and zebrafish dbEST, respectively. Of the matched genes, 197 and 115 genes obtained Unigene IDs in medaka dbEST and zebrafish dbEST, respectively. Many of the annotated genes are potential biomarkers for environmental stresses. In a differential display reverse transcriptase-polymerase chain reaction (DD RT-PCR) study, 56 differential expressed genes were obtained from fish liver exposed to bisphenol A. Of these, 16 genes were identified after BLAST search to GenBank, and the annotated genes were mainly involved in catalytic activity and binding. The expression patterns of these 16 genes were validated by real-time RT-PCR of liver tissue from fish exposed to bisphenol A. Our findings suggest that expression of these 16 genes is modulated by endocrine disrupting chemicals, and therefore that they are potential biomarkers for environmental stress including EDCs exposure. [Abstract/Link to Full Text]

Cho CW, Lee HJ, Chung E, Kim KM, Heo JE, Kim JI, Chung J, Ma Y, Fukui K, Lee DW, Kim DH, Chung YS, Lee JH
Molecular characterization of the soybean L-asparaginase gene induced by low temperature stress.
Mol Cells. 2007 Jun 30;23(3):280-6.
L-asparaginase (EC 3.5.1.1) catalyzes the hydrolysis of the amide group of L-asparagine, releasing aspartate and NH4+. We isolated a low temperature-inducible cDNA sequence encoding L-asparaginase from soybean leaves. The full-length L-asparaginase cDNA, designated GmASP1, contains an open reading frame of 1,258 bp coding for a protein of 326 amino acids. Genomic DNA blotting and fluorescence in situ hybridization showed that the soybean genome has two copies of GmASP1. GmASP1 mRNA was induced by low temperature, ABA and NaCl, but not by heat shock or drought stress. E. coli cells expressing recombinant GmASP1 had 3-fold increased L-asparaginase activity. A possible function of L-asparaginase in the early response to low temperature stress is discussed. [Abstract/Link to Full Text]

Suh SH, Paik IY, Jacobs K
Regulation of blood glucose homeostasis during prolonged exercise.
Mol Cells. 2007 Jun 30;23(3):272-9.
The maintenance of normal blood glucose levels at rest and during exercise is critical. The maintenance of blood glucose homeostasis depends on the coordination and integration of several physiological systems, including the sympathetic nervous system and the endocrine system. During prolonged exercise increased demand for glucose by contracting muscle causes to increase glucose uptake to working skeletal muscle. Increase in glucose uptake by working skeletal muscle during prolonged exercise is due to an increase in the translocation of insulin and contraction sensitive glucose transporter-4 (GLUT4) proteins to the plasma membrane. However, normal blood glucose level can be maintained by the augmentation of glucose production and release through the stimulation of liver glycogen breakdown, and the stimulation of the synthesis of glucose from other substances, and by the mobilization of other fuels that may serve as alternatives. Both feedback and feedforward mechanisms allow glycemia to be controlled during exercise. This review focuses on factors that control blood glucose homeostasis during prolonged exercise. [Abstract/Link to Full Text]

Zhuo M
A synaptic model for pain: long-term potentiation in the anterior cingulate cortex.
Mol Cells. 2007 Jun 30;23(3):259-71.
Investigation of molecular and cellular mechanisms of synaptic plasticity is the major focus of many neuroscientists. There are two major reasons for searching new genes and molecules contributing to central plasticity: first, it provides basic neural mechanism for learning and memory, a key function of the brain; second, it provides new targets for treating brain-related disease. Long-term potentiation (LTP), mostly intensely studies in the hippocampus and amygdala, is proposed to be a cellular model for learning and memory. Although it remains difficult to understand the roles of LTP in hippocampus-related memory, a role of LTP in fear, a simplified form of memory, has been established. Here, I will review recent cellular studies of LTP in the anterior cingulate cortex (ACC) and then compare studies in vivo and in vitro LTP by genetic/ pharmacological approaches. I propose that ACC LTP may serve as a cellular model for studying central sensitization that related to chronic pain, as well as pain-related cognitive emotional disorders. Understanding signaling pathways related to ACC LTP may help us to identify novel drug target for various mental disorders. [Abstract/Link to Full Text]

Kim HB, Lee H, Oh CJ, Lee NH, An CS
Expression of EuNOD-ARP1 encoding auxin-repressed protein homolog is upregulated by auxin and localized to the fixation zone in root nodules of Elaeagnus umbellata.
Mol Cells. 2007 Feb 28;23(1):115-21.
Root nodule formation is controlled by plant hormones such as auxin. Auxin-repressed protein (ARP) genes have been identified in various plant species but their functions are not clear. We have isolated a full-length cDNA clone (EuNOD-ARP1) showing high sequence homology to previously identified ARP genes from root nodules of Elaeagnus umbellata. Genomic Southern hybridization showed that there are at least four ARP-related genes in the genome of E. umbellata. The cDNA clone encodes a polypeptide of 120 amino acid residues with no signal peptide or organelle-targeting signals, indicating that it is a cytosolic protein. Its cytosolic location was confirmed using Arabidopsis protoplasts expressing a EuNOD-ARP1:smGFP fusion protein. Northern hybridization showed that EuNOD-ARP1 expression was higher in root nodules than in leaves or uninoculated roots. Unlike the ARP genes of strawberry and black locust, which are negatively regulated by exogenous auxin, EuNOD-ARP1 expression is induced by auxin in leaf tissue of E. umbellata. In situ hybridization revealed that EuNOD-ARP1 is mainly expressed in the fixation zone of root nodules. [Abstract/Link to Full Text]

You MK, Oh SI, Ok SH, Cho SK, Shin HY, Jeung JU, Shin JS
Identification of putative MAPK kinases in Oryza minuta and O. sativa responsive to biotic stresses.
Mol Cells. 2007 Feb 28;23(1):108-14.
The mitogen-activated protein kinase (MAPK) signaling cascade is critical for regulating plant defense systems against various kinds of pathogen and environmental stresses. One component of this cascade, the MAP kinase kinases (MAPKK), has not yet been shown to be induced in plants following biotic attacks, such as those by insects and fungi. We describe here a gene coding for a blast (Magnaporthe grisea)- and insect (Nilaparvata lugens)-responsive putative MAPK kinase, OmMKK1 (Oryza minuta MAPKK 1), which was identified in a library of O. minuta expressed sequence tags (ESTs). Two copies of OmMKK1 are present in the O. minuta genome. They encode a predicted protein with molecular mass 39 kDa and pI of 6.2. Transcript patterns following imbibition of plant hormones such as methyl jasmonic acid (MeJA), ethephone, salicylic acid (SA) and abscisic acid (ABA), as well as exposure to methyl viologen (MV), revealed that the expression of OmMKK1 is related to defense response signaling pathways. A comparative analysis of OmMKK1 and its O. sativa ortholog OsMKK1 showed that both were induced by stress-related hormones and biotic stresses, but that the kinetics of their responses differed despite their high amino acid sequence identity (96%). [Abstract/Link to Full Text]

Cho SH, Hoang Q, Phee JW, Kim YY, Shin HY, Shin JS
Modified suppression subtractive hybridization identifies an AP2-containing protein involved in metal responses in Physcomitrella patens.
Mol Cells. 2007 Feb 28;23(1):100-7.
The moss Physcomitrella patens has two life cycles, filamentous protonema and leafy gametophore. A modified from of suppression subtractive hybridization (SSH), mirror orientation selection (MOS), was applied to screen genes differentially expressed in the P. patens protonema. Using reverse Northern blot analysis, differentially expressed clones were identified. The identified genes were involved mainly in metal binding and detoxification. One of these genes was an AP2 (APETALA2) domain-containing protein (PpACP1), which was highly up-regulated in the protonema. Alignment with other AP2/EREBPs (Ethylene Responsive Element Binding Proteins) revealed significant sequence homology of the deduced amino acid sequence in the AP2/EREBP DNA binding domain. Northern analysis under various stress conditions showed that PpACP1 was induced by ethephon, cadmium, copper, ABA, IAA, and cold. In addition, it was highly expressed in suspension-cultured protonema. We suggest that PpACP1 is involved in responses to metals, and that suspension culture enhance the expression of genes responding to metals. [Abstract/Link to Full Text]

Hwang MN, Kim KS, Choi YW, Jou I, Yoon S
PMA activates Stat3 in the Jak/Stat pathway and induces SOCS5 in rat brain astrocytes.
Mol Cells. 2007 Feb 28;23(1):94-9.
Suppressors of cytokine signaling (SOCS) family members are negative feedback regulators of the Jak/Stat pathway, which is an essential inflammatory signaling pathway. We investigated expression of eight members of the SOCS family in rat astrocytes, using two inflammatory stimulants, PMA and IFN-gamma. Only a few SOCS genes were induced by both stimulants, and we detected an increase in SOCS5 protein with PMA. PMA activated the Jnk, Erk, p38, and Jak/Stat signal pathways. In addition, it increased the level of activated-Stat3 resulting from tyrosine phosphorylation. A gel-shift assay showed that a protein in nuclear extracts from PMA-treated cells was able to bind to Stat binding elements. These results suggest that activated Stat3 binds to SOCS promoters and leads to their transcriptional induction. [Abstract/Link to Full Text]

Park JJ, Lee HK, Shin MW, Kim SJ, Noh SY, Shin J, Yu WS
Short-term cold exposure may cause a local decrease of neuropeptide Y in the rat hypothalamus.
Mol Cells. 2007 Feb 28;23(1):88-93.
Neuropeptide Y (NPY) is an orexigenic and hypothermic peptide. To understand its role in hypothermic conditions, male rats were placed in a 24 degrees C or 4 degrees C air chamber for 1.5 h. The expression of c-Fos protein, and NPY mRNA and protein, was analyzed in the hypothalamus 1 h-2 h later. The cold treatment increased the number of c-Fos-immunoreactive cells in the paraventricular hypothalamic nucleus (PVN) and arcuate nucleus (ARC). At the same time it decreased the density of NPY-immunoreactive components in the PVN, dorsomedial hypothalamic nucleus and ARC, as well as of NPY transcripts in the PVN and ARC. No colocalization of c-Fos with NPY was detected. These results suggest that short-term cold exposure should reduce indirectly NPY production in some hypothalamic nuclei to facilitate thermogenesis without inducing feeding behavior. [Abstract/Link to Full Text]

---

Recent Articles in Cell Research

---

Shi YH, Liu J, Xia JH, Gui JF
Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp.
Cell Res. 2002 Jun;12(2):133-42.
A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169 TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control of gene expression in the gynogenetic fish embryogenesis. [Abstract/Link to Full Text]

Zhang M, Zhang BH, Chen L, An W
Overexpression of heme oxygenase-1 protects smooth muscle cells against oxidative injury and inhibits cell proliferation.
Cell Res. 2002 Jun;12(2):123-32.
To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we established an in vitro transfection of human HO-1 gene into rat VSMC mediated by a retroviral vector. The results showed that the profound expression of HO-1 protein as well as HO activity was 1.8- and 2.0-fold increased respectively in the transfected cells compared to the non-transfected ones. The treatment of VSMC with different concentrations of H2O2 led to the remarkable cell damage as indicated by survival rate and LDH leakage. However, the resistance of the HO-1 transfected VSMC against H2O2 was significantly raised. This protective effect was dramatically diminished when the transfected VSMC were pretreated with ZnPP-IX, a specific inhibitor of HO, for 24 h. In addition, we found that the growth potential of the transfected cells was significantly inhibited directly by increased activity of HO-1, and this effect might be related to decreased phosphorylation of MAPK. These results suggest that the overexpression of introduced hHO-1 is potentially able to reduce the risk factors of atherosclerosis, partially due to its cellular protection against oxidative injury and to its inhibitory effect on cellular proliferation. [Abstract/Link to Full Text]

Li H, Chen XY, Kong QY, Liu J
Cytopathological evaluations combined RNA and protein analyses on defined cell regions using single frozen tissue block.
Cell Res. 2002 Jun;12(2):117-21.
The co-existence of multiple cell components in tissue samples is the main obstacle for precise molecular evaluation on defined cell types. Based on morphological examination, we developed an efficient approach for paralleled RNA and protein isolations from an identical histological region in frozen tissue section. The RNA and protein samples prepared were sufficient for RT-PCR and Western blot analyses, and the results obtained were well coincident each other as well as with the corresponding parameters revealed from immunohistochemical examinations. By this way, the sampling problem caused by cell-cross contamination can be largely avoided, committing the experimental data more specific to a defined cell type. These novel methods thus allow us to use single tissue block for a comprehensive study by integration of conventional cytological evaluations with nucleic acid and protein analyses. [Abstract/Link to Full Text]

Ye SQ, Lavoie T, Usher DC, Zhang LQ
Microarray, SAGE and their applications to cardiovascular diseases.
Cell Res. 2002 Jun;12(2):105-15.
The wealth of DNA data generated by the human genome project coupling with recently invented high-throughput gene expression profiling techniques has dramatically sped up the process for biomedical researchers on elucidating the role of genes in human diseases. One powerful method to reveal insight into gene functions is the systematic analysis of gene expression. Two popular high-throughput gene expression technologies, microarray and Serial Analysis of Gene Expression (SAGE) are capable of producing large amounts of gene expression data with the potential of providing novel insights into fundamental disease processes, especially complex syndromes such as cardiovascular disease, whose etiologies are due to multiple genetic factors and their interplay with the environment. Microarray and SAGE have already been used to examine gene expression patterns of cell-culture, animal and human tissues models of cardiovascular diseases. In this review, we will first give a brief introduction of microarray and SAGE technologies and point out their limitations. We will then discuss the major discoveries and the new biological insights that have emerged from their applications to cardiovascular diseases. Finally we will touch upon potential challenges and future developments in this area. [Abstract/Link to Full Text]

Zhang W, Liu HT
MAPK signal pathways in the regulation of cell proliferation in mammalian cells.
Cell Res. 2002 Mar;12(1):9-18.
MAPK families play an important role in complex cellular programs like proliferation, differentiation, development, transformation, and apoptosis. At least three MAPK families have been characterized: extracellular signal-regulated kinase (ERK), Jun kinase (JNK/SAPK) and p38 MAPK. The above effects are fulfilled by regulation of cell cycle engine and other cell proliferation related proteins. In this paper we discussed their functions and cooperation with other signal pathways in regulation of cell proliferation. [Abstract/Link to Full Text]

Hou CH, Huang J, Qian RL
Identification of a NF-kappaB site in the negative regulatory element (epsilon-NRAII) of human epsilon-globin gene and its binding protein NF-kappaB p50 in the nuclei of K562 cells.
Cell Res. 2002 Mar;12(1):79-82.
The developmental control of the human epsilon-globin gene expression is mediated by transcription regulatory elements in the 5' flanking DNA of this gene. Sequence analysis has revealed a DNA motif (GGGGAATTTGCT) similar to NF-kappaB consensus sequence resides in the negative regulatory element (-3028bp approximately -2902bp, termed e-NRAII) 5' to the cap site of this gene. NRF DNA fragment (-3010bp approximately -2986bp) containing the NF-kappaB motif similar sequence was synthesized and used in electrophoresis mobility shift assay (EMSA) and competitive analysis. Data showed that a protein factor from nuclear extracts of K562 cells specifically interacted with NRF DNA fragment. The synthetic NF DNA fragment (containing NF-kappaB consensus sequence) could competed for the protein binding, but MNF DNA fragment (mutated NF-kappaB motif) could not, suggesting that the binding protein is a member of NF-kappaB/Rel family. Western blot assay demonstrated that the molecular weight of NF-kappaB protein in the nuclei of K562 cells is 50ku. We suggested that NF-kappaB p50 may play an important role in the regulation of human epsilon-globin gene expression. [Abstract/Link to Full Text]

Yong P, Gu Z, Luo JP, Wang JR, Tso JK
Antibodies against the C-terminal peptide of rabbit oviductin inhibit mouse early embryo development to pass 2-cell stage.
Cell Res. 2002 Mar;12(1):69-78.
A full-length rabbit oviductin cDNA(1909bp) was cloned. It consists of a 5'-UTR of 52bp, an open reading frame (ORF) of 1374bp and a 3'-UTR of 483bp and has more than 80% homology with that of other mammal oviductins. N-terminal peptide (NTP) (384 residues) and C-terminal peptide (CTP) (73 residues) of deduced protein precursor has about 80% and 50% identity with that of other mammals respectively. Fusion proteins GST-NTP 368(1R-368N)and GST-CTP73 (369F-441A) were expressed and purified. NH2-terminal of CTP sequencing reveals that the purified protein is consistent with the deduced one. In order to study the function of NTP and CTP the mouse anti-NTP and rabbit anti-CTP antisera were prepared. Tissue-specific (skeleton muscle, oviduct, uterus, ovary, liver, heart and brain) analysis indicated that rabbit oviductin was only found in oviduct. The conditioned medium derived from the rabbit oviduct mucosa epithelial cells has a function of overcoming the early embryonic development block of Kunming mouse cultured in vitro. Anti-CTP antiserum could totally inhibit the early embryo development at 2-cell stage cultured in the conditioned culture medium, but anti-NTP antiserum couldn't. There was a positive relationship between the ratio of early embryos at development block and the dosage of anti-CTP antiserum added in the conditioned culture medium. These results suggest that oviductin has a function not only on fertilization, but also on the release of early embryonic development block, and the later function domain of rabbit oviductin may be situate in its C-terminal. [Abstract/Link to Full Text]

Qi ZX, Zeng H, Li XL, Chen CB, Song WQ, Chen RY
The molecular characterization of maize B chromosome specific AFLPs.
Cell Res. 2002 Mar;12(1):63-8.
The origin and evolution of B chromosomes could be explained by the specific DNA sequence on them. But the specific sequences known were quite limited. To investigate maize B chromosome sqicific DNA sequeces, maize genomes with and without B chromosomes were analyzed by AFLP. Only 5 markers were found specific to genomes with B chromosomes among about 2000 AFLP markers. Southern hybridization and sequence analysis revealed that only the sequence of M8-2D was a B chromosome specific sequence. This sequence contained the telomeric repeat unit AGG [Abstract/Link to Full Text]

Zhang RG, Zhang RP, Wang XW, Xie H
Effects of cisplatin on telomerase activity and telomere length in BEL-7404 human hepatoma cells.
Cell Res. 2002 Mar;12(1):55-62.
Telomerase activity was inhibited in a dose and time-dependent manner with the treatment of cisplatin for 24, 48, or 72 h in a concentration ranged from 0.8 to 50 microM in BEL-7404 human hepatoma cells. There were no changes in expression pattern of three telomerase subunits, its catalytic reverse transcriptase subunit (hTERT), its RNA component (hTR) or the associated protein subunit (TP1), after cisplatin treated for 72 h with indicated concentrations. Mean telomere lengths were decreased by the cisplatin treatment. Cell growth inhibition and cell cycle accumulation in G2/M phase were found to be correlated with telomerase inhibition in the present study, but percentages of cell apoptosis did not change markedly during the process. [Abstract/Link to Full Text]

Chen L, Li G, Tang L, Wang J, Ge XR
The inhibition of lung cancer cell growth by intracellular immunization with LC-1 ScFv.
Cell Res. 2002 Mar;12(1):47-54.
A monoclonal antibody, LC-1, recognizing lung cancer associated common antigens was obtained in authors' laboratory. Its single chain Fv fragment (ScFv) named LC-1 ScFv was constructed based on recombinant phage displayed techniques. For expression on cell membrane, LC-1 ScFv was cloned into pDisplay vector, which directed the cloned gene to express as cell membrane bound protein. The resulting plasmid was sequenced and then introduced by the lipofectin method into a lung adenocarcinoma cell line SPC-A-1. G418 resistant cells were obtained by G418 selection. After transfection, LC-1 ScFv expression was observed by Western blot analysis and the expression of cognate antigens was down-regulated as shown in ELISA assay. SPC-A-1-pDisplay-ScFv cells grew in vitro at lower speed than the control intact cells and the cells transfected with vacant vector. Flow cytometry analysis detected a substantial increase in G1 phase and decrease in S phase in population of SPC-A-1-pDisplay-ScFv cells compared to SPC-A-1 and SPC-A-1-pDisplay cells. Semi-quantitative RT-PCR analysis showed that c-myc expression was down-regulated in SPC-A-1-pDisplay-ScFv cells. It seems that the antigens recognized by LC-1 may be in some way involved in a growth stimulating pathway and the antibody blocking of the function of the antigens shut down the pathway and thus down-regulate the expression of c-myc and growth of the cells. [Abstract/Link to Full Text]

Li B, Sun M, He B, Yu J, Zhang YD, Zhang YL
Identification of differentially expressed genes in human uterine leiomyomas using differential display.
Cell Res. 2002 Mar;12(1):39-45.
In searching of differentially expressed genes in human uterine leiomyomas, differential display was used with twelve pairs of primers to compare human uterine leiomyomas with matched myometrium. False positives were eliminated by reverse Northern analysis. Positives were confirmed by Northern blot analysis. RESULTS: Four of 69 cDNA fragments (3 up-regulated named L1, L2 and L3 and 1 down-regulated named M1 in leiomyoma) were confirmed by Northern analysis. Sequence comparison and Northern analysis proved that L1 is exactly the human ribosomal protein S19. It was present ubiquitously in 13 tissues tested but in various levels and even in different size. L1 was highly expressed in parotidean cystadenocarcinoma, pancreatic cancer and breast cancer examined. No mutations have been found in human uterine leiomyomas (n=6). CONCLUSIONS: hRPS19 overexpression might be a universal signal in rapid cell growth tissues. [Abstract/Link to Full Text]

Hu JH, Yan YC
Identification of gamma1 subunit of GABA(A) receptor in rat testis.
Cell Res. 2002 Mar;12(1):33-7.
The isoform type of gamma subunits of GABA(A) receptor is a molecular determinant of its pharmacological characteristics. At present, the existence of GABA(A) receptor in mammalian sperm is still a controversy. By using degenerate primers designed according to highly conserved region in all three gamma (gamma1, gamma2 and gamma3) subunits cloned in rat brain, we performed reverse transcription polymerase chain reaction (RT-PCR) to examine the expression pattern of gamma subunits of GABA(A) receptor in rat testis. Only one 370 bp fragment was obtained from RT-PCR in rat testis and sequencing results showed that it represented gamma1 subunit, but not gamma2 or gamma3 subunit. Using the cloned fragment as probe, a 3.8 kb transcript which in size as same as gamma1 subunit in rat brain was detected in rat testis mRNA by performing Northern blot assay. Furthermore, results of in situ hybridization assay confirmed that gamma1 subunit was expressed in round spermatids and spermatozoa, maybe also in secondary spermatocyte. These evidences proved that gamma1 subunit of GABA(A) receptor is exclusively expressed in rat testis and this feature may be the structural basis of the specific function of GABA(A) receptors in sperm acrosome reaction. [Abstract/Link to Full Text]

Kiani C, Chen L, Wu YJ, Yee AJ, Yang BB
Structure and function of aggrecan.
Cell Res. 2002 Mar;12(1):19-32.
Aggrecan is the major proteoglycan in the articular cartilage. This molecule is important in the proper functioning of articular cartilage because it provides a hydrated gel structure (via its interaction with hyaluronan and link protein) that endows the cartilage with load-bearing properties. It is also crucial in chondroskeletal morphogenesis during development. Aggrecan is a multimodular molecule expressed by chondrocytes. Its core protein is composed of three globular domains (G1, G2, and G3) and a large extended region (CS) between G2 and G3 for glycosaminoglycan chain attachment. G1 comprises the amino terminus of the core protein. This domain has the same structural motif as link protein. Functionally, the G1 domain interacts with hyaluronan acid and link protein, forming stable ternary complexes in the extracellular matrix. G2 is homologous to the tandem repeats of G1 and of link protein and is involved in product processing. G3 makes up the carboxyl terminus of the core protein. It enhances glycosaminoglycan modification and product secretion. Aggrecan plays an important role in mediating chondrocyte-chondrocyte and chondrocyte-matrix interactions through its ability to bind hyaluronan. [Abstract/Link to Full Text]

Mu J, Wei LX
Telomere and telomerase in oncology.
Cell Res. 2002 Mar;12(1):1-7.
Shortening of the telomeric DNA at the chromosome ends is presumed to limit the lifespan of human cells and elicit a signal for the onset of cellular senescence. To continually proliferate across the senescent checkpoint, cells must restore and preserve telomere length. This can be achieved by telomerase, which has the reverse transcriptase activity. Telomerase activity is negative in human normal somatic cells but can be detected in most tumor cells. The enzyme is proposed to be an essential factor in cell immortalization and cancer progression. In this review we discuss the structure and function of telomere and telomerase and their roles in cell immortalization and oncogenesis. Simultaneously the experimental studies of telomerase assays for cancer detection and diagnosis are reviewed. Finally, we discuss the potential use of inhibitors of telomerase in anti-cancer therapy. [Abstract/Link to Full Text]

Hu YJ, Zang L, Wu YD, Sun B
High IFN-alpha expression is associated with the induction of experimental autoimmune uveitis (EAU) in Fischer 344 rat.
Cell Res. 2001 Dec;11(4):293-300.
Th1-response plays a crucial role in determining pathogenesis of organ-specific autoimmune diseases. It is believed that both IL-12 and INF-alpha are initiators to regulate Th1-response. In our experimental autoimmune uveitis (EAU) model, both Lewis and Fischer 344 rats share the same MHC class II molecules, while Lewis rat is EAU susceptible and Fischer 344 rat is EAU resistant. However, under the same condition of immunization, if pertussis toxin (PTX) was injected intraperitoneally as an additional adjuvant, Fischer 344 rat can develop EAU. In this study we investigate which mechanisms are involved in the induction of EAU in CFA+R16+PTX-treated (CRP-treated) Fischer 344 rats. In vivo and in vitro data demonstrated that Th1-cytokine, IFN-gamma mRNA expression was significantly increased in disease target tissue-eyes and in draining lymph node cells of CRP-treated Fischer 344 rat. When IL-12 and IFN-alpha mRNA expression were compared in the experimental groups, only IFN-alpha mRNA expression was associated with EAU development. To distinguish the sources of IFN-alpha producing cells, it was observed that IFN-alpha expression was mainly produced by macrophages. It was further confirmed that normal macrophage from Fischer 344 rat was able to produce significant IFN-alpha in the presence of PTX. The data strongly suggested that IFN-alpha might be involved in initiating Th1-cell differentiation and in turn contribute to the induction of EAU. High IFN-alpha expression induced by PTX may represent a novel pathway to initiate Th1 response in Fischer 344 rat. [Abstract/Link to Full Text]

Guo XZ, Su JD, Sun QW, Jiao BH
Expression of estrogen receptor (ER) -alpha and -beta transcripts in the neonatal and adult rat cerebral cortex, cerebellum, and olfactory bulb.
Cell Res. 2001 Dec;11(4):321-4.
In the present study expression of estrogen receptor subtype -alpha (ERalpha) and -beta (ERbeta) in the cerebral cortex, cerebellum, and olfactory bulb was investigated and compared between neonatal (1 to approximately 3-days-old) and adult (250 to approximately 350 g) rats, using reverse transcription-polymerase chain reaction (RT-PCR). No ERalpha transcripts were detectable in the adult cerebellum and olfactory bulb, whereas very weak expression of ERalpha was present in the adult cerebral cortex. No significant difference in ERbeta transcripts was detectable between the neonatal and adult rats. While transcripts for both ER subtypes were co-expressed in these brain areas of neonatal rats, although ERalpha expression was significantly weaker than ERbeta. Even in the cerebral cortex known to contain both ER subtypes in adult rats, ERalpha transcripts in neonatal rats were much higher than in adult. These observations provide evidence for the existence of different expression patterns of ERalpha/ERbeta transcripts in these three brain areas between the neonatal and adult rats, suggesting that each ER subtype may play a distinct role in the regulation of differentiation, development, and functions of the brain by estrogen. [Abstract/Link to Full Text]

Li H, Ma SK, Hu XP, Zhang GY, Fei J
Norepinephrine transporter (NET) is expressed in cardiac sympathetic ganglia of adult rat.
Cell Res. 2001 Dec;11(4):317-20.
The sympathetic nervous system plays a cardinal role in regulating cardiac function through releasing the neurotransmitter norepinephrine (NE). In comparison with central nervous system, the molecular mechanism of NE uptake in myocardium is not clear. In present study, we proved that in rat the CNS type of NE transporter (NET) was also expressed in middle cervical-stellate ganglion complex (MC-SG complex) which is considered to control the activity of heart, but not expressed in myocardium. The results also showed that NET expression level in right ganglion was significantly higher than in the left, rendering the greater capacity of NE uptake in right ventricle, a fact which may contribute to the maintenance of right ventricular function under pathologic state. [Abstract/Link to Full Text]

Li HL, Ye KH, Zhang HW, Luo YR, Ren XD, Xiong AH, Situ R
Effect of heparin on apoptosis in human nasopharyngeal carcinoma CNE2 cells.
Cell Res. 2001 Dec;11(4):311-5.
In order to study the mechanism of the effect of heparin on apoptosis in carcinoma cells, the nasopharyngeal carcinoma cell line CNE2 was used to identify the effect of heparin on apoptosis associated with the expression of c-myc, bax, bcl-2 proteins by use of Hoechst 33258 staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), agarose gel electrophoresis, and flow cytometry, as well as Western blot analysis. The results showed that heparin induced apoptosis of CNE2 cells including the morphologic changes such as reduction in the volume, and the nuclear chromatin condensation, as well as the "ladder pattern" revealed by agarose gel electrophoresis of DNA in a concentration-dependent manner. The number of TUNEL-positive cells was dramatically increased to 33.6+/-1.2% from 2.8+/-0.3% by treatment with heparin in different concentrations (10 to approximately 40 kU/L). The apoptotic index was increased to 32.5% from 3.5% by detecting SubG1 peaks on flow cytometry. Western blot analysis showed that levels of bcl-2, bax and c-myc were significantly overexpressed by treatment with the increase of heparin concentrations. These results suggest that heparin induces apoptosis of CNE2 cells, which may be regulated by differential expression of apoptosis-related genes. [Abstract/Link to Full Text]

Zhang SB, He QY, Zhao H, Gui CY, Jiang C, Qian RL
Function of GATA transcription factors in hydroxyurea-induced HEL cells.
Cell Res. 2001 Dec;11(4):301-10.
HEL cells, a human erythroleukemia cell line, mainly express the fetal (gamma) globin gene and trace amount of the embryonic (epsilon) globin gene, but not adult (beta) globin gene. Here we show that hydroxyurea (HU) can induce HEL cells to express adult (beta) globin gene and lead these cells to terminal differentiation. Results showed in Gel mobility shift assays that GATA factors could specifically bind to the regulatory elements of human beta-globin gene, including the proximal regulatory element (the beta-promoter) and the distal regulatory elements (the DNase I hypersensitive sites in the LCR, HS2-HS4 core sequences). However, the DNA binding patterns of GATA factors were quite different between HU-induced and uninduced HEL cells. Western-blot analysis of nuclear extracts from both the uninduced and HU-induced HEL cells revealed that the level of GATA-2 transcription factor decreased, whereas the level of GATA-1 transcription factor increased following the time of hydroxyurea induction. Furthermore, using RT-PCR analysis the expression of human beta-globin gene in HU-induced HEL cells could be blocked again when HEL cells were incubated in the presence of antisense oligonucleotides for hGATA-1, suggesting that the upregulation of hGATA-1 transcription factor might be critical for the expression of human beta-globin gene in HU-induced HEL cells. [Abstract/Link to Full Text]

Wang JW, Wu JR
Overexpression of a novel gene, Cms1, can rescue the growth arrest of a Saccharomyces cerevisiae mcm10 suppressor.
Cell Res. 2001 Dec;11(4):285-91.
MCM10 protein is an essential replication factor involved in the initiation of DNA replication. A mcm10 mutant (mcm10-1) of budding yeast shows a growth arrest at 37 degrees C. In the present work, we have isolated a mcm10-1 suppressor strain, which grows at 37 degrees C. Interestingly, this mcm10-1 suppressor undergoes cell cycle arrest at 14 degrees C. A novel gene, YLR003c, is identified by high-copy complementation of this suppressor. We called it as Cms1 (Complementation of Mcm 10 Suppressor). Furthermore, the experiments of transformation show that cells of mcm10-1 suppressor with high-copy plasmid but not low-copy plasmid grow at 14 degrees C, indicating that overexpression of Cms1 can rescue the growth arrest of this mcm10 suppressor at non-permissive temperature. These results suggest that CMS1 protein may functionally interact with MCM10 protein and play a role in the regulation of DNA replication and cell cycle control. [Abstract/Link to Full Text]

Wang YM, Wang JB, Luo D, Jia JF
Regeneration of plants from callus tissues of hairy roots induced by Agrobacterium rhizogenes on Alhagi pseudoalhagi.
Cell Res. 2001 Dec;11(4):279-84.
The legume forage Alhagi pseudoalhagi was transformed by the Agrobacterium rhizogenes strain A4 using cotyledon and hypocotyl segments as infection materials. Regenerated plants were achieved from sterile calli derived from hairy roots, which occurred at or near the infection sites. The regenerated plants from hairy root were characterized by normal leaf morphology and stem growth but a shallow and more extensive root system than normal plants. Opine synthesis, PCR and Southern blot confirmed that T-DNA had been integrated into the A. pseudoalhagi genome. Acetosyringone (AS) was found to be vital for successful transformation of A. pseudoalhagi. [Abstract/Link to Full Text]

Ni DA, Wang LJ, Ding CH, Xu ZH
Auxin distribution and transport during embryogenesis and seed germination of Arabidopsis.
Cell Res. 2001 Dec;11(4):273-8.
Auxin distribution during embryogenesis and seed germination were studied with transgenic Arabidopsis plants expressing GUS gene driven by a synthetic DR5 promoter, an auxin responsive promoter. The results showed that GUS activity is higher in ends of hypophysis and cotyledon primordia of heart-, torpedo- and cotyledon-stage embryos, leaf tip area, lateral root primordia, root apex and cotyledon of young seedlings. And GUS accumulated in root apex of the seedlings grown on auxin transport inhibitor containing media. All these suggested that above-mentioned part of the organs and tissues have a higher level of auxin, and auxin polar transport inhibitor could cause the accumulation of auxin in root apex. And auxin transport inhibitor also resulted in aberration of Arabidopsis leaf pattern formation, root gravitropism and elongation. [Abstract/Link to Full Text]

Jia SQ, Yong WD, Xu WZ, Xu YY, Wu JS, Chong K, Tan KH, Xu ZH
Existence of homologous sequences corresponding to cDNA of the ver gene in diverse higher plant species.
Cell Res. 2001 Dec;11(4):265-71.
The presence of DNA homologues corresponding to verc203 (vernalization-related cDNA clone) was investigated by molecular hybridization techniques. The genes were detected in 16 plant species that cover 12 subclasses of the Takhtajan system of angiosperms classification including diverse model species. The results of Southern blot analysis showed a low copy number of this gene existed in rice, wheat, barley and Arabidopsis. The hybridization result of PCR products demonstrated the conservation of the gene corresponding to ver203 in diverse plants. The phylogenetic tree of the ver203 gene in tested plants was supported by evolution relationship of species. The ver203 gene expressed in a vernalized plumule winter wheat, instead of the root. And the endosperm before the treatment was essential for the ver203 expression during vernalization in wheat. In Arabidopsis thaliana, the pattern of expression showed that the gene corresponding to ver203 was expressed at low temperature for 14 days. Gibberellin (GA3) may accelerate the expression of ver203 gene in Arabidopsis exposed to low temperature. However, it could not replace vernalization treatment to initiate the gene expression. [Abstract/Link to Full Text]

Geng YJ
Molecular signal transduction in vascular cell apoptosis.
Cell Res. 2001 Dec;11(4):253-64.
Apoptosis is a form of genetically programmed cell death, which plays a key role in regulation of cellularity in a variety of tissue and cell types including the cardiovascular tissues. Under both physiological and pathophysiological conditions, various biophysiological and biochemical factors, including mechanical forces, reactive oxygen and nitrogen species, cytokines, growth factors, oxidized lipoproteins, etc., may influence apoptosis of vascular cells. The Fas/Fas ligand/caspase death-signaling pathway, Bcl-2 protein family/mitochondria, the tumor suppressive gene p53, and the proto-oncogene c-myc may be activated in atherosclerotic lesions, and mediates vascular apoptosis during the development of atherosclerosis. Abnormal expression and dysfunction of these apoptosis-regulating genes may attenuate or accelerate vascular cell apoptosis and affect the integrity and stability of atherosclerotic plaques. Clarification of the molecular mechanism that regulates apoptosis may help design a new strategy for treatment of atherosclerosis and its major complication, the acute vascular syndromes. [Abstract/Link to Full Text]

Shi YB, Fu L, Hsia SC, Tomita A, Buchholz D
Thyroid hormone regulation of apoptotic tissue remodeling during anuran metamorphosis.
Cell Res. 2001 Dec;11(4):245-52.
Anuran metamorphosis involves systematic transformations of individual organs in a thyroid hormone (TH)-dependent manner. Morphological and cellular studies have shown that the removal of larval organs/tissues such the tail and the tadpole intestinal epithelium is through programmed cell death or apoptosis. Recent molecular investigations suggest that TH regulates metamorphosis by regulating target gene expression through thyroid hormone receptors (TRs), which are DNA-binding transcription factors. Cloning and characterization of TH response genes show that diverse groups of early response genes are induced by TH. The products of these TH response genes are believed to directly or indirectly affect the expression and/or functions of cell death genes, which are conserved at both sequence and function levels in different animal species. A major challenge for future research lies at determining the signaling pathways leading to the activation of apoptotic processes and whether different death genes are involved in the regulation of apoptosis in different tissues/organs to effect tissue-specific transformations. [Abstract/Link to Full Text]

Tang W
Agrobacterium-mediated transformation and assessment of factors influencing transgene expression in loblolly pine (Pinus taeda L.).
Cell Res. 2001 Sep;11(3):237-43.
This investigation reports a protocol for transfer and expression of foreign chimeric genes in loblolly pine (Pinus taeda L.). Transformation was achieved by co-cultivation of mature zygotic embryos with Agrobacterium tumefaciens strain LBA4404 which harbored a binary vector (pBI121) including genes for beta-glucuronidase (GUS) and neomycin phosphotransferase (NPTII). Factors influencing transgene expression including seed sources of loblolly pine, concentration of bacteria, and the wounding procedures of target explants were investigated. The expression of foreign gene was confirmed by the ability of mature zygotic embryos to produce calli in the presence of kanamycin, by histochemical assays of GUS activity, by PCR analysis, and by Southern blot. The successful expression of the GUS gene in different families of loblolly pine suggests that this transformation system is probably useful for the production of the genetically modified conifers. [Abstract/Link to Full Text]

He YK, Sun JG, Feng XZ, Czakó M, Márton L
Differential mercury volatilization by tobacco organs expressing a modified bacterial merA gene.
Cell Res. 2001 Sep;11(3):231-6.
Mercury pollution is a major environmental problem accompanying industrial activities. Most of the mercury released ends up and retained in the soil as complexes of the toxic ionic mercury (Hg2+), which then can be converted by microbes into the even more toxic methylmercury which tends to bioaccumulate. Mercury detoxification of the soil can also occur by microbes converting the ionic mercury into the least toxic metallic mercury (Hg0) form, which then evaporates. The remediation potential of transgenic plants carrying the MerA gene from E. coli encoding mercuric ion reductase could be evaluated. A modified version of the gene, optimized for plant codon preferences (merApe9, Rugh et al. 1996), was introduced into tobacco by Agrobacterium-mediated leaf disk transformation. Transgenic seeds were resistant to HgCl2 at 50 microM, and some of them (10-20% ) could germinate on media containing as much as 350 microM HgCl2, while the control plants were fully inhibited or died on 50 microM HgCl2. The rate of elemental mercury evolution from Hg2+ (added as HgCl2) was 5-8 times higher for transgenic plants than the control. Mercury volatilization by isolated organs standardized for fresh weight was higher (up to 5 times) in the roots than in shoots or the leaves. The data suggest that it is the root system of the transgenic plants that volatilizes most of the reduced mercury (Hg0). It also suggests that much of the mercury need not enter the vascular system to be transported to the leaves for volatilization. Transgenic plants with the merApe9 gene may be used to mercury detoxification for environmental improvement in mercury-contaminated regions more efficiently than it had been predicted based on data on volatilization of whole plants via the upper parts only (Rugh et al. 1996). [Abstract/Link to Full Text]

Fang CM, Xu YH
Down-regulated expression of atypical PKC-binding domain deleted asip isoforms in human hepatocellular carcinomas.
Cell Res. 2001 Sep;11(3):223-9.
Asip is a mammalian homologue of polarity protein Par-3 of Caenorhabditis elegans and Bazooka of Drosophila melanogaster. Asip/Par-3/Bazooka are PDZ-motif containing proteins that localize asymmetrically to the cell periphery and play a pivotal role in cell polarity and asymmetric cell division. In the present study, we have cloned human asip cDNA and its splicing variants by 5'-RACE and RT-PCR using candidate human EST clones which have a high homology to rat asip cDNA. The full-length cDNA of human asip encodes a 1,353 aa protein exhibiting 88% similarity to the rat one. Human asip is a single copy gene consisting of at least 26 exons and localizing in human chromosome 10, band p11.2, with some extraordinarily long introns. All exon/intron boundary nucleotides conform to the "gt-ag" rule. Three main transcripts were detected by Northern blot analysis, and at least five variants, from alternative splicing and polyadenylation, have been identified by RT-PCR and liver cDNA library screening. Exon 17b deleted asip mRNAs expressed ubiquitously in normal human tissues, including liver, on RT-PCR analysis. However, they were absent from most human liver cancer cell lines examined. More interestingly, the expression of exon 17b deleted variants was down regulated in 52.6% (10/19) clinic specimens of human hepatocellular carcinomas (HCCs), compared with the surrounding nontumorous liver tissues from the same patients. The presence of various splicing transcripts, the variation of their distribution among different tissues and cells, and their differential expressions in human HCCs suggest that human Asip isoforms may function in different context. [Abstract/Link to Full Text]

Fang L, Fang J, Chen CQ
TNF receptor-associated factor-2 binding site is involved in TNFR75-dependent enhancement of TNFR55-induced cell death.
Cell Res. 2001 Sep;11(3):217-22.
TNF recepter-55 is the main mediator of TNF-induced apoptosis. TNF receptor-75-dependent induction or enhancement of cytotoxicity has been explained by intracellular signaling, "ligand passing", or induction of endogenous TNF. To study the function of human TNF receptor-75 (hTR75) and the interaction between human TNF receptor-55 (hTR55) and hTR75 in hTNFalpha-induced cytotoxicity, HEp-2 cells were transfected with bicistronic expression vector of hTR75 and its deletion mutants genes. hTNFalpha-induced cytotoxicity was determined by crystal violet colorimetric method. The expression of hTR75 and its deletion mutants in HEp-2 cells was demonstrated by RT-PCR and indirect ELISA. We found that the overexpressed hTR75 could significantly increase the susceptibility of HEp-2 cells to hTNFalpha which especially required TRAF2 binding site. hTR75 could not only mediate partial hTNFalpha-induced cytotoxicity independently but also fulfill an accessory role in enhancing or synergizing hTR55-mediated cytotoxicity. [Abstract/Link to Full Text]

Qin WX, Wan F, Sun FY, Zhang PP, Han LW, Huang Y, Jiang HQ, Zhao XT, He M, Ye Y, Cong WM, Wu MC, Zhang LS, Yang NW, Gu JR
Cloning and characterization of a novel gene (C17orf25) from the deletion region on chromosome 17p13.3 in hepatocelular carcinoma.
Cell Res. 2001 Sep;11(3):209-16.
Using a combination of hybridization of PAC to a cDNA library and RACE technique, we isolated a novel cDNA, designated as C17orf25 (Chromosome 17 open reading frame 25, previously named it HC71A), from the deletion region on chromosome 17p13.3. The cDNA encodes a protein of 313 amino acids with a calculated molecular mass of 34.8 kDa. C17orf25 is divided into 10 exons and 9 introns, spanning 23 kb of genomic DNA. Northern blot analysis showed that the mRNA expression of C17orf25 was decreased in hepatocellular carcinoma samples as compared to adjacent noncancerous liver tissues from the same patients. The transfection of C17orf25 into the hepatocellular carcinoma cell SMMC7721 and overexpression could inhibit the cell growth. The above results indicate that C17orf25 is a novel human gene, and the cloning and preliminary characterization of C17orf25 is a prerequisite for further functional analysis of this novel gene in human hepatocellular carcinoma. [Abstract/Link to Full Text]

---

Recent Articles in Cellular & Molecular Biology Letters

---

Goessler UR, Bugert P, Bieback K, Deml M, Sadick H, Hormann K, Riedel F
In-vitro analysis of the expression of TGFbeta -superfamily-members during chondrogenic differentiation of mesenchymal stem cells and chondrocytes during dedifferentiation in cell culture.
Cell Mol Biol Lett. 2005;10(2):345-62.
Traditional surgical methods for the reconstruction of cartilage defects rely on the transplantation of autologous and allogenous tissues. The disadvantages of these techniques are the limited availability of suitable tissues and the donor site morbidity of transplants. In addition, in cultured chondrocytes, the dedifferentiation of cells seems unavoidable during multiplication. In this study, we investigated the expression of distinct markers during the dedifferentiation of human chondrocytes (HC) and human mesenchymal stem cells (MSC) in cell culture using microarray technique, immunohistochemistry and RT-PCR. Transforming growth factor beta (TGFbeta) is a multifunctional peptide that plays play a crucial role in inducing and maintaining chondrogenic differentiation. In dedifferentiating chondrocytes, the gene for TGFbeta1 was constantly expressed, while the gene for TGFbeta2 was never expressed. The genes for TGFalpha, TGFbeta4 and TGFbetai were activated with ongoing dedifferentiation. TGFbeta-receptor 3 was constantly expressed, while the genes for the TGFbeta-receptors 1 and 2 were never expressed. Immunohistochemical staining for TGFbeta beta 3 revealed upregulation in the course of dedifferentiation. The genes for LTBP1 and LTBP2 were activated with ongoing dedifferentiation, whereas the gene for LTBP3 was constantly expressed, and negative results were obtained for the gene for LTBP4. The genes for LTBP1 and LTBP2 were activated with ongoing dedifferentiation. During chondrogenic differentiation, the MSCs constantly expressed TGFbeta1, beta2, beta3 and beta4. LTBP1, LTBP2 and TGFbeta-R3 were downregulated. In conclusion, TGFbeta3, TGFbeta4, TGFbetai, LTBP1 and LTBP2 may assist the process of dedifferentiation, while TGFbeta1 and beta2 might not be involved in this process. Of the TGFbeta-receptors, only type 3 might be involved in dedifferentiation. [Abstract/Link to Full Text]

Boersma JG, Pallotta M, Li C, Buirchell BJ, Sivasithamparam K, Yang H
Construction of a genetic linkage map using MFLP and identification of molecular markers linked to domestication genes in narrow-leafed lupin (Lupinus angustifolius L.).
Cell Mol Biol Lett. 2005;10(2):331-44.
A mapping population of F(8)derived recombinant inbred lines (RILs) was established from a cross between a domesticated breeding line 83A:476 and a wild type P27255 in narrow-leaf lupin (Lupinus angustifolius L.). The parents together with the 89 RILs were subjected to DNA fingerprinting using microsatellite-anchored fragment length polymorphism (MFLP) to rapidly generate DNA markers to construct a linkage map. Five hundred and twenty two unique markers of which 21% were co-dominant, were generated and mapped. Phenotypic data for the domestication traits: mollis (soft seeds), leucospermus (white flower and seed colour); Lentus (reduced pod-shattering), iucundis (low alkaloid), Ku (early flowering) and moustache pattern on seed coats; were included. Three to 7 molecular markers were identified within 5 cM of each of these domestication genes. The anthracnose resistance gene Lanr1 was also mapped. Linkage groups were constructed using MapManager version QTXb20, resulting in 21 linkage groups consisting of 7 or more markers. The total map length was 1543 cM, with an average distance of 3.4 cM between adjacent markers. This is the first published map for a lupin species. The map can be exploited for marker assisted selection for genetic improvement in lupin breeding programs. [Abstract/Link to Full Text]

Akcay YD, Yalcin A, Sozmen EY
The effect of melatonin on lipid peroxidation and nitrite/nitrate levels, and on superoxide dismutase and catalase activities in kainic acid-induced injury.
Cell Mol Biol Lett. 2005;10(2):321-9.
Kainic acid (KA) initiates neuronal injury and death by inducing oxidative stress and nitric oxide release from various regions of the brain. It was recently shown that melatonin has free radical-scavenging action and may protect against kainate-induced toxicity. In order to assess the possible supportive effect of melatonin treatment in KA-induced injury in the rat brain cortex, we determined malondialdehyde (MDA) levels as an index of lipid peroxidation, and assessed the activities of catalase (CAT) and superoxide dismutase (SOD) and the levels of nitrite/nitrate 35 male rats were divided into five groups, each receiving a different intraperitoneal treatment: saline solution (0.2 ml), kainic acid (15 mg/kg), melatonin (20 mg/kg), KA then melatonin (each as above, 15 min apart), or melatonin then KA (each as above, 30 min apart). Administration of KA caused an about five-fold increase in the catalase activity and an increase in the SOD activity in the cortex relative to the activities for the controls. Treatment with melatonin 15 min after KA injection kept malondialdehyde levels and catalase and superoxide dismutase activities at the normal levels, and led to an increase in the levels of nitrite/nitrate. Our data suggests that melatonin treatment following KA administration has a protective effect on antioxidant enzyme activities and thus supports the role of melatonin and oxidative stress in the regulation of antioxidative enzyme activity. [Abstract/Link to Full Text]

Ahlström M, Pekkinen M, Huttunen M, Lamberg-Allardt C
Cyclic nucleotide phosphodiesterases (PDEs) in human osteoblastic cells; the effect of PDE inhibition on cAMP accumulation.
Cell Mol Biol Lett. 2005;10(2):305-19.
The regulation of the secondary messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), is crucial in the hormonal regulation of bone metabolism. Both cAMP and cGMP are inactivated by cyclic nucleotide phosphodiesterases (PDEs), a superfamily of enzymes divided into 11 families (PDE1-11). We compared the PDEs of cultured human osteoblasts (NHOst) and SaOS-2 osteosarcoma cells. The PDE activity of NHOst cells consisted of PDE1, PDE3 and PDE7, whereas PDE1, PDE7 and PDE4, but no PDE3 activity was detected in SaOS-2 cells. In line with the difference in the PDE profiles, rolipram, a PDE4 inhibitor, increased the accumulation of cAMP in SaOS-2, but not in NHOst cells. Expression of PDE subtypes PDE1C, PDE3A, PDE4A, PDE4B, PDE7A and PDE7B was detected in both cell types. NHOst cells additionally expressed PDE1A. [Abstract/Link to Full Text]

Ko?cz J, Druka?a J, Bzowska M, Rajwa B, Korohoda W, Malec E
The expression of connexin 43 in children with Tetralogy of Fallot.
Cell Mol Biol Lett. 2005;10(2):287-303.
Abnormalities in the expression and distribution of Connexin 43 (Cx43) in cardiomyocytes may lead to anomalous conotruncal embryogenesis and disturbances in the maturation and function of the heart. Tetralogy of Fallot (TOF) is the most frequent, cyanotic congenital heart defect in which conotruncal anomalies, right ventricle dysfunction and life-threatening arrhythmias occur. In this study, age-related changes in the expression and spatial distribution of Cx43 in cardiomyocytes from TOF children compared to patients without right ventricular outflow tract pathology were determined Confocal microscopy and flow cytometry were used to assess the changes. Disturbances in both the expression and distribution of Cx43 were found. In the group of infants with TOF, a lower level of expression of the protein was determined. Cardiomyocytes from TOF hearts were found to have Cx43 distributed over their entire surface, which is the pattern seen in immature tissue. In the controls, Cx43 was located within the intercalated disks. Expression of Cx43 in TOF hearts increases with the age of the subject, whereas its spatial distribution remains the same in both infants and older children. Disturbances in Cx43 expression and localization may influence heart embryogenesis and maturation, contribute to hypertrophy and dysfunction of the right ventricle and induce arrhythmias in children with TOF. Early redistribution of Cx43 and functional maturation of the heart muscle support a strategy of early total correction of the defect. [Abstract/Link to Full Text]

Qi Y, Lin F
Parallelisation of the blast algorithm.
Cell Mol Biol Lett. 2005;10(2):281-5.
Retrieving homologous DNA and protein sequences from existing databases is a fundamental routine in bioinformatics research. Programs of the NCBI BLAST family are widely used for this purpose. We evaluated paraBLAST, a parallelised version of the NCBI BLAST algorithm, using a Message Passing Interface (MPI) on a multi-node compute cluster. Here, we propose static and dynamic database-partitioning schemes based on the availability of the cluster. We evaluated the application of the algorithm in querying nucleotide sequences against a large-scale sequence database with different numbers of database partitions, and hence, different numbers of CPUs. Since the program's tasks are performed independently of each other, each available CPU can run its own copy of BLAST queries, resulting in reduced interference between processes and leading to a highly scalable solution. [Abstract/Link to Full Text]

Saker MM
A biological and molecular characterization of some Egyptian barley genotypes which are resistant to net blotch disease.
Cell Mol Biol Lett. 2005;10(2):265-80.
A survey for resistance against net blotch disease (caused by Pyrenophora teres) was performed on some Egyptian barley landraces and some selected resistance and susceptible standard German barley genotypes. The results indicated that most of the Egyptian barley landraces are extremely resistant to the disease. Molecular analysis using RAPD and AFLP showed unique banding profiles for the different genotypes, and specific AFLP markers for the Egyptian genotypes were identified. The effectiveness of RAPD and AFLP for identifying different barley genotypes of different origins and with different reactions against P. teres was discussed. The results of the biological evaluation and molecular characterization done in this study can be seen as the starting point needed to identify the valuable net blotch resistant Egyptian barley germplasm at both the phenotype and genotype levels and draw the attention of breeders and banks of natural plant genetic resources towards this valuable yet neglected germplasm. [Abstract/Link to Full Text]

Panjamurthy K, Manoharan S, Ramachandran CR
Lipid peroxidation and antioxidant status in patients with periodontitis.
Cell Mol Biol Lett. 2005;10(2):255-64.
Our aim was to assess the degree of oxidative stress in patients with periodontitis by measuring their levels of thiobarbituric acid reactive substances (TBARS), enzymatic antioxidants (superoxide dismutase (SOD), catalase (CAT), glutathione peroxide (GSHPx)), and non-enzymatic antioxidants (vitamins E and C, reduced glutathione (GSH)). This study was conducted on 25 adult chronic periodontitis sufferers who were patients in Rajah Muthiah Dental College and Hospital, Annamalai University. The levels of TBARS and non-enzymatic antioxidants, and the activities of enzymatic antioxidants in the patients' plasma, erythrocytes and gingival tissues were assayed using specific colorimetric methods. The periodontitis sufferers had a significantly higher TBARS level than the healthy subjects. In the plasma, erythrocytes, erythrocyte membranes and gingival tissues of the periodontitis sufferers, enzymatic antioxidant activities were found to be significantly higher, whereas the levels of non-enzymatic antioxidants were significantly lower (except for reduced glutathione in the gingival tissues) relative to the parameters found for healthy subjects. The disturbance in the endogenous antioxidant defense system due to over-production of lipid peroxidation products at inflammatory sites can be related to a higher level of oxidative stress in patients with periodontitis. [Abstract/Link to Full Text]

Khan HA
The effect of DNA labeling with the fluorescent dyes R110 and R6G on genotype analysis using capillary electrophoresis.
Cell Mol Biol Lett. 2005;10(2):247-53.
We investigated the mobility of DNA fragments labeled with the fluorescent dyes R110 and R6G, specifically for use in genotyping using capillary electrophoresis. Genomic DNA was isolated from blood, and a highly polymorphic region of the HLA-C gene was amplified by PCR with the use of either [F]-dNTP-[R110] or [F]-dNTP-[R6G]. Pre-diluted (30-fold) PCR products were mixed with formamide, denatured (at 95 degrees C for 2 min.), and rapidly cooled on ice before being subjected to electrophoresis. The results showed that the number and mobility of allele-specific DNA fragments were independent of the two dyes used. Both dyes were equally efficient at differentiating homozygous or heterozygous allelic presentation. An additional dye-specific peak of 132-base-pair mobility was observed with the use of [F]-dNTP-[R110]; it significantly impaired the resolution of one allele-specific peak. The electropherograms obtained with [F]-dNTP-[R6G] were free from any interfering peaks within the target region, thus the [R6G]-based procedure is more preferable for genotype analysis. As this procedure does not involve any post-PCR cleanup, it is simple, rapid and cost-effective. [Abstract/Link to Full Text]

Salimullah M, Hamano K, Tachibana M, Inoue K, Nishigaki K
Efficient SNP analysis enabled by joint application of the muTGGE and heteroduplex methods.
Cell Mol Biol Lett. 2005;10(2):237-45.
Gene science-based diagnoses have become an increasingly realistic option as the state of knowledge has improved regarding the genetic basis of disease. To facilitate the creation of this potential diagnostic tool, researchers have made large-scale detection of point mutations a key issue. Here, we propose an inexpensive and convenient method with a high performance level for this purpose: micro temperature gradient gel electrophoresis (muTGGE)-empowered heteroduplex analysis (muTG-HD). First, muTGGE was shown to separate double-stranded DNA containing single nucleotide polymorphism (SNP) with sufficiently high resolution when used in the mode of perpendicular TGGE. Using human c-Ki-ras and rat p53 DNA, point mutations could be unequivocally detected by muTG-HD when parallel TGGE was employed. The mutation type (such as G/C to A/T), the position of the point mutation (centre or not) and the DNA size (around 100 or 200 bp) were examined and found to be detectable. Thus, muTG-HD could detect point mutations efficiently at a much lower cost by having multiple lanes per gel. [Abstract/Link to Full Text]

Paziewska A, Wyrwicz LS, Ostrowski J
The binding activity of yeast RNAs to yeast Hek2p and mammalian hnRNP K proteins, determined using the three-hybrid system.
Cell Mol Biol Lett. 2005;10(2):227-35.
K homology (KH) domains are scaffolds for the binding of RNAs by the heterogeneous nuclear ribonucleoprotein (hnRNP) K protein and its yeast ortholog, Hek2p. KH domains are remarkably conserved between mammals and yeast. To assess the binding activity for yeast RNA of the two proteins, we used full-length K protein and Hek2p as baits in the yeast three-hybrid system. All the unique RNA sequences bound by Hek2p and all but two bound by K protein represented different fragments of only two transcripts, encoded by the 18S and 25S ribosomal RNA genes. Most of them were transcribed from the antisense strand. The RNA-binding activity of K protein was significantly higher than that of Hek2p. These results and those from our previously published reports demonstrate that the specificity of target RNA recognition by both the K protein and Hek2p depends on both RNA-specific sequences and the structure of the protein. Both mammalian K protein and its yeast ortholog may be involved in the regulation of gene expression. [Abstract/Link to Full Text]

Baranowska-Bosiacka I, Machali?ski B, Tarasiuk J
The purine nucleotide content in human leukemia cell lines.
Cell Mol Biol Lett. 2005;10(2):217-26.
The HPLC method was used to determine the purine nucleotide (ATP, ADP, AMP, GTP, GDP, GMP, NAD(+)) contents and the values of the adenylate energy charge (AEC) and guanylate energy charge (GEC) for three human acute myelogenous leukemia (AML) cell lines: HL60 (M3 subtype of AML), THP1 (M5 subtype of AML), and HEL (M6 subtype of AML) in French-American-British classification (FAB) and for one chronic myelogenous leukemia (CML) cell line: K562. The results showed that the examined leukemic cells had some significant changes in their purine nucleotide concentrations relative to healthy cells. On the basis of the obtained results, it seems that two of the tested acute myelogenous leukemia cell lines, HL60 and HEL, have similar purine nucleotide metabolisms, while the third AML cell line, THP1, has a purine nucleotide metabolism like that of the chronic myelogenous leukemia cell line, K562. [Abstract/Link to Full Text]

Prasad TK, Rao NM
The role of plasmid constructs containing the SV40 DNA nuclear-targeting sequence in cationic lipid-mediated DNA delivery.
Cell Mol Biol Lett. 2005;10(2):203-15.
One of the steps that limit transfection efficiency in non-viral gene delivery is inefficient nuclear import of plasmid DNA, once it has been delivered into the cytoplasm. Recently, via microinjection into the cytoplasm and in situ hybridizations into a few cell types, it was shown that a region of Simian virus 40(SV40), specifically a c. 372-bp fragment of SV40 genomic DNA encompassing the SV40 promoter-enhancer-origin of replication (SV40 DTS), could enable the nuclear import of a plasmid carrying these sequences (Dean D.A. Exp. Cell Res. 230 (1997) 293). In this report, we address the issue of the suitability of the SV40 DTS for cationic lipid-mediated gene delivery, and its capacity to improve the efficiency of the transfection process. For this study, we used transient reporter gene expression assays on various cell types. The gene expression from the plasmid constructs carrying the SV40 DTS varied with cell type and plasmid construct used. Such cell-type and plasmid-construct dependency on gene expression from plasmids containing the SV40 DTS suggests that the gene expression from plasmids is not entirely dependent on its ability to enhance the nuclear import of said plasmids. [Abstract/Link to Full Text]

Li J, Ji C, Zheng H, Fei X, Zheng M, Dai J, Gu S, Xie Y, Mao Y
Molecular cloning and characterization of a novel human gene containing 4 ankyrin repeat domains.
Cell Mol Biol Lett. 2005;10(1):185-93.
Ankyrin repeat, one of the most important protein motifs, plays a wide variety of roles in protein-protein interactions and in the signal pathways. Via large-scale sequencing, a novel 941-bp gene was isolated from an 18-week old human fetal brain cDNA library. It encodes a putative protein of 158 amino acid residues with four conserved ankyrin repeat domains. It displays a high degree of homology with rat low-density lipoprotein receptor-related protein 2-binding protein (Lrp2bp), and was therefore was named hLrp2bp (human Lrp2bp). The hLrp2bp gene was located in chromosome 4q35 and the conserved ankyrin repeat domains were located between amino acid residues 10 and 116. RT-PCR revealed that hLrp2bp was mainly expressed in the human testis, small intestine, colon and blood leukocytes, and in human pancreatic adenocarcinoma cells. A HEK293 cell was transfected with the ORF of hLrp2bp, and analyses showed that the protein was distributed both in the cytoplasm and nucleus. [Abstract/Link to Full Text]

Fatouros DG, Bouropoulos N, Ioannou PV, Antimisiaris SG
Stability and aggregation studies of non-sonicated arsonolipid-containing vesicles.
Cell Mol Biol Lett. 2005;10(1):173-83.
In this study, we investigated non-sonicated arsonolipid-containing liposomes (arsonoliposomes) in terms of the influence of lipid composition on their stability, assessed as membrane integrity and physical stability [size]. Vesicles consisting of plain arsonolipids or mixtures of arsonolipids with cholesterol [Chol] or with distearoyl-phospatidylcholine [DSPC] were studied. Membrane integrity was evaluated by measuring the retention of incorporated 5-(6)carboxyfluorescein [CF] during incubation of the vesicles in Tris buffer, pH 7.4. Photon correlation spectroscopy was used to investigate the time-dependent aggregation of arsonoliposomes in the absence and presence of Ca(2+)ions. Vesicles composed of plain C18 (acyl fatty chain) arsonolipids were found to be unstable, with only 15% of the initially incorporated CF remaining in the vesicles after 24 hours. The addition of Chol to the membrane (1:1 mol/mol) significantly increased the stability of arsonoliposomes, while the addition of DSPC to the lipid bilayer (1:1 mol/mol) increased vesicle stability to a lower extent. The results of particle size analysis showed that non-sonicated arsonoliposomes consisting of plain arsonolipid Ars/Stearic are highly and rapidly aggregated, while calcium-induced aggregation is also significant, but slower. Aggregation could not be always explained on the basis of zeta potential changes, indicating that the process is complex. [Abstract/Link to Full Text]

Konovalov F, Toshchakova E, Gostimsky S
A CAPS marker set for mapping in linkage group III of pea (Pisum sativum L.).
Cell Mol Biol Lett. 2005;10(1):163-71.
A set of twelve CAPS markers was mapped for linkage group III of pea (Pisum sativum L.). New primers were designed to use a polymerase chain reaction to amplify fragments of sequenced pea genes containing at least one large intron. Amplification products were tested for polymorphism across three pea lines (Chi115, Flagman and WL1238) using eleven four-base restriction endonucleases. Nine STS markers for linkage group III from the literature were also tested for polymorphism, and five of these were used in this mapping study as anchor points. All polymorphic loci were located by genetic analysis of the F(2)population from the cross Chi115 x WL1238, and a map of linkage group III consisting of one morphological and twelve CAPS markers was created. The map covers the full length of the chromosome and is about 162 cM long. All the CAPS markers in a set were used to test for polymorphism among 10 additional pea DNA samples extracted from different marker lines and cultivars. [Abstract/Link to Full Text]

Bo?towicz D, Szczerbakowa A, Wielgat B
RAPD analysis of the interspecific somatic hybrids: Solanum bulbocastanum (+) S. tuberosum.
Cell Mol Biol Lett. 2005;10(1):151-62.
The diploid Mexican species S. bulbocastanum (blb) was used as a source of late blight resistance in somatic hybridization with the potato (S. tuberosum, tbr) dihaploid H-8105. The produced 2x blb (+) 2x tbr H-8105 somatic hybrids did not retain the blb parent's characteristic high resistance to P. infestans. The revealed aneuploidy of blb (+) tbr H-8105 hybrids indicated a possible loss of individual blb chromosome(s) carrying the resistance genes. Four hybrid clones differing in terms of their ploidy, morphology and growth potential were analysed for the presence of all twelve blb chromosomes (linkage groups). The RAPD markers assigned to particular chromosomes were selected on the basis of the linkage map of S. bulbocastanum constructed by Naess et al., Mol. Gen. Genom. 265 (2001) 694-704. Of the 86 markers analysed, twelve (14%) were common for blb and H-8105, while 34 (40%) and 40 (46%) markers were specific for the blb and H-8105 genome, respectively; this confirms the differences between the nuclear genomes of the two species. Seventeen markers (20%) present in one or the other parent were absent in the hybrids, and only one new marker was found in the hybrids. The poorly growing, aneuploid and chimeric hybrids had the same band profiles as the well growing, morphologically normal hybrids, except for two bands that were present only in normal hybrids. The presence of eleven blb linkage groups in the blb (+) tbr H-8105 hybrids was confirmed. The markers specific for the second linkage group (chromosome 2) of blb were not present in the RAPD patterns of the somatic hybrids analysed, suggesting a loss or rearrangement of this chromosome in the combined nuclear genome of the hybrids. [Abstract/Link to Full Text]

Taylor-Harris PM, Felkin LE, Birks EJ, Franklin RC, Yacoub MH, Baines AJ, Barton PJ, Pinder JC
Expression of human membrane skeleton protein genes for protein 4.1 and betaIISigma2-spectrin assayed by real-time RT-PCR.
Cell Mol Biol Lett. 2005;10(1):135-49.
The proteins, spectrin and 4.1 confer support and resilience to animal cell membranes, and promote assembly of multimeric, membrane-bound signalling complexes. Protein 4.1 also plays important roles in tumour suppression and the regulation of cell proliferation. To assess relative tissue expression of the four genes encoding human protein 4.1, we measured mRNA levels using quantitative real-time polymerase chain reaction. We compared 4.1 expression with that of a major splice variant of spectrin, betaIISigma2 that has a shortened C-terminus lacking a pleckstrin homology domain. mRNA for 4.1R is four-fold higher in bone marrow than in tissues with the next highest prevalence: cerebellum, lung, testis and thymus. 4.1G mRNA is highly expressed in brain, spinal cord and testis; 4.1N in brain, spinal cord and adrenal gland; 4.1B in testis, brain, spinal cord, and kidney. Thus, 4.1N, 4.1B and 4.1G all show high accumulation in nervous tissues. mRNA for betaIISigma2-spectrin is ubiquitous, but most abundant in cardiac and nervous tissues. Comparative transcript abundance was analysed in heart and brain. betaIISigma2-spectrin was the most abundant transcript in heart with levels 5 fold greater than 4.1G or 4.1N and at least 9 fold greater than 4.1B. In brain, 4.1N was the most abundant transcript, with levels 2.4 fold greater than 4.1B and at least 4 fold greater than 4.1G or betaIISigma2-spectrin. 4.1R abundance was very low in both tissues. Whilst we expected that 4.1 mRNAs would feature highly in muscle and nerve, we note their high abundance in testis, indicating previously unsuspected functions in reproduction. [Abstract/Link to Full Text]

You M, Boersma JG, Buirchell BJ, Sweetingham MW, Siddique KH, Yang H
A PCR-based molecular marker applicable for marker-assisted selection for anthracnose disease resistance in lupin breeding.
Cell Mol Biol Lett. 2005;10(1):123-34.
Selection for anthracnose disease resistance is one of the major objectives in lupin breeding programs. The aim of this study was to develop a molecular marker linked to a gene conferring anthracnose resistance in narrow-leafed lupin (Lupinus angustifolius L.), which can be widely used for MAS in lupin breeding. A F(8)derived RIL population from a cross between cultivar Tanjil (resistant to anthracnose) and Unicrop (susceptible) was used for marker development. DNA fingerprinting was conducted on 12 representative plants by combining the AFLP method with primers designed based on conserved sequences of plant disease resistance genes. A co-dominant candidate marker was detected from a DNA fingerprint. The candidate marker was cloned, sequenced, and converted into a sequence-specific, simple PCR based marker. Linkage analysis based on a segregating population consisting of 184 RILs suggested that the marker, designated as AntjM2, is located 2.3 cM away from the R gene conferring anthracnose resistance in L. angustifolius. The marker has now being implemented for MAS in the Australian national lupin breeding program. [Abstract/Link to Full Text]

Gummadi SN, Kumar KS
The mystery of phospholipid flip-flop in biogenic membranes.
Cell Mol Biol Lett. 2005;10(1):101-21.
Phospholipid flip-flop is required for bilayer assembly and the maintenance of biogenic (self-synthesizing) membranes such as the eukaryotic endoplasmic reticulum and the bacterial cytoplasmic membrane. Due to the membrane topology of phospholipid biosynthesis, newly synthesized phospholipids are initially located in the cytoplasmic leaflet of biogenic membranes and must be translocated to the exoplasmic leaflet to give uniform bilayer growth. It is clear from many studies that phospholipid flip-flop in biogenic membranes occurs very rapidly, within a period of a few minutes. These studies also reveal that phospholipid translocation in biogenic membranes occurs bi-directionally, independently of the phospholipid head group, via a facilitated diffusion process in the absence of metabolic energy input, and that this type of transport requires specific membrane proteins. These translocators have been termed biogenic membrane flippases, and they differ from metabolic energy-dependent transporters (ABC transporters and MDR proteins). No biogenic membrane flippases have been characterized. This review briefly discusses the importance of biogenic membrane flippases, the various assay methods used for measuring the rate of phospholipid flip-flop, and the progress that has been made towards identifying these proteins. [Abstract/Link to Full Text]

Kowalczy?ska HM, Nowak-Wyrzykowska M, Inkielman M, Sto?owska L, Marciniak E
The shape of cells adhering to sulfonated copolymer surfaces.
Cell Mol Biol Lett. 2005;10(1):91-9.
We studied the shape of L1210 leukaemia cells adhering in a protein-free medium to sulfonated (styrene/methyl methacrylate) copolymer surfaces of two sulfonic group densities, and thus of differing wettability. The use of our image analysis method and the mathematical procedure [Kowalczynska, H.M. et al, Colloids Surfaces B: Biointerfaces, 30 (2003) 193-206.] allowed us to calculate the values of the so-called shape parameter, which quantitatively determines the three-dimensional cell shape. Here, we show that the values of the shape parameter of the adhering cells and the F-actin concentration, in the region near the cell-substratum interface, depend on the density of sulfonic groups present on the substratum surface. [Abstract/Link to Full Text]

Plewczy?ski D, Tkacz A, Godzik A, Rychlewski L
A support vector machine approach to the identification of phosphorylation sites.
Cell Mol Biol Lett. 2005;10(1):73-89.
We describe a bioinformatics tool that can be used to predict the position of phosphorylation sites in proteins based only on sequence information. The method uses the support vector machine (SVM) statistical learning theory. The statistical models for phosphorylation by various types of kinases are built using a dataset of short (9-amino acid long) sequence fragments. The sequence segments are dissected around post-translationally modified sites of proteins that are on the current release of the Swiss-Prot database, and that were experimentally confirmed to be phosphorylated by any kinase. We represent them as vectors in a multidimensional abstract space of short sequence fragments. The prediction method is as follows. First, a given query protein sequence is dissected into overlapping short segments. All the fragments are then projected into the multidimensional space of sequence fragments via a collection of different representations. Those points are classified with pre-built statistical models (the SVM method with linear, polynomial and radial kernel functions) either as phosphorylated or inactive ones. The resulting list of plausible sites for phosphorylation by various types of kinases in the query protein is returned to the user. The efficiency of the method for each type of phosphorylation is estimated using leave-one-out tests and presented here. The sensitivities of the models can reach over 70%, depending on the type of kinase. The additional information from profile representations of short sequence fragments helps in gaining a higher degree of accuracy in some phosphorylation types. The further development of an automatic phosphorylation site annotation predictor based on our algorithm should yield a significant improvement when using statistical algorithms in order to quantify the results. [Abstract/Link to Full Text]

Khan RH, Naeem A, Baig MA
Spectroscopic studies on the protective effect of a specific sugar on concanavalin A at acidic, neutral and alkaline pH.
Cell Mol Biol Lett. 2005;10(1):61-72.
A Systematic investigation of the effect of pH on concanavalin A in the presence of specific and non-specific sugars is made using CD (circular dichroism) and fluorescence. The specific and non-specific sugars for concanavalin A were methyl alpha-D-glucopyranoside and methyl alpha-D-galactopyranoside respectively. Far-UV CD showed changes in the MRE value at 217 nm in the presence of the above-mentioned sugars. At pH 7, the CD and fluorescence spectra obtained in the presence of methyl alpha-D-glucopyranoside were slightly different from those for the native state and a significant difference was obtained in the presence of methyl alpha-D-galactopyranoside. Near-UV CD spectra showed the retention of a native-like tertiary structure in the presence of the specific sugar upon pH denaturation. Tryptophan fluorescence studies indicated a change in the tryptophan enviornment. The results obtained from our CD data are consistent with those obtained from fluorescence studies. Upon pH exposure of concanavalin A in the presence of methyl alpha-D-glucopyranoside and methyl alpha-D-galactopyranoside, the former acted as a protector preventing conformational alteration at different pH while the presence of latter induced a different stable conformational state and this state persists over the pH range from 2 to 10. [Abstract/Link to Full Text]

Sjakste T, Röder M, Labeikyte D, Bagdoniene L, Levina A, Juodka B, Sjakste N
Distribution of tight DNA-protein complexes along the barley chromosome 1H, as revealed by microsatellite marker analysis.
Cell Mol Biol Lett. 2005;10(1):49-59.
The distribution of DNA complexes with proteins resistant to routine deproteinisation procedures (tightly bound proteins, TBP) was studied on the barley chromosome 1H by means of microsatellite analysis. The polypeptide spectrum of the barley shoot TBP was similar to that formerly described for other organisms. In order to reveal developmental changes in the distribution of the TBP, DNA was extracted from dry grains, coleoptiles, root tips, and young and old leaves. In the seeds, all the studied DNA sites were evenly distributed between free DNA and DNA containing the tight DNA-protein complexes. Germination made the interaction between TBP and chromosomal loci specific. In coleoptile DNA, sites containing microsatellites located in the distal part of the long arm of the chromosome were not bound to the TBP anymore, however, the centromeric markers were found exclusively in the tight DNA-protein complexes. A similar but not identical distribution of markers was observed in the root tips and young leaves. Leaf senescence was accompanied by a loss in interaction specificity between chromosomal loci and tightly bound proteins. These results are considered to reflect changes in chromatin domain interaction with the nuclear matrix during plant development. [Abstract/Link to Full Text]

Sroda K, Rydlewski J, Langner M, Kozubek A, Grzybek M, Sikorski AF
Repeated injections of PEG-PE liposomes generate anti-PEG antibodies.
Cell Mol Biol Lett. 2005;10(1):37-47.
Liposomes containing the polyethylene glycol (PEG) derivative of phosphatidyl ethanolamine (PE) have recently been found to be promising drug carriers, as they facilitate controlled and target-oriented release of therapeutics. They also reduce the side effects of many drugs. Here, we present the results of a study on antiliposomal properties of rabbit sera obtained after weekly injections of small liposomes containing 20% PEG-PE. The effect was analysed as the level of induced carboxyfluorescein release from these liposomes in vitro. The incubation of liposomes with rabbit serum taken after the injections induced the release of carboxyfluorescein at a higher level than was seen for incubation with untreated animal's serum. The strongest effect was observed for serum obtained after the second injection, i.e. during the second week of the study. The effect was much smaller after the serum samples were preheated at 56 degrees C. The binding of serum proteins by PEGylated liposomes was analysed via gel filtration and via the immunoblot technique using goat anti-rabbit IgG; this revealed that the serum protein which bound to the liposomes in vitro had a molecular weight of 55 kD and reacted with the anti-IgG antibody. Competition with PEG or lipids indicate that this IgG has an anti-PEG activity. We therefore assume that these antibodies are responsible for the activation of complement and leakage induction of PEG-liposomes. Such antibodies could be responsible for increased phagocytosis by RES macrophages (in particular liver macrophages) and decreased circulation time. [Abstract/Link to Full Text]

Saulis G
The loading of human erythrocytes with small molecules by electroporation.
Cell Mol Biol Lett. 2005;10(1):23-35.
The technology for loading the cell with membrane-impermeable substances by means of electroporation consists of the following three stages: (i) the creation of pores permeable for the desired substance; (ii) the introduction of a substance into the cell cytosol; and (iii) the restoration of the membrane barrier function. In this paper, the experimental data on the loading of human erythrocytes with small molecules (molecular weight below 500 Da) is presented. The results obtained show that increasing the intensity of the electric field pulse increases the fraction of electroporated cells. The pores through which the molecules of ascorbic acid and mannitol (radius below 0.5 nm) can enter the erythrocytes appear when the field strength exceeds 2.5 kV/cm. The concentration of ascorbic acid inside the cells increases linearly. At 4 degrees C, the rate of ascorbic acid influx was constant for at least 4 hours. The original permeability of most of the cells towards ascorbic acid and mannitol was restored after about 6-7 min at 37 degrees C, and the characteristic time for complete resealing was about 20-40 min. The procedure described here can be used for loading cells with membrane-impermeable substances. [Abstract/Link to Full Text]

B?aszczyk A, Skolimowski J
Apoptosis and cytotoxicity caused by ethoxyquin and two of its salts.
Cell Mol Biol Lett. 2005;10(1):15-21.
In our study, we analyzed the cytotoxicity of ethoxyquin (EQ) and its two salts, ethoxyquin hydrochloride (EQ-HCL) and ethoxyquin phosphate (EQ-P). It was shown that EQ was the most cytotoxic compound (IC(50) = 0.09 mM), while the lowest cytotoxic effect was observed for EQ-P (IC(50) = 0.8 mM). The properties of ethoxyquin and its salts were also analyzed with the TUNEL method, which evaluates their ability to induce apoptosis. It was shown that EQ induced apoptosis in cultured human lymphocytes, especially at concentrations of 0.25 and 0.5 mM. [Abstract/Link to Full Text]

Kondracki S, Bonaszewska D, Mielnicka C
The effect of age on the morphometric sperm traits of domestic pigs (Sus scrofa domestica).
Cell Mol Biol Lett. 2005;10(1):3-13.
The experiment was conducted using 20 male domestic pigs, which were in 2 (equal-sized) age groups: under 14 months old and over 18 months old. At least 5 ejaculates from each male were taken, and in each ejaculate, morphometric measurements of 50 spermatozoa were made. The measured parameters were head area, head length and width, and flagellum length. For each ejaculate, the basic physical traits and the frequency of occurrence of developmental anomalies of the spermatozoa were examined. It was found that sperm dimensions tended to increase along with the boars' age. Considerable and statistical differences in head area and flagellum length were proved. Spermatozoa collected from older boars (above 18 months of age) had a head area larger by 0.49 microm(2)(P< or = 0.01) and a flagellum longer by 0.67 microm (P< or =0.01) than spermatozoa collected from younger boars (under 14 months of age). The differences in head length and width between the spermatozoa of the tested boars were considerably smaller and were not statistically provable. Significant correlations between the head area of spermatozoa and the head length (r = 0.56, P< or =0.05) and head width (r = 0.36, P< or =0.05) were found. However, the head length was not significantly directly correlated with its width (r = 0.18, P< or =0.05), and flagellum length was negatively correlated with spermatozoan head width (r = -0.71, P< or =0.05). Slight correlations between the morphometric traits of the sperm and the physical traits of the ejaculates (r = -0.11 to 0.16) were found, although in most cases, the correlations were not statistically provable. [Abstract/Link to Full Text]

Paw?owicz I, Grygorowicz WJ, Rorat T
DHN10 dehydrin is not expressed in transgenic Solanum species plants when the Dhn10 gene is fused to a glucosyl transferase promoter.
Cell Mol Biol Lett. 2004;9(4B):947-61.
A gene fusion system was used to study the expression pattern of the Dhn10 gene, encoding the DHN10 dehydrin protein in transgenic Solanum tuberosum plants carrying a combined GT-Dhn10 transgen in which the glucosyl transferase (GT) promoter region was fused to the coding sequence of the Dhn10 gene. Expression of the native Dhn10 gene and the GT-Dhn10 constructs was analysed in regenerated S. tuberosum transgenic plants, both at the transcript accumulation and protein levels. We showed that the expression of both the GT-Dhn10 transgen and the Dhn10 gene was regulated in the regenerated plants at the transcriptional level in an independent way, but only the protein product of the native Dhn10 expression was detected. The transcription product of the GT-Dhn10 transgen did not affect the expression of the Dhn10 gene either at the transcription level or at the protein level. The GT-Dhn10 plants did not show changes in freezing capacity compared to the control, non-transgenic ones. [Abstract/Link to Full Text]

Lorenc-Kuku?a K, Korobczak A, Aksamit-Stachurska A, Kosty? K, Lukaszewicz M, Szopa J
Glucosyltransferase: the gene arrangement and enzyme function.
Cell Mol Biol Lett. 2004;9(4B):935-46.
Glucosyltransferases were isolated and characterised from many plant sources. The enzymes show middle amino acid similarity and broad substrate specificity. The promoter of the potato 5-UGT gene reveals strong environmental induction. The activation of the gene expression by UV radiation, ABA and cold treatments was detected. Overexpression of 5-UGT resulted in the accumulation of the diglucoside derivative of petunidin in transgenic tubers; the latter is most probably the reason for plant resistance to pathogen infection. Overexpressing plants produced more tubers, and the overall yield was higher when compared to nontransformants. [Abstract/Link to Full Text]

---

Recent Articles in BMC Cell Biology

---

Pauly B, Lasi M, MacKintosh C, Morrice N, Imhof A, Regula J, Rudd S, David CN, Böttger A
Proteomic screen in the simple metazoan Hydra identifies 14-3-3 binding proteins implicated in cellular metabolism, cytoskeletal organisation and Ca2+ signalling.
BMC Cell Biol. 2007;831.
BACKGROUND: 14-3-3 proteins have been implicated in many signalling mechanisms due to their interaction with Ser/Thr phosphorylated target proteins. They are evolutionarily well conserved in eukaryotic organisms from single celled protozoans and unicellular algae to plants and humans. A diverse array of target proteins has been found in higher plants and in human cell lines including proteins involved in cellular metabolism, apoptosis, cytoskeletal organisation, secretion and Ca2+ signalling. RESULTS: We found that the simple metazoan Hydra has four 14-3-3 isoforms. In order to investigate whether the diversity of 14-3-3 target proteins is also conserved over the whole animal kingdom we isolated 14-3-3 binding proteins from Hydra vulgaris using a 14-3-3-affinity column. We identified 23 proteins that covered most of the above-mentioned groups. We also isolated several novel 14-3-3 binding proteins and the Hydra specific secreted fascin-domain-containing protein PPOD. In addition, we demonstrated that one of the 14-3-3 isoforms, 14-3-3 HyA, interacts with one Hydra-Bcl-2 like protein in vitro. CONCLUSION: Our results indicate that 14-3-3 proteins have been ubiquitous signalling components since the start of metazoan evolution. We also discuss the possibility that they are involved in the regulation of cell numbers in response to food supply in Hydra. [Abstract/Link to Full Text]

Lawless MW, Mankan AK, White M, O'Dwyer MJ, Norris S
Expression of hereditary hemochromatosis C282Y HFE protein in HEK293 cells activates specific endoplasmic reticulum stress responses.
BMC Cell Biol. 2007;830.
BACKGROUND: Hereditary Hemochromatosis (HH) is a genetic disease associated with iron overload, in which individuals homozygous for the mutant C282Y HFE associated allele are at risk for the development of a range of disorders particularly liver disease. Conformational diseases are a class of disorders associated with the expression of misfolded protein. HFE C282Y is a mutant protein that does not fold correctly and consequently is retained in the Endoplasmic Reticulum (ER). In this context, we sought to identify ER stress signals associated with mutant C282Y HFE protein expression, which may have a role in the molecular pathogenesis of HH. RESULTS: Vector constructs of Wild type HFE and Mutant C282Y HFE were made and transfected into HEK293 cell lines. We have shown that expression of C282Y HFE protein triggers both an unfolded protein response (UPR), as revealed by the increased GRP78, ATF6 and CHOP expression, and an ER overload response (EOR), as indicated by NF-kappaB activation. Furthermore, C282Y HFE protein induced apoptotic responses associated with activation of ER stress. Inhibition studies demonstrated that tauroursodeoxycholic acid, an endogenous bile acid, downregulates these events. Finally, we found that the co-existence of both C282Y HFE and Z alpha 1-antitrypsin protein (the protein associated with the liver disease of Z alpha 1-antitrypsin deficiency) expression on ER stress responses acted as potential disease modifiers with respect to each other. CONCLUSION: Our novel observations suggest that both the ER overload response (EOR) and the unfolded protein response (UPR) are activated by mutant C282Y HFE protein. [Abstract/Link to Full Text]

Koo TH, Eipper BA, Donaldson JG
Arf6 recruits the Rac GEF Kalirin to the plasma membrane facilitating Rac activation.
BMC Cell Biol. 2007;829.
BACKGROUND: Many studies implicate Arf6 activity in Rac-mediated membrane ruffling and cytoskeletal reorganization. Although Arf6 facilitates the trafficking of Rac1 to the plasma membrane and in many cases Arf6 activation leads to the activation of Rac1, the details of how Arf6 influences Rac function remain to be elucidated. RESULTS: We demonstrate in binding assays and by co-immunoprecipitation that GDP-bound Arf6 binds to Kalirin5, a Rho family guanine nucleotide exchange factor, through interaction with the spectrin repeat region. In cells, expression of wild type Arf6 recruits spectrin repeat 5 and Kalirin to the plasma membrane and leads to enhanced Kalirin5-induced ruffling. By contrast, expression of an Arf6 mutant that cannot become activated, Arf6 T27N, still recruits spectrin repeat 5 and Kalirin to membranes but inhibits Kalirin5-induced ruffling in HeLa cells. Kalirin5-induced Rac1 activation is increased by the expression of wild type Arf6 and decreased by Arf6T27N. Furthermore, expression of a catalytically-inactive mutant of Kalirin5 inhibits cytoskeletal changes observed in cells expressing EFA6, an Arf6 guanine nucleotide exchange factor that leads to activation of Rac. CONCLUSION: We show here with over-expressed proteins that the GDP-bound form of Arf6 can bind to the spectrin repeat regions in Kalirin Rho family GEFs thereby recruiting Kalirin to membranes. Although Kalirin is recruited onto membranes by Arf6-GDP, subsequent Rac activation and membrane ruffling requires Arf6 activation. From these results, we suggest that Arf6 can regulate through its GTPase cycle the activation of Rac. [Abstract/Link to Full Text]

Hao le T, Fuller HR, Lam le T, Le TT, Burghes AH, Morris GE
Absence of gemin5 from SMN complexes in nuclear Cajal bodies.
BMC Cell Biol. 2007;828.
BACKGROUND: Spinal muscular atrophy is caused by reduced levels of the survival of motor neurons (SMN) protein. SMN is found in large complexes with Sm proteins and at least eight other proteins, including seven "gemins". These complexes are involved in the assembly of snRNPs in the cytoplasm and their transport into the nucleus, but the precise roles of the individual protein components are largely unknown. RESULTS: We have investigated the subcellular distribution of gemins using novel antibodies against gemins 3-7, and existing mAbs against SMN, gemin2, unrip, fibrillarin and profilin II. Most gemins were equally distributed between nuclear and cytoplasmic fractions of HeLa cells, but gemin5 and unrip were more abundant in the cytoplasm. In a cytoplasmic extract obtained by mild disruption of HeLa cells, nearly all the SMN and gemins 2-4 were in large complexes, but most of the gemin5 sedimented separately with a lower S value. Most of the unrip sedimented with gemins 6 and 7 near the top of the sucrose density gradients, separate from both SMN and gemin5. Anti-SMN mAbs pulled down gemin5 from cytoplasmic extracts, but not from nuclear extracts, and gemin5 did not co-sediment with large SMN complexes in nuclear extracts. These data suggest that gemin5 is easily detached from SMN-gemin complexes in the nucleus. By immuno-histochemistry, gemin5 was rarely detectable in nuclear gems/Cajal bodies, although it was accessible to antibody and easily detectable when present. This suggests that gemin5 is normally absent from SMN complexes in these nuclear storage sites. CONCLUSION: We conclude that SMN complexes usually exist without gemin5 in nuclear gems/Cajal bodies. Gemin5 is believed to be involved in capturing snRNA into SMN complexes in the cytoplasm for transport into the nucleus. We hypothesize that gemin5, though present in the nucleus, is no longer needed for SMN complex function during the time these complexes are stored in gems/Cajal bodies. [Abstract/Link to Full Text]

Ciani L, Salinas PC
c-Jun N-terminal kinase (JNK) cooperates with Gsk3beta to regulate Dishevelled-mediated microtubule stability.
BMC Cell Biol. 2007;827.
BACKGROUND: Wnt factors are a large family of signaling molecules that play important roles in the regulation of cell fate specification, tissue polarity and cell movement. In the nervous system, Wnts also regulates the formation of neuronal connection acting as retrograde signals that regulate the remodeling of the axons prior to the assembly of the presynaptic apparatus. The scaffold protein Dishevelled (Dvl) mimics the effect of Wnt on the neuronal cytoskeleton by increasing the number of stable microtubule along the axon shaft and inducing the formation of looped microtubules (MT) at enlarged growth cones. A divergent Wnt-Dvl canonical pathway which bifurcates downstream of Gsk3beta regulates MT dynamics. RESULTS: Here we show that the Wnt pathway also activates c-Jun N-terminal kinase (JNK) to regulate MT stabilization. Although in the Wnt planar cell polarity (PCP) pathway, JNK lays downstream of Rho GTPases, these GTPases are not required for Wnt-mediated MTs stability. Epistatic analyses and pharmacological studies suggest that the Wnt-Dvl signalling regulates the dynamic of the cytoskeleton through two different pathways that lead to inhibition of Gsk3beta and activation of JNK in the same cell. CONCLUSION: We demonstrate a novel role for JNK in Wnt-mediated MT stability. Wnt-Dvl pathway increases MT stability through a transcription independent mechanism that requires the concomitant inhibition of Gsk3beta and activation of JNK. These studies demonstrate that Wnts can simultaneously activate different signalling pathways to modulate cytoskeleton dynamics. [Abstract/Link to Full Text]

Tanaka T, Huang X, Jorgensen E, Gietl D, Traganos F, Darzynkiewicz Z, Albino AP
ATM activation accompanies histone H2AX phosphorylation in A549 cells upon exposure to tobacco smoke.
BMC Cell Biol. 2007;826.
BACKGROUND: In response to DNA damage or structural alterations of chromatin, histone H2AX may be phosphorylated on Ser139 by phosphoinositide 3-kinase related protein kinases (PIKKs) such as ataxia telangiectasia mutated (ATM), ATM-and Rad-3 related (ATR) kinase, or by DNA dependent protein kinase (DNA-PKcs). When DNA damage primarily involves formation of DNA double-strand breaks (DSBs), H2AX is preferentially phosphorylated by ATM rather than by the other PIKKs. We have recently reported that brief exposure of human pulmonary adenocarcinoma A549 cells or normal human bronchial epithelial cells (NHBE) to cigarette smoke (CS) induced phosphorylation of H2AX. RESULTS: We report here that H2AX phosphorylation in A549 cells induced by CS was accompanied by activation of ATM, as revealed by ATM phosphorylation on Ser1981 (ATM-S1981P) detected immunocytochemically and by Western blotting. No cell cycle-phase specific differences in kinetics of ATM activation and H2AX phosphorylation were observed. When cells were exposed to CS from cigarettes with different tobacco and filter combinations, the expression levels of ATM-S1981P correlated well with the increase in expression of phosphorylated H2AX (gammaH2AX) (R = 0.89). In addition, we note that while CS-induced gammaH2AX expression was localized within discrete foci, the activated ATM was distributed throughout the nucleoplasm. CONCLUSION: These data implicate ATM as the PIKK that phosphorylates H2AX in response to DNA damage caused by CS. Based on current understanding of ATM activation, expression and localization, these data would suggest that, in addition to inducing potentially carcinogenic DSB lesions, CS may also trigger other types of DNA lesions and cause chromatin alterations. As checkpoint kinase (Chk) 1, Chk2 and the p53 tumor suppressor gene are known to be phosphorylated by ATM, the present data indicate that exposure to CS may lead to their phosphorylation, with the downstream consequences related to the halt in cell cycle progression and increased propensity to undergo apoptosis. Defining the nature and temporal sequence of molecular events that are disrupted by CS through activation and eventual dysregulation of normal defense mechanisms such as ATM and its downstream effectors may allow a more precise understanding of how CS promotes cancer development. [Abstract/Link to Full Text]

Rajagopal R, Ishii S, Beebe DC
Intracellular mediators of transforming growth factor beta superfamily signaling localize to endosomes in chicken embryo and mouse lenses in vivo.
BMC Cell Biol. 2007;825.
BACKGROUND: Endocytosis is a key regulator of growth factor signaling pathways. Recent studies showed that the localization to endosomes of intracellular mediators of growth factor signaling may be required for their function. Although there is substantial evidence linking endocytosis and growth factor signaling in cultured cells, there has been little study of the endosomal localization of signaling components in intact tissues or organs. RESULTS: Proteins that are downstream of the transforming growth factor-beta superfamily signaling pathway were found on endosomes in chicken embryo and postnatal mouse lenses, which depend on signaling by members of the TGFbeta superfamily for their normal development. Phosphorylated Smad1 (pSmad1), pSmad2, Smad4, Smad7, the transcriptional repressors c-Ski and TGIF and the adapter molecules Smad anchor for receptor activation (SARA) and C184M, localized to EEA-1- and Rab5-positive vesicles in chicken embryo and/or postnatal mouse lenses. pSmad1 and pSmad2 also localized to Rab7-positive late endosomes. Smad7 was found associated with endosomes, but not caveolae. Bmpr1a conditional knock-out lenses showed decreased nuclear and endosomal localization of pSmad1. Many of the effectors in this pathway were distributed differently in vivo from their reported distribution in cultured cells. CONCLUSION: Based on the findings reported here and data from other signaling systems, we suggest that the localization of activated intracellular mediators of the transforming growth factor-beta superfamily to endosomes is important for the regulation of growth factor signaling. [Abstract/Link to Full Text]

Fang Y, Ferrie AM
Optical biosensor differentiates signaling of endogenous PAR1 and PAR2 in A431 cells.
BMC Cell Biol. 2007;824.
BACKGROUND: Protease activated receptors (PARs) consist of a family of four G protein-coupled receptors. Many types of cells express several PARs, whose physiological significance is mostly unknown. RESULTS: Here, we show that non-invasive resonant waveguide grating (RWG) biosensor differentiates signaling of endogenous protease activated receptor subtype 1 (PAR1) and 2 (PAR2) in human epidermoid carcinoma A431 cells. The biosensor directly measures dynamic mass redistribution (DMR) resulted from ligand-induced receptor activation in adherent cells. In A431, both PAR1 and PAR2 agonists, but neither PAR3 nor PAR4 agonists, trigger dose-dependent Ca2+ mobilization as well as Gq-type DMR signals. Both Ca2+ flux and DMR signals display comparable desensitization patterns upon repeated stimulation with different combinations of agonists. However, PAR1 and PAR2 exhibit distinct kinetics of receptor re-sensitization. Furthermore, both trypsin- and thrombin-induced Ca2+ flux signals show almost identical dependence on cell surface cholesterol level, but their corresponding DMR signals present different sensitivities. CONCLUSION: Optical biosensor provides an alternative readout for examining receptor activation under physiologically relevant conditions, and differentiates the signaling of endogenous PAR1 and PAR2 in A431. [Abstract/Link to Full Text]

Mondal S, Neelamegan D, Rivero F, Noegel AA
GxcDD, a putative RacGEF, is involved in Dictyostelium development.
BMC Cell Biol. 2007;823.
BACKGROUND: Rho subfamily GTPases are implicated in a large number of actin-related processes. They shuttle from an inactive GDP-bound form to an active GTP-bound form. This reaction is catalysed by Guanine nucleotide exchange factor (GEFs). GTPase activating proteins (GAPs) help the GTPase return to the inactive GDP-bound form. The social amoeba Dictyostelium discoideum lacks a Rho or Cdc42 ortholog but has several Rac related GTPases. Compared to our understanding of the downstream effects of Racs our understanding of upstream mechanisms that activate Rac GTPases is relatively poor. RESULTS: We report on GxcDD (Guanine exchange factor for Rac GTPases), a Dictyostelium RacGEF. GxcDD is a 180-kDa multidomain protein containing a type 3 CH domain, two IQ motifs, three PH domains, a RhoGEF domain and an ArfGAP domain. Inactivation of the gene results in defective streaming during development under different conditions and a delay in developmental timing. The characterization of single domains revealed that the CH domain of GxcDD functions as a membrane association domain, the RhoGEF domain can physically interact with a subset of Rac GTPases, and the ArfGAP-PH tandem accumulates in cortical regions of the cell and on phagosomes. Our results also suggest that a conformational change may be required for activation of GxcDD, which would be important for its downstream signaling. CONCLUSION: The data indicate that GxcDD is involved in proper streaming and development. We propose that GxcDD is not only a component of the Rac signaling pathway in Dictyostelium, but is also involved in integrating different signals. We provide evidence for a Calponin Homology domain acting as a membrane association domain. GxcDD can bind to several Rac GTPases, but its function as a nucleotide exchange factor needs to be studied further. [Abstract/Link to Full Text]

Lyly A, von Schantz C, Salonen T, Kopra O, Saarela J, Jauhiainen M, Kyttälä A, Jalanko A
Glycosylation, transport, and complex formation of palmitoyl protein thioesterase 1 (PPT1)--distinct characteristics in neurons.
BMC Cell Biol. 2007;822.
BACKGROUND: Neuronal ceroid lipofuscinoses (NCLs) are collectively the most common type of recessively inherited childhood encephalopathies. The most severe form of NCL, infantile neuronal ceroid lipofuscinosis (INCL), is caused by mutations in the CLN1 gene, resulting in a deficiency of the lysosomal enzyme, palmitoyl protein thioesterase 1 (PPT1). The deficiency of PPT1 causes a specific death of neocortical neurons by a mechanism, which is currently unclear. To understand the function of PPT1 in more detail, we have further analyzed the basic properties of the protein, especially focusing on possible differences in non-neuronal and neuronal cells. RESULTS: Our study shows that the N-glycosylation of N197 and N232, but not N212, is essential for PPT1's activity and intracellular transport. Deglycosylation of overexpressed PPT1 produced in neurons and fibroblasts demonstrates differentially modified PPT1 in different cell types. Furthermore, antibody internalization assays showed differences in PPT1 transport when compared with a thoroughly characterized lysosomal enzyme aspartylglucosaminidase (AGA), an important observation potentially influencing therapeutic strategies. PPT1 was also demonstrated to form oligomers by size-exclusion chromatography and co-immunoprecipitation assays. Finally, the consequences of disease mutations were analyzed in the perspective of our new results, suggesting that the mutations increase both the degree of glycosylation of PPT1 and its ability to form complexes. CONCLUSION: Our current study describes novel properties for PPT1. We observe differences in PPT1 processing and trafficking in neuronal and non-neuronal cells, and describe for the first time the ability of PPT1 to form complexes. Understanding the basic characteristics of PPT1 is fundamental in order to clarify the molecular pathogenesis behind neurodegeneration in INCL. [Abstract/Link to Full Text]

Baumann A, Lange C, Soppa J
Transcriptome changes and cAMP oscillations in an archaeal cell cycle.
BMC Cell Biol. 2007;821.
BACKGROUND: The cell cycle of all organisms includes mass increase by a factor of two, replication of the genetic material, segregation of the genome to different parts of the cell, and cell division into two daughter cells. It is tightly regulated and typically includes cell cycle-specific oscillations of the levels of transcripts, proteins, protein modifications, and signaling molecules. Until now cell cycle-specific transcriptome changes have been described for four eukaryotic species ranging from yeast to human, but only for two prokaryotic species. Similarly, oscillations of small signaling molecules have been identified in very few eukaryotic species, but not in any prokaryote. RESULTS: A synchronization procedure for the archaeon Halobacterium salinarum was optimized, so that nearly 100% of all cells divide in a time interval that is 1/4th of the generation time of exponentially growing cells. The method was used to characterize cell cycle-dependent transcriptome changes using a genome-wide DNA microarray. The transcript levels of 87 genes were found to be cell cycle-regulated, corresponding to 3% of all genes. They could be clustered into seven groups with different transcript level profiles. Cluster-specific sequence motifs were detected around the start of the genes that are predicted to be involved in cell cycle-specific transcriptional regulation. Notably, many cell cycle genes that have oscillating transcript levels in eukaryotes are not regulated on the transcriptional level in H. salinarum.Synchronized cultures were also used to identify putative small signaling molecules. H. salinarum was found to contain a basal cAMP concentration of 200 microM, considerably higher than that of yeast. The cAMP concentration is shortly induced directly prior to and after cell division, and thus cAMP probably is an important signal for cell cycle progression. CONCLUSION: The analysis of cell cycle-specific transcriptome changes of H. salinarum allowed to identify a strategy of transcript level regulation that is different from all previously characterized species. The transcript levels of only 3% of all genes are regulated, a fraction that is considerably lower than has been reported for four eukaryotic species (6%-28%) and for the bacterium C. crescentus (19%).It was shown that cAMP is present in significant concentrations in an archaeon, and the phylogenetic profile of the adenylate cyclase indicates that this signaling molecule is widely distributed in archaea. The occurrence of cell cycle-dependent oscillations of the cAMP concentration in an archaeon and in several eukaryotic species indicates that cAMP level changes might be a phylogenetically old signal for cell cycle progression. [Abstract/Link to Full Text]

Oh HY, Jin X, Kim JG, Oh MJ, Pian X, Kim JM, Yoon MS, Son CI, Lee YS, Hong KC, Kim H, Choi YJ, Whang KY
Characteristics of primary and immortalized fibroblast cells derived from the miniature and domestic pigs.
BMC Cell Biol. 2007;820.
BACKGROUND: The pig, Sus scrofa domestica includes both the miniature and commercial domestic breed. These animals have influenced the human life and economies and have been studied throughout history. Although the miniature breeds are more recent and have increasingly been used in a variety of biomedical studies, their cell lines have rarely been established. Therefore, we sought to establish primary and immortal cell lines derived from both the miniature and domestic pig to better enable insight into possible in vivo growth differences. RESULTS: The in vitro lifespan of primary domestic pig fibroblast (PF) and miniature pig fibroblast (MPF) cells using a standard 3T3 protocol was determined. Both of the primary PF and MPF cells were shown to have a two-step replicative senescence barrier. Primary MPF cells exhibited a relatively shorter lifespan and slower proliferation rate compared to those of primary PF cells. Beyond senescence barriers, lifespan-extended PF and MPF cells were eventually established and indicated spontaneous cellular immortalization. In contrast to the immortalized PF cells, immortal MPF cells showed a transformed phenotype and possessed more frequent chromosomal abnormalities and loss of p53 regulatory function. The lifespan of primary MPF and PF cells was extended by inactivation of the p53 function using transduction by SV40LT without any detectable senescent phenotype. CONCLUSION: These results suggest that p53 signaling might be a major determinant for the replicative senescence in the MPF cells that have the shorter lifespan and slower growth rate compared to PF cells in vitro. [Abstract/Link to Full Text]

Szebenyi G, Wigley WC, Hall B, Didier A, Yu M, Thomas P, Krämer H
Hook2 contributes to aggresome formation.
BMC Cell Biol. 2007;819.
BACKGROUND: Aggresomes are pericentrosomal accumulations of misfolded proteins, chaperones and proteasomes. Their positioning near the centrosome, like that of other organelles, requires active, microtubule-dependent transport. Linker proteins that can associate with the motor protein dynein, organelles, and microtubules are thought to contribute to the active maintenance of the juxtanuclear localization of many membrane bound organelles and aggresomes. Hook proteins have been proposed to serve as adaptors for the association of cargos with dynein for transport on microtubules. Hook2 was shown to localize to the centrosome, bind centriolin, and contribute to centrosomal function. RESULTS: Here we show that overexpression of hook2 promotes the accumulation of the cystic fibrosis transmembrane regulator in aggresomes without altering its biochemical properties or its steady state level. A dominant negatively acting form of hook2 that lacks the centriolin binding C-terminal inhibits aggresome formation. CONCLUSION: We propose that hook2 contributes to the establishment and maintenance of the pericentrosomal localization of aggresomes by promoting the microtubule-based delivery of protein aggregates to pericentriolar aggresomes. [Abstract/Link to Full Text]

Noer A, Boquest AC, Collas P
Dynamics of adipogenic promoter DNA methylation during clonal culture of human adipose stem cells to senescence.
BMC Cell Biol. 2007;818.
BACKGROUND: Potential therapeutic use of mesenchymal stem cells (MSCs) is likely to require large-scale in vitro expansion of the cells before transplantation. MSCs from adipose tissue can be cultured extensively until senescence. However, little is known on the differentiation potential of adipose stem cells (ASCs) upon extended culture and on associated epigenetic alterations. We examined the adipogenic differentiation potential of clones of human ASCs in early passage culture and upon senescence, and determined whether senescence was associated with changes in adipogenic promoter DNA methylation. RESULTS: ASC clones cultured to senescence display reduced adipogenic differentiation capacity in vitro, on the basis of limited lipogenesis and reduced transcriptional upregulation of FABP4 and LPL, two adipogenic genes, while LEP and PPARG2 transcription remains unaffected. In undifferentiated senescent cells, PPARG2 and LPL expression is unaltered, whereas LEP and FABP4 transcript levels are increased but not in all clones. Bisulfite sequencing analysis of DNA methylation reveals overall relative stability of LEP, PPARG2, FABP4 and LPL promoter CpG methylation during senescence and upon differentiation. Mosaicism in methylation profiles is maintained between and within ASC clones, and any CpG-specific methylation change detected does not necessarily relate to differentiation potential. One exception to this contention is CpG No. 21 in the LEP promoter, whose senescence-related methylation may impair upregulation of the gene upon adipogenic stimulation. CONCLUSION: Senescent ASCs display reduced in vitro differentiation ability and transcriptional activation of adipogenic genes upon differentiation induction. These restrictions, however, cannot in general be attributed to specific changes in DNA methylation at adipogenic promoters. There also seems to be a correlation between CpGs that are hypomethylated and important transcription factor binding sites. [Abstract/Link to Full Text]

Gao X, Ray R, Xiao Y, Barker PE, Ray P
Inhibition of sulfur mustard-induced cytotoxicity and inflammation by the macrolide antibiotic roxithromycin in human respiratory epithelial cells.
BMC Cell Biol. 2007;817.
BACKGROUND: Sulfur mustard (SM) is a potent chemical vesicant warfare agent that remains a significant military and civilian threat. Inhalation of SM gas causes airway inflammation and injury. In recent years, there has been increasing evidence of the effectiveness of macrolide antibiotics in treating chronic airway inflammatory diseases. In this study, the anti-cytotoxic and anti-inflammatory effects of a representative macrolide antibiotic, roxithromycin, were tested in vitro using SM-exposed normal human small airway epithelial (SAE) cells and bronchial/tracheal epithelial (BTE) cells. Cell viability, expression of proinflammatory cytokines including interleukin (IL)-1beta, IL-6, IL-8 and tumor necrosis factor (TNF), and expression of inducible nitric oxide synthase (iNOS) were examined, since these proinflammatory cytokines/mediators are import indicators of tissue inflammatory responses. We suggest that the influence of roxithromycin on SM-induced inflammatory reaction could play an important therapeutic role in the cytotoxicity exerted by this toxicant. RESULTS: MTS assay and Calcein AM/ethidium homodimer (EthD-1) fluorescence staining showed that roxithromycin decreased SM cytotoxicity in both SAE and BTE cells. Also, roxithromycin inhibited the SM-stimulated overproduction of the proinflammatory cytokines IL-1beta, IL-6, IL-8 and TNF at both the protein level and the mRNA level, as measured by either enzyme-linked immunosorbent assay (ELISA) or real-time RT-PCR. In addition, roxithromycin inhibited the SM-induced overexpression of iNOS, as revealed by immunocytochemical analysis using quantum dots as the fluorophore. CONCLUSION: The present study demonstrates that roxithromycin has inhibitory effects on the cytotoxicity and inflammation provoked by SM in human respiratory epithelial cells. The decreased cytotoxicity in roxithromycin-treated cells likely depends on the ability of the macrolide to down-regulate the production of proinflammatory cytokines and/or mediators. The results obtained in this study suggest that macrolide antibiotics may serve as potential vesicant respiratory therapeutics through mechanisms independent of their antibacterial activity. [Abstract/Link to Full Text]

Parikh N, Koshy C, Dhayabaran V, Perumalsamy LR, Sowdhamini R, Sarin A
The N-terminus and alpha-5, alpha-6 helices of the pro-apoptotic protein Bax, modulate functional interactions with the anti-apoptotic protein Bcl-xL.
BMC Cell Biol. 2007;816.
BACKGROUND: Bcl-2 family proteins are key regulators of mitochondrial integrity and comprise both pro- and anti-apoptotic proteins. Bax a pro-apoptotic member localizes as monomers in the cytosol of healthy cells and accumulates as oligomers in mitochondria of apoptotic cells. The Bcl-2 homology-3 (BH3) domain regulates interactions within the family, but regions other than BH3 are also critical for Bax function. Thus, the N-terminus has been variously implicated in targeting to mitochondria, interactions with BH3-only proteins as well as conformational changes linked to Bax activation. The transmembrane (TM) domains (alpha5-alpha6 helices in the core and alpha9 helix in the C-terminus) in Bax are implicated in localization to mitochondria and triggering cytotoxicity. Here we have investigated N-terminus modulation of TM function in the context of regulation by the anti-apoptotic protein Bcl-xL. RESULTS: Deletion of 29 amino acids in the Bax N-terminus (Bax 30-192) caused constitutive accumulation at mitochondria and triggered high levels of cytotoxicity, not inhibited by Bcl-xL. Removal of the TM domains (Bax 30-105) abrogated mitochondrial localization but resulted in Bcl-xL regulated activation of endogenous Bax and Bax-Bak dependent apoptosis. Inclusion of the alpha5-alpha6 helices/TMI domain (Bax 30-146) phenocopied Bax 30-192 as it restored mitochondrial localization, Bcl-xL independent cytotoxicity and was not dependent on endogenous Bax-Bak. Inhibition of function and localization by Bcl-xL was restored in Bax 1-146, which included the TM1 domain. Regardless of regulation by Bcl-xL, all N-terminal deleted constructs immunoprecipitated Bcl-xLand converged on caspase-9 dependent apoptosis consistent with mitochondrial involvement in the apoptotic cascade. Sub-optimal sequence alignments of Bax and Bcl-xL indicated a sequence similarity between the alpha5-alpha6 helices of Bax and Bcl-xL. Alanine substitutions of three residues (T14A-S15A-S16A) in the N-terminus (Bax-Ala3) attenuated regulation by the serine-threonine kinase Akt/PKB but not by Bcl-xL indicative of distinct regulatory mechanisms. CONCLUSION: Collectively, the analysis of Bax deletion constructs indicates that the N-terminus drives conformational changes facilitating inhibition of cytotoxicity by Bcl-xL. We speculate that the TM1 helices may serve as 'structural antagonists' for BH3-Bcl-xL interactions, with this function being regulated by the N-terminus in the intact protein. [Abstract/Link to Full Text]

Wong R, Fabian L, Forer A, Brill JA
Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesis.
BMC Cell Biol. 2007;815.
BACKGROUND: Phosphatidylinositol 4,5-bisphosphate (PIP2) is required for successful completion of cytokinesis. In addition, both PIP2 and phosphoinositide-specific phospholipase C (PLC) have been localized to the cleavage furrow of dividing mammalian cells. PLC hydrolyzes PIP2 to yield diacylglycerol (DAG) and inositol trisphosphate (IP3), which in turn induces calcium (Ca2+) release from the ER. Several studies suggest PIP2 must be hydrolyzed continuously for continued cleavage furrow ingression. The majority of these studies employ the N-substituted maleimide U73122 as an inhibitor of PLC. However, the specificity of U73122 is unclear, as its active group closely resembles the non-specific alkylating agent N-ethylmaleimide (NEM). In addition, the pathway by which PIP2 regulates cytokinesis remains to be elucidated. RESULTS: Here we compared the effects of U73122 and the structurally unrelated PLC inhibitor ET-18-OCH3 (edelfosine) on cytokinesis in crane-fly and Drosophila spermatocytes. Our data show that the effects of U73122 are indeed via PLC because U73122 and ET-18-OCH3 produced similar effects on cell morphology and actin cytoskeleton organization that were distinct from those caused by NEM. Furthermore, treatment with the myosin light chain kinase (MLCK) inhibitor ML-7 caused cleavage furrow regression and loss of both F-actin and phosphorylated myosin regulatory light chain from the contractile ring in a manner similar to treatment with U73122 and ET-18-OCH3. CONCLUSION: We have used multiple inhibitors to examine the roles of PLC and MLCK, a predicted downstream target of PLC regulation, in cytokinesis. Our results are consistent with a model in which PIP2 hydrolysis acts via Ca2+ to activate myosin via MLCK and thereby control actin dynamics during constriction of the contractile ring. [Abstract/Link to Full Text]

Wang L, Luo J, He S
Induction of MMP-9 release from human dermal fibroblasts by thrombin: involvement of JAK/STAT3 signaling pathway in MMP-9 release.
BMC Cell Biol. 2007;814.
BACKGROUND: It has been recognized that dermal fibroblasts and matrix metalloproteases (MMP) play crucial roles in wound healing process in skin. Thrombin was found to stimulate IL-8 release from human dermal fibroblasts (HDFs). However, little is known of the effect of thrombin on secretion of MMPs from dermal fibroblasts. In the present study, the influence of thrombin on proMMP-2 and proMMP-9 activity release from primary cultured HDFs, and its potential signaling pathways were investigated. RESULTS: The results showed that thrombin induced proMMP-9, but not proMMP-2 release from HDFs in a dose dependent manner at 6 h following incubation. Thrombin also upregulated expression of proMMP-9 mRNA in HDFs. Hirudin completely abolished the action of thrombin on HDFs. An agonist peptide of protease-activated receptor-1, SFLLR-NH2 stimulated an enhanced release of proMMP-9 from HDFs. AG490, an inhibitor of STAT3 inhibited basal and thrombin-provoked proMMP-9 release and phosphorylation of STAT3. PD98059, an inhibitor of MAPK and LY294002, an inhibitor PI3K failed to significantly inhibit thrombin induced proMMP-9 release. CONCLUSION: Thrombin is a potent stimulus of proMMP-9 release from HDFs. Thrombin induced proMMP-9 release is most likely through activation of PAR-1. JAK/STAT3 signaling pathway is involved in proMMP-9 release from HDFs. [Abstract/Link to Full Text]

Durand M, Kolpak A, Farrell T, Elliott NA, Shao W, Brown M, Volkert MR
The OXR domain defines a conserved family of eukaryotic oxidation resistance proteins.
BMC Cell Biol. 2007;813.
BACKGROUND: The NCOA7 gene product is an estrogen receptor associated protein that is highly similar to the human OXR1 gene product, which functions in oxidation resistance. OXR genes are conserved among all sequenced eukaryotes from yeast to humans. In this study we examine if NCOA7 has an oxidation resistance function similar to that demonstrated for OXR1. We also examine NCOA7 expression in response to oxidative stress and its subcellular localization in human cells, comparing these properties with those of OXR1. RESULTS: We find that NCOA7, like OXR1 can suppress the oxidative mutator phenotype when expressed in an E. coli strain that exhibits an oxidation specific mutator phenotype. Moreover, NCOA7's oxidation resistance function requires expression of only its carboxyl-terminal domain and is similar in this regard to OXR1. We find that, in human cells, NCOA7 is constitutively expressed and is not induced by oxidative stress and appears to localize to the nucleus following estradiol stimulation. These properties of NCOA7 are in striking contrast to those of OXR1, which is induced by oxidative stress, localizes to mitochondria, and appears to be excluded, or largely absent from nuclei. CONCLUSION: NCOA7 most likely arose from duplication. Like its homologue, OXR1, it is capable of reducing the DNA damaging effects of reactive oxygen species when expressed in bacteria, indicating the protein has an activity that can contribute to oxidation resistance. Unlike OXR1, it appears to localize to nuclei and interacts with the estrogen receptor. This raises the possibility that NCOA7 encodes the nuclear counterpart of the mitochondrial OXR1 protein and in mammalian cells it may reduce the oxidative by-products of estrogen metabolite-mediated DNA damage. [Abstract/Link to Full Text]

Planutis K, Planutiene M, Moyer MP, Nguyen AV, Pérez CA, Holcombe RF
Regulation of norrin receptor frizzled-4 by Wnt2 in colon-derived cells.
BMC Cell Biol. 2007;812.
BACKGROUND: Norrin is a potent Wnt pathway ligand. Aberrant activation of this signaling pathway can result in colon tumors but the role of norrin-based signaling in the genesis of colon cancer, and its relationship to activation of the pathway by traditional Wnt ligands, is not defined. RESULTS: Fresh normal human colon tissue and all the cell lines studied expressed mRNA for Fz4, LRP5 and norrin, except Colo205 which lacked Fz4 expression. Canonical Wnt pathway throughput was increased slightly in NCM460 following treatment with Wnt3a CM but was inhibited by Wnt2 and Wnt1. The colon cancer cell line, RKO, responded to Wnt3a CM, Wnt2 and Wnt1 by increasing canonical Wnt pathway throughput. Wnt5a did not affect Wnt pathway throughput in either cell line. Wnt2, but not Wnt3a, abrogated Fz4 expression in NCM460, but not in RKO or another colon cancer cell line, HCT116. This effect on Fz4 was confirmed at both the RNA and protein levels via RT-PCR and a norrin binding assay. The expression of all others 9 Fz receptors did not change after treatment of NCM460 cells with Wnt2. CONCLUSION: The data suggests that colonic mucosa and colon tumors may possess two autoregulatory positive Wnt feedback loops, one through canonical signals induced by Wnt:Fz interactions and one through signals resulting from norrin:Fz4 interactions. The latter interactions may be modulated via regulation of Fz4 expression by Wnt2. Retention of Fz4 by cancers, in contrast to the loss of Fz4 by normal mucosal cells, could provide a selective advantage to the tumor cells. Fz4 expression may play a critical role in responses to Wnt signaling in the tumor microenvironment. [Abstract/Link to Full Text]

Sardăo VA, Oliveira PJ, Holy J, Oliveira CR, Wallace KB
Vital imaging of H9c2 myoblasts exposed to tert-butylhydroperoxide--characterization of morphological features of cell death.
BMC Cell Biol. 2007;811.
BACKGROUND: When exposed to oxidative conditions, cells suffer not only biochemical alterations, but also morphologic changes. Oxidative stress is a condition induced by some pro-oxidant compounds, such as by tert-butylhydroperoxide (tBHP) and can also be induced in vivo by ischemia/reperfusion conditions, which is very common in cardiac tissue. The cell line H9c2 has been used as an in vitro cellular model for both skeletal and cardiac muscle. Understanding how these cells respond to oxidative agents may furnish novel insights into how cardiac and skeletal tissues respond to oxidative stress conditions. The objective of this work was to characterize, through vital imaging, morphological alterations and the appearance of apoptotic hallmarks, with a special focus on mitochondrial changes, upon exposure of H9c2 cells to tBHP. RESULTS: When exposed to tBHP, an increase in intracellular oxidative stress was detected in H9c2 cells by epifluorescence microscopy, which was accompanied by an increase in cell death that was prevented by the antioxidants Trolox and N-acetylcysteine. Several morphological alterations characteristic of apoptosis were noted, including changes in nuclear morphology, translocation of phosphatidylserine to the outer leaflet of the cell membrane, and cell blebbing. An increase in the exposure period or in tBHP concentration resulted in a clear loss of membrane integrity, which is characteristic of necrosis. Changes in mitochondrial morphology, consisting of a transition from long filaments to small and round fragments, were also detected in H9c2 cells after treatment with tBHP. Bax aggregates near mitochondrial networks were formed after short periods of incubation. CONCLUSION: Vital imaging of alterations in cell morphology is a useful method to characterize cellular responses to oxidative stress. In the present work, we report two distinct patterns of morphological alterations in H9c2 cells exposed to tBHP, a pro-oxidant agent frequently used as model to induce oxidative stress. In particular, dynamic changes in mitochondrial networks could be visualized, which appear to be centrally involved in how these cells respond to oxidative stress. The data also indicate that the cause of H9c2 cell death following tBHP exposure is increased intracellular oxidative stress. [Abstract/Link to Full Text]

Nguyen VP, Chen SH, Trinh J, Kim H, Coomber BL, Dumont DJ
Differential response of lymphatic, venous and arterial endothelial cells to angiopoietin-1 and angiopoietin-2.
BMC Cell Biol. 2007;810.
BACKGROUND: The lymphatic system complements the blood circulatory system in absorption and transport of nutrients, and in the maintenance of homeostasis. Angiopoietins 1 and 2 (Ang1 and Ang2) are regulators of both angiogenesis and lymphangiogenesis through the Tek/Tie-2 receptor tyrosine kinase. The response of endothelial cells to stimulation with either Ang1 or Ang2 is thought to be dependent upon the origin of the endothelial cells. In this study, we examined the effects of the angiopoietins on lymphatic, venous and arterial primary endothelial cells (bmLEC, bmVEC and bmAEC, respectively), which were isolated and cultured from bovine mesenteric vessels. RESULTS: BmLEC, bmVEC and bmAEC cell populations all express Tie-2 and were shown to express the appropriate cellular markers Prox-1, VEGFR3, and Neuropilin-1 that define the particular origin of each preparation. We showed that while bmLECs responded slightly more readily to angiopoietin-2 (Ang2) stimulation, bmVECs and bmAECs were more sensitive to Ang1 stimulation. Furthermore, exposure of bmLECs to Ang2 induced marginally higher levels of proliferation and survival than did exposure to Ang1. However, exposure to Ang1 resulted in higher levels of migration in bmLECs than did to Ang2. CONCLUSION: Our results suggest that although both Ang1 and Ang2 can activate the Tie-2 receptor in bmLECs, Ang1 and Ang2 may have distinct roles in mesenteric lymphatic endothelial cells. [Abstract/Link to Full Text]

Holla ŘL, Cameron J, Berge KE, Ranheim T, Leren TP
Degradation of the LDL receptors by PCSK9 is not mediated by a secreted protein acted upon by PCSK9 extracellularly.
BMC Cell Biol. 2007;89.
BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) post-transcriptionally degrades the low density lipoprotein receptors (LDLR). However, it is unknown whether PCSK9 acts directly on the LDLR or if PCSK9 activates another protein that in turn causes degradation of the LDLR. RESULTS: We have transiently transfected HepG2 cells with wild-type and mutant D374Y-PCSK9 plasmids to study the effect of the conditioned medium on the LDLR of untransfected HepG2 cells. The ability of the conditioned medium to reduce the internalization of LDL was abolished by removal of recombinant PCSK9 from the conditioned medium by affinity chromatography. Thus, PCSK9 is the only factor in the conditioned medium able to mediate degradation of the LDLR. Moreover, fractionation of the conditioned medium by gel filtration showed that the ability of the fractions to reduce the internalization of LDL, closely paralleled the amount of D374Y-PCSK9 in the fractions. Incubation of a secreted, truncated LDLR without cytoplasmic and transmembrane domains, as well as membrane fractions from HepG2 cells, with conditioned medium containing PCSK9, did not reduce the amount of LDLR as determined by western blot analysis. Thus, the LDLR is not degraded by PCSK9 on the cell surface. The LDLR of HepG2 cells incubated with conditioned medium was protected from PCSK9-mediated degradation by the addition of nocodazole or ammonium chloride, but was not protected when the conditioned medium was made hypertonic. These findings indicate that the intracellular degradation of the LDLR involves intracellular transport along microtubules, an acidic intracellular compartment and that it occurs even when endocytosis through clathrin-coated pits has been blocked. CONCLUSION: Degradation of the LDLR by PCSK9 is not mediated by a secreted protein acted upon by PCSK9 extracellularly. Also the PCSK9-mediated degradation of the LDLR does not take place on the cell surface. Rather, the PCSK9-mediated degradation of the LDLR appears to take place intracellularly and occurs even when endocytosis through clathrin-coated pits is blocked by hypertonic medium. [Abstract/Link to Full Text]

Ulmasov B, Bruno J, Woost PG, Edwards JC
Tissue and subcellular distribution of CLIC1.
BMC Cell Biol. 2007;88.
BACKGROUND: CLIC1 is a chloride channel whose cellular role remains uncertain. The distribution of CLIC1 in normal tissues is largely unknown and conflicting data have been reported regarding the cellular membrane fraction in which CLIC1 resides. RESULTS: New antisera to CLIC1 were generated and were found to be sensitive and specific for detecting this protein. These antisera were used to investigate the distribution of CLIC1 in mouse tissue sections and three cultured cell lines. We find CLIC1 is expressed in the apical domains of several simple columnar epithelia including glandular stomach, small intestine, colon, bile ducts, pancreatic ducts, airway, and the tail of the epididymis, in addition to the previously reported renal proximal tubule. CLIC1 is expressed in a non-polarized distribution in the basal epithelial cell layer of the stratified squamous epithelium of the upper gastrointesitinal tract and the basal cells of the epididymis, and is present diffusely in skeletal muscle. Distribution of CLIC1 was examined in Panc1 cells, a relatively undifferentiated, non-polarized human cell line derived from pancreatic cancer, and T84 cells, a human colon cancer cell line which can form a polarized epithelium that is capable of regulated chloride transport. Digitonin extraction was used to distinguish membrane-inserted CLIC1 from the soluble cytoplasmic form of the protein. We find that digitonin-resistant CLIC1 is primarily present in the plasma membrane of Panc1 cells. In T84 cells, we find digitonin-resistant CLIC1 is present in an intracellular compartment which is concentrated immediately below the apical plasma membrane and the extent of apical polarization is enhanced with forskolin, which activates transepithelial chloride transport and apical membrane traffic in these cells. The sub-apical CLIC1 compartment was further characterized in a well-differentiated mouse renal proximal tubule cell line. The distribution of CLIC1 was found to overlap that of megalin and the sodium-phosphate cotransporter, NaPi-II, which are markers of the apical endocytic/recycling compartment in proximal tubule. CONCLUSION: The cell and tissue specific patterns of CLIC1 expression suggest it may play distinct roles in different cell types. In certain polarized columnar epithelia, it may play a role in apical membrane recycling. [Abstract/Link to Full Text]

König HG, Rehm M, Gudorf D, Krajewski S, Gross A, Ward MW, Prehn JH
Full length Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons.
BMC Cell Biol. 2007;87.
BACKGROUND: Bcl-2 homology domain (BH) 3-only proteins are pro-apoptotic proteins of the Bcl-2 family that couple stress signals to the mitochondrial cell death pathways. The BH3-only protein Bid can be activated in response to death receptor activation via caspase 8-mediated cleavage into a truncated protein (tBid), which subsequently translocates to mitochondria and induces the release of cytochrome-C. Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid) from the cytosol to mitochondria. We induced a delayed excitotoxic cell death in cultured rat hippocampal neurons by a 5-min exposure to the glutamate receptor agonist N-methyl-D-aspartate (NMDA; 300 microM). RESULTS: Western blot experiments confirmed a translocation of FL-Bid to the mitochondria during excitotoxic apoptosis that was associated with the release of cytochrome-C from mitochondria. These results were confirmed by immunofluorescence analysis of Bid translocation during excitotoxic cell death using an antibody raised against the amino acids 1-58 of mouse Bid that is not able to detect tBid. Finally, inducible overexpression of FL-Bid or a Bid mutant that can not be cleaved by caspase-8 was sufficient to induce apoptosis in the hippocampal neuron cultures. CONCLUSION: Our data suggest that translocation of FL-Bid is sufficient for the activation of mitochondrial cell death pathways in response to glutamate receptor overactivation. [Abstract/Link to Full Text]

Andersson M, Warolén M, Nilsson J, Selander M, Sterky C, Bergdahl K, Sörving C, James SR, Doverskog M
Baculovirus-mediated gene transfer and recombinant protein expression do not interfere with insulin dependent phosphorylation of PKB/Akt in human SHSY-5Y and C3A cells.
BMC Cell Biol. 2007;86.
BACKGROUND: Recombinant adenovirus vectors and transfection agents comprising cationic lipids are widely used as gene delivery vehicles for functional expression in cultured cells. Consequently, these tools are utilized to investigate the effects of functional over-expression of proteins on insulin mediated events. However, we have previously reported that cationic lipid reagents cause a state of insulin unresponsiveness in cell cultures. In addition, we have found that cultured cells often do not respond to insulin stimulation following adenovirus treatment. Infection with adenovirus compromises vital functions of the host cell leading to the activation of protein kinases central to insulin signalling, such as protein kinase B/Akt. Therefore, we investigated the effect of adenovirus infection on insulin unresponsiveness by means of Akt activation in cultured cells. Moreover, we investigated the use of baculovirus as a heterologous viral gene delivery vehicle to circumvent these phenomena. Since the finding that baculovirus can efficiently transduce mammalian cells, the applications of this viral system in gene delivery has greatly expanded and one advantage is the virtual absence of cytotoxicity in mammalian cells. RESULTS: We show that infection of human neuroblastoma SHSY-5Y and liver C3A cells with recombinant adenovirus results in the activation of Akt in a dose dependent manner. In addition, this activation makes treated cells unresponsive to insulin stimulation as determined by an apparent lack of differential phosphorylation of Akt on serine-473. Our data further indicate that the use of recombinant baculovirus does not increase the phosphorylation of Akt in SHSY-5Y and C3A cells. Moreover, following infection with baculovirus, SHSY-5Y and C3A cells respond to insulin by means of phosphorylation of Akt on serine-473 in the same manner as uninfected cells. CONCLUSION: Widely-used adenovirus vectors for gene delivery cause a state of insulin unresponsiveness in human SHSY-5Y and C3A cells in culture due to the activation of central protein kinases of the insulin signalling pathway. This phenomenon can be avoided when studying insulin signalling by using recombinant baculovirus as a heterologous viral expression system. In addition, our data may contribute to an understanding of the molecular mechanisms underlying baculovirus infection of human cells. [Abstract/Link to Full Text]

Kaur H, Stiff AC, Date DA, Taylor WR
Analysis of mitotic phosphorylation of borealin.
BMC Cell Biol. 2007;85.
BACKGROUND: The main role of the chromosomal passenger complex is to ensure that Aurora B kinase is properly localized and activated before and during mitosis. Borealin, a member of the chromosomal passenger complex, shows increased expression during G2/M phases and is involved in targeting the complex to the centromere and the spindle midzone, where it ensures proper chromosome segregation and cytokinesis. Borealin has a consensus CDK1 phosphorylation site, threonine 106 and can be phosphorylated by Aurora B Kinase at serine 165 in vitro. RESULTS: Here, we show that Borealin is phosphorylated during mitosis in human cells. Dephosphorylation of Borealin occurs as cells exit mitosis. The phosphorylated form of Borealin is found in an INCENP-containing complex in mitosis. INCENP-containing complexes from cells in S phase are enriched in the phosphorylated form suggesting that phosphorylation may encourage entry of Borealin into the chromosomal passenger complex. Although Aurora B Kinase is found in complexes that contain Borealin, it is not required for the mitotic phosphorylation of Borealin. Mutation of T106 or S165 of Borealin to alanine does not alter the electrophoretic mobility shift of Borealin. Experiments with cyclohexamide and the phosphatase inhibitor sodium fluoride suggest that Borealin is phosphorylated by a protein kinase that can be active in interphase and mitosis and that the phosphorylation may be regulated by a short-lived phosphatase that is active in interphase but not mitosis. CONCLUSION: Borealin is phosphorylated during mitosis. Neither residue S165, T106 nor phosphorylation of Borealin by Aurora B Kinase is required to generate the mitotic, shifted form of Borealin. Suppression of phosphorylation during interphase is ensured by a labile protein, possibly a cell cycle regulated phosphatase. [Abstract/Link to Full Text]

Evans KE, Fox SW
Interleukin-10 inhibits osteoclastogenesis by reducing NFATc1 expression and preventing its translocation to the nucleus.
BMC Cell Biol. 2007;84.
BACKGROUND: IL-10 has a potent inhibitory effect on osteoclastogenesis. In vitro and in vivo studies confirm the importance of this cytokine in bone metabolism, for instance IL-10-deficient mice develop the hallmarks of osteoporosis. Although it is known that IL-10 directly inhibits osteoclastogenesis at an early stage, preventing differentiation of osteoclast progenitors to preosteoclasts, the precise mechanism of its action is not yet clear. Several major pathways regulate osteoclastogenesis, with key signalling genes such as p38, TRAF6, NF-kappaB and NFATc1 well established as playing vital roles. We have looked at gene expression in eleven of these genes using real-time quantitative PCR on RNA extracted from RANKL-treated RAW264.7 monocytes. RESULTS: There was no downregulation by IL-10 of DAP12, FcgammaRIIB, c-jun, RANK, TRAF6, p38, NF-kappaB, Gab2, Pim-1, or c-Fos at the mRNA level. However, we found that IL-10 significantly reduces RANKL-induced NFATc1 expression. NFATc1 is transcribed from two alternative promoters in Mus musculus and, interestingly, only the variant transcribed from promoter P1 and beginning with exon 1 was downregulated by IL-10 (isoform 1). In addition, immunofluorescence studies showed that IL-10 reduces NFATc1 levels in RANKL-treated precursors and suppresses nuclear translocation. The inhibitory effect of IL-10 on tartrate-resistant acid phosphatase-positive cell number and NFATc1 mRNA expression was reversed by the protein kinase C agonist phorbol myristate acetate, providing evidence that interleukin-10 disrupts NFATc1 activity through its effect on Ca2+ mobilisation. CONCLUSION: IL-10 acts directly on mononuclear precursors to inhibit NFATc1 expression and nuclear translocation, and we provide evidence that the mechanism may involve disruption of Ca2+ mobilisation. We detected downregulation only of the NFATc1 isoform 1 transcribed from promoter P1. This is the first report indicating that one of the ways in which IL-10 directly inhibits osteoclastogenesis is by suppressing NFATc1 activity. [Abstract/Link to Full Text]

George M, Ying G, Rainey MA, Solomon A, Parikh PT, Gao Q, Band V, Band H
Shared as well as distinct roles of EHD proteins revealed by biochemical and functional comparisons in mammalian cells and C. elegans.
BMC Cell Biol. 2007;83.
BACKGROUND: The four highly homologous human EHD proteins (EHD1-4) form a distinct subfamily of the Eps15 homology domain-containing protein family and are thought to regulate endocytic recycling. Certain members of this family have been studied in different cellular contexts; however, a lack of concurrent analyses of all four proteins has impeded an appreciation of their redundant versus distinct functions. RESULTS: Here, we analyzed the four EHD proteins both in mammalian cells and in a cross-species complementation assay using a C. elegans mutant lacking the EHD ortholog RME-1. We show that all human EHD proteins rescue the vacuolated intestinal phenotype of C. elegans rme-1 mutant, are simultaneously expressed in a panel of mammalian cell lines and tissues tested, and variably homo- and hetero-oligomerize and colocalize with each other and Rab11, a recycling endosome marker. Small interfering RNA (siRNA) knock-down of EHD1, 2 and 4, and expression of dominant-negative EH domain deletion mutants showed that loss of EHD1 and 3 (and to a lesser extent EHD4) but not EHD2 function retarded transferrin exit from the endocytic recycling compartment. EH domain deletion mutants of EHD1 and 3 but not 2 or 4, induced a striking perinuclear clustering of co-transfected Rab11. Knock-down analyses indicated that EHD1 and 2 regulate the exit of cargo from the recycling endosome while EHD4, similar to that reported for EHD3 (Naslavsky et al. (2006) Mol. Biol. Cell 17, 163), regulates transport from the early endosome to the recycling endosome. CONCLUSION: Altogether, our studies suggest that concurrently expressed human EHD proteins perform shared as well as discrete functions in the endocytic recycling pathway and lay a foundation for future studies to identify and characterize the molecular pathways involved. [Abstract/Link to Full Text]

Bathe FS, Rommelaere H, Machesky LM
Phenotypes of myopathy-related actin mutants in differentiated C2C12 myotubes.
BMC Cell Biol. 2007;82.
BACKGROUND: About 20 % of nemaline myopathies are thus far related to skeletal muscle alpha-actin. Seven actin mutants located in different parts of the actin molecule and linked to different forms of the disease were selected and expressed as EGFP-tagged constructs in differentiated C2C12 mytoubes. Results were compared with phenotypes in patient skeletal muscle fibres and with previous expression studies in fibroblasts and C2C12 myoblasts/myotubes. RESULTS: Whereas EGFP wt-actin nicely incorporated into endogenous stress fibres and sarcomeric structures, the mutants showed a range of phenotypes, which generally changed upon differentiation. Many mutants appeared delocalized in myoblasts but integrated into endogenous actin structures after 4-6 days of differentiation, demonstrating a poor correlation between the appearance in myotubes and the severity of the disease. However, for some mutants, integration into stress fibres induced aberrant structures in differentiated cells, like thickening or fragmentation of stress fibres. Other mutants almost failed to integrate but formed huge aggregates in the cytoplasm of myotubes. Those did not co-stain with alpha-actinin, a main component of nemaline bodies found in patient muscle. Interestingly, nuclear aggregates as formed by two of the mutants in myoblasts were found less frequently or not at all in differentiated cells. CONCLUSION: Myotubes are a suitable system to study the capacity of a mutant to incorporate into actin structures or to form or induce pathological changes. Some of the phenotypes observed in undifferentiated myoblasts may only be in vitro effects. Other phenotypes, like aberrant stress fibres or rod formation may be more directly correlated with disease phenotypes. Some mutants did not induce any changes in the cellular actin system, indicating the importance of additional studies like functional assays to fully characterize the pathological impact of a mutant. [Abstract/Link to Full Text]

---

Recent Articles in Cell Communication and Signaling

---

Girăo H, Catarino S, Pereira P
7-Ketocholesterol modulates intercellular communication through gap-junction in bovine lens epithelial cells.
Cell Commun Signal. 2004 Jun 1;2(1):2.
BACKGROUND: Connexin43 (Cx43) is an integral membrane protein that forms intercellular channels called gap junctions. Intercellular communication in the eye lens relies on an extensive network of gap junctions essential for the maintenance of lens transparency. The association of Cx43 with cholesterol enriched lipid raft domains was recently demonstrated. The objective of this study is to assess if products of cholesterol oxidation (oxysterols) affect gap junction intercellular communication (GJIC). RESULTS: Primary cultures of lens epithelial cells (LEC) were incubated with 7-ketocholesterol (7-Keto), 25-hydroxycholesterol (25-OH) or cholesterol and the subcellular distribution of Cx43 was evaluated by immunofluorescence confocal microscopy. The levels of Cx43 present in gap junction plaques were assessed by its insolubility in Triton X-100 and quantified by western blotting. The stability of Cx43 at the plasma membrane following incubation with oxysterols was evaluated by biotinylation of cell surface proteins. Gap junction intercellular communication was evaluated by transfer of the dye Lucifer yellow. The results obtained showed that 7-keto induces an accumulation of Cx43 at the plasma membrane and an increase in intercellular communication through gap junction. However, incubation with cholesterol or 25-OH did not lead to significant alterations on subcellular distribution of Cx43 nor in intercellular communication. Data further suggests that increased intercellular communication results from increased stability of Cx43 at the plasma membrane, presumably forming functional gap-junctions, as suggested by decreased solubility of Cx43 in 1% Triton X-100. The increased stability of Cx43 at the plasma membrane seems to be specific and not related to disruption of endocytic pathway, as demonstrated by dextran uptake. CONCLUSIONS: Results demonstrate, for the first time, that 7-keto induces an increase in gap junction intercellular communication, that is most likely due to an increased stability of protein at the plasma membrane and to increased abundance of Cx43 assembled in gap junction plaques. [Abstract/Link to Full Text]

Iyú D, Atucha NM, Martínez-Prieto C, Ortiz MC, García-Estań J
Altered calcium signaling in platelets from nitric oxide-deficient hypertensive rats.
Cell Commun Signal. 2004 May 10;2(1):1.
BACKGROUND: In the present study we have analyzed the mechanisms of calcium entry and mobilization in platelets obtained from rats chronically treated with the nitric oxide synthesis inhibitor, N-nitro L-arginine methyl ester [L-NAME, 40 mg/kg/day, 5 days). The platelets were obtained the day of the experiment, washed and loaded with fura-2. The intracellular calcium levels were determined in suspension of cells by means of fluorescence spectroscopy. RESULTS: Basal calcium levels were always elevated in the platelets of the L-NAME-treated rats, both in the presence and in the absence of extracellular calcium. The administration of thrombin in the absence and in the presence of extracellular calcium induced important elevations in calcium levels that were always of greater magnitude in the platelets of the L-NAME-treated rats than in those of the controls. The addition of calcium to thapsigargin-treated platelets produced a massive elevation in calcium levels in both groups that was significantly greater in the platelets obtained from the hypertensive rats than in those of the controls. CONCLUSIONS: It is concluded that the arterial hypertension induced by the reduction of nitric oxide alters the regulation of platelet calcium levels so that elevated baseline levels and calcium entry and mobilization are enhanced. This could be the result of direct or indirect effects of the lack of nitric oxide synthesis in platelets or in other tissues. [Abstract/Link to Full Text]

Lake AC, Castellot JJ
CCN5 modulates the antiproliferative effect of heparin and regulates cell motility in vascular smooth muscle cells.
Cell Commun Signal. 2003 Nov 24;1(1):5.
BACKGROUND: Vascular smooth muscle cell (VSMC) hyperplasia plays an important role in both chronic and acute vascular pathologies including atherosclerosis and restenosis. Considerable work has focused on the mechanisms regulating VSMC proliferation and motility. Earlier work in our lab revealed a novel growth arrest-specific (gas) gene induced in VSMC exposed to the antiproliferative agent heparin. This gene is a member of the CCN family and has been given the name CCN5. The objective of the present study is to elucidate the function of CCN5 protein and to explore its mechanism of action in VSMC. RESULTS: Using RNA interference (RNAi), we first demonstrate that CCN5 is required for the antiproliferative effect of heparin in VSMC. We also use this gene knockdown approach to show that CCN5 is an important negative regulator of motility. To explore the mechanism of action of CCN5 on VSMC motility, we use RNAi to demonstrate that knock down of CCN5 up regulates expression of matrix metalloproteinase-2 (MMP-2), an important stimulator of motility in VSMC. In addition, forced expression of CCN5 via adenovirus results in reduced MMP-2 activity, this also corroborates the gene knock down results. Finally, we show that loss of CCN5 expression in VSMC causes changes in VSMC morphology and cytoskeletal organization, including a reduction in the amount and macromolecular assembly of smooth muscle cell alpha-actin. CONCLUSIONS: This work provides important new insights into the regulation of smooth muscle cell proliferation and motility by CCN5 and may aid the development of therapies for vascular diseases. [Abstract/Link to Full Text]

Chrisman TD, Perkins DT, Garbers DL
Identification of a potent serum factor that causes desensitization of the receptor for C-Type natriuretic peptide.
Cell Commun Signal. 2003 Nov 19;1(1):4.
BACKGROUND: Guanylyl cyclase-B (GC-B; NPR-B), the receptor for C-type natriuretic peptide (CNP) is rapidly and effectively desensitized by a factor(s) in serum. Given the potential importance of this receptor in remodeling after tissue injury, identification of the serum factor(s) is of significant medical importance. RESULTS: Partial purification of desensitization activity in serum by DEAE-Sepharose and reverse phase C18 chromatography, followed by mass spectroscopy, identified peptide sequences identical to those of apolipoprotein A2 (Apo A2), a known component of high density lipoprotein (HDL). Apo A2, however, could be eliminated as the active desensitization factor. Never the less, substantial desensitization activity was associated with purified preparations of bovine or human HDL. Since HDL is a well-known transporter of various lipids and phospholipids, we extracted either HDL or partially purified serum preparations with butanol and all activity extracted into the solvent. Of various lipophilic signaling molecules known to be associated with HDL, a prominent component is sphingosine-1-phosphate (S1P). We therefore tested authentic S1P as well as other known components of HDL (sphingosylphosphorylcholine; platelet activating factor) for activity; only S1P caused desensitization of GC-B. S1P was relatively potent, causing one-half maximal desensitization of GC-B at concentrations of 5-10 nM. These effects were seen within a few minutes after addition. Lysophosphatidic acid, another component of serum capable of desensitizing GC-B, was only effective at Micromolar concentrations. The pathway by which serum or S1P desensitizes GC-B seems unique in that pertussis toxin failed to inhibit GC-B desensitization, and yet blocked serum or S1P activation of extracellular signal-regulated kinase (ERK) or Akt/protein kinase B (Akt/PKB). CONCLUSION: Since the concentrations of S1P that desensitize GC-B are well within serum physiological ranges, this mitogenic signaling molecule likely functions as a strong adversary of the CNP/GC-B signaling pathway in the regulation of cell proliferation and other growth factor-induced phenotypes. The mechanism by which S1P desensitizes GC-B appears different than the known S1P signaling pathways. [Abstract/Link to Full Text]

Perbal B
Communication is the key.
Cell Commun Signal. 2003 Oct 27;1(1):3.
All forms of communication between human beings have long been recognized as a requirement for reciprocal understanding, transfer of knowledge, and productive development of societies. This also applies to living cells who are organized in <microsocietiest; that constantly adjust to their environment through a complex network of signaling pathways. The chemical communication which occurs at various levels results in an integrated exchange of information that is essential for coordinated responses.We wish to present a few features of Cell Communication and Signaling: an open access, peer-reviewed journal devoted to the publication of manuscripts covering all aspects of cell communication, with a particular focus on molecular processes that govern intercellular signaling and events that sustain cellular communication, both in normal and pathological conditions.The launching of Cell Communication and Signaling provides us the opportunity to present a brief overview of basic processes underlying cell communication and the signaling processes that take place within and between cells to permit an efficient communication. [Abstract/Link to Full Text]

Lombet A, Planque N, Bleau AM, Li CL, Perbal B
CCN3 and calcium signaling.
Cell Commun Signal. 2003 Aug 15;1(1):1.
The CCN family of genes consists presently of six members in human (CCN1-6) also known as Cyr61 (Cystein rich 61), CTGF (Connective Tissue Growth Factor), NOV (Nephroblastoma Overexpressed gene), WISP-1, 2 and 3 (Wnt-1 Induced Secreted Proteins). Results obtained over the past decade have indicated that CCN proteins are matricellular proteins, which are involved in the regulation of various cellular functions, such as proliferation, differentiation, survival, adhesion and migration. The CCN proteins have recently emerged as regulatory factors involved in both internal and external cell signaling. CCN3 was reported to physically interact with fibulin-1C, integrins, Notch and S100A4. Considering that, the conformation and biological activity of these proteins are dependent upon calcium binding, we hypothesized that CCN3 might be involved in signaling pathways mediated by calcium ions.In this article, we review the data showing that CCN3 regulates the levels of intracellular calcium and discuss potential models that may account for the biological effects of CCN3. [Abstract/Link to Full Text]

Yeger H
Building a Solid Foundation: CCS in Developing Skeleton and the CCN Family Role.
Cell Commun Signal. 2003 Oct 2;1(1):2. [Abstract/Link to Full Text]

---

Recent Articles in Eukaryotic Cell

---

Meléndez-López SG, Herdman S, Hirata K, Choi MH, Choe Y, Craik C, Caffrey CR, Hansell E, Chávez-Munguía B, Chen YT, Roush WR, McKerrow J, Eckmann L, Guo J, Stanley SL, Reed SL
Use of recombinant Entamoeba histolytica cysteine proteinase 1 to identify a potent inhibitor of amebic invasion in a human colonic model.
Eukaryot Cell. 2007 Jul;6(7):1130-6.
Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis. [Abstract/Link to Full Text]

Nikko E, André B
Split-ubiquitin two-hybrid assay to analyze protein-protein interactions at the endosome: application to Saccharomyces cerevisiae Bro1 interacting with ESCRT complexes, the Doa4 ubiquitin hydrolase, and the Rsp5 ubiquitin ligase.
Eukaryot Cell. 2007 Aug;6(8):1266-77.
Targeting of membrane proteins into the lysosomal/vacuolar lumen for degradation requires their prior sorting into multivesicular bodies (MVB). The MVB sorting pathway depends on ESCRT-0, -I, -II, and -III protein complexes functioning on the endosomal membrane and on additional factors, such as Bro1/Alix and the ubiquitin ligase Rsp5/Nedd4. We used the split-ubiquitin two-hybrid assay to analyze the interaction partners of yeast Bro1 at its natural cellular location. We show that Bro1 interacts with ESCRT-I and -III components, including Vps23, the Saccharomyces cerevisiae homologue of human Tsg101. These interactions do not require the C-terminal proline-rich domain (PRD) of Bro1. Rather, this PRD interacts with the Doa4 deubiquitinating enzyme to recruit it to the endosome. This interaction is disrupted by a single amino acid substitution in the conserved ELC box motif in Doa4. The PRD of Bro1 also mediates an association with Rsp5, and this interaction appears to be conserved, as Alix, the human homologue of Bro1, coimmunoprecipitates with Nedd4 in yeast lysates. We further show that the Bro1 PRD domain is essential to MVB sorting of only cargo proteins whose sorting to the vacuolar lumen is dependent on their own ubiquitination and Doa4. The Bro1 region preceding the PRD, however, is required for MVB sorting of proteins irrespective of whether their targeting to the vacuole is dependent on their ubiquitination and Doa4. Our data indicate that Bro1 interacts with several ESCRT components and contributes via its PRD to associating ubiquitinating and deubiquitinating enzymes with the MVB sorting machinery. [Abstract/Link to Full Text]

Rasmussen CG, Glass NL
Localization of RHO-4 indicates differential regulation of conidial versus vegetative septation in the filamentous fungus Neurospora crassa.
Eukaryot Cell. 2007 Jul;6(7):1097-107.
Rho-4 mutants of the filamentous fungus Neurospora crassa lack septa and asexual spores (conidia) and grow slowly. In this report, localization of green fluorescent protein-tagged RHO-4 is used to elucidate the differences in factors controlling RHO-4 localization during vegetative growth versus asexual development. RHO-4 forms a ring at incipient vegetative septation sites that constricts with the formation of the septum toward the septal pore; RHO-4 persists around the septal pore after septum completion. During the formation of conidia, RHO-4 localizes to the primary septum but subsequently is relocalized to the cytoplasm after the placement of the secondary septum. Cytoplasmic localization and inactivation of RHO-4 are mediated by a direct physical interaction with RDI-1, a RHO guanosine nucleotide dissociation inhibitor. Inappropriate activation of the cyclic AMP-dependent protein kinase A pathway during vegetative growth causes mislocalization of RHO-4 away from septa to the cytoplasm, a process which was dependent upon RDI-1. An adenylate cyclase cr-1 mutant partially suppresses the aconidial defect of rho-4 mutants but only rarely suppresses the vegetative septation defect, indicating that conidial septation is negatively regulated by CR-1. These data highlight the differences in the regulation of septation during conidiation versus vegetative septation in filamentous fungi. [Abstract/Link to Full Text]

Maxwell PH, Curcio MJ
Host factors that control long terminal repeat retrotransposons in Saccharomyces cerevisiae: implications for regulation of mammalian retroviruses.
Eukaryot Cell. 2007 Jul;6(7):1069-80. [Abstract/Link to Full Text]

van der Kaaij RM, Yuan XL, Franken A, Ram AF, Punt PJ, van der Maarel MJ, Dijkhuizen L
Two novel, putatively cell wall-associated and glycosylphosphatidylinositol-anchored alpha-glucanotransferase enzymes of Aspergillus niger.
Eukaryot Cell. 2007 Jul;6(7):1178-88.
In the genome sequence of Aspergillus niger CBS 513.88, three genes were identified with high similarity to fungal alpha-amylases. The protein sequences derived from these genes were different in two ways from all described fungal alpha-amylases: they were predicted to be glycosylphosphatidylinositol anchored, and some highly conserved amino acids of enzymes in the alpha-amylase family were absent. We expressed two of these enzymes in a suitable A. niger strain and characterized the purified proteins. Both enzymes showed transglycosylation activity on donor substrates with alpha-(1,4)-glycosidic bonds and at least five anhydroglucose units. The enzymes, designated AgtA and AgtB, produced new alpha-(1,4)-glycosidic bonds and therefore belong to the group of the 4-alpha-glucanotransferases (EC 2.4.1.25). Their reaction products reached a degree of polymerization of at least 30. Maltose and larger maltooligosaccharides were the most efficient acceptor substrates, although AgtA also used small nigerooligosaccharides containing alpha-(1,3)-glycosidic bonds as acceptor substrate. An agtA knockout of A. niger showed an increased susceptibility towards the cell wall-disrupting compound calcofluor white, indicating a cell wall integrity defect in this strain. Homologues of AgtA and AgtB are present in other fungal species with alpha-glucans in their cell walls, but not in yeast species lacking cell wall alpha-glucan. Possible roles for these enzymes in the synthesis and/or maintenance of the fungal cell wall are discussed. [Abstract/Link to Full Text]

Krajaejun T, Gauthier GM, Rappleye CA, Sullivan TD, Klein BS
Development and application of a green fluorescent protein sentinel system for identification of RNA interference in Blastomyces dermatitidis illuminates the role of septin in morphogenesis and sporulation.
Eukaryot Cell. 2007 Aug;6(8):1299-309.
A high-throughput strategy for testing gene function would accelerate progress in our understanding of disease pathogenesis for the dimorphic fungus Blastomyces dermatitidis, whose genome is being completed. We developed a green fluorescent protein (GFP) sentinel system of gene silencing to rapidly study genes of unknown function. Using Gateway technology to efficiently generate RNA interference plasmids, we cloned a target gene, "X," next to GFP to create one hairpin to knock down the expression of both genes so that diminished GFP reports target gene expression. To test this approach in B. dermatitidis, we first used LACZ and the virulence gene BAD1 as targets. The level of GFP reliably reported interference of their expression, leading to rapid detection of gene-silenced transformants. We next investigated a previously unstudied gene encoding septin and explored its possible role in morphogenesis and sporulation. A CDC11 septin homolog in B. dermatitidis localized to the neck of budding yeast cells. CDC11-silenced transformants identified with the sentinel system grew slowly as flat or rough colonies on agar. Microscopically, they formed ballooned, distorted yeast cells that failed to bud, and they sporulated poorly as mold. Hence, this GFP sentinel system enables rapid detection of gene silencing and has revealed a pronounced role for septin in morphogenesis, budding, and sporulation of B. dermatitidis. [Abstract/Link to Full Text]

Liang J, Fantes P
The Schizosaccharomyces pombe Cdc7 protein kinase required for septum formation is a client protein of Cdc37.
Eukaryot Cell. 2007 Jul;6(7):1089-96.
Cdc37 is an essential molecular chaperone found in fungi and metazoa whose main specificity is for certain protein kinases. Cdc37 can act as an Hsp90 cochaperone or alone; in yeasts, the interaction with Hsp90 is weak and appears not to be essential for Cdc37 function. Numerous genetic interactions between Cdc37 and likely client proteins have been observed in yeasts, but biochemical confirmation has been reported in only a few cases. We and others have generated and characterized temperature-sensitive cdc37 alleles in S. pombe and have used them to investigate the cellular roles of Cdc37: previous work has shown that mitotic Cdc2 is a major client. In this paper, we describe a screen for mutations synthetically lethal with a cdc37ts mutant with the aim of identifying genes encoding further client proteins of Cdc37. Ten such strains were isolated, and genomic libraries were screened for rescuing plasmids. In one case, a truncated cdc7 gene was identified. Further experiments showed that the mutation in this strain was indeed in cdc7. Cdc7 is a protein kinase required for septum initiation, and we show that its kinase activity is greatly reduced when Cdc37 function is impaired. Cdc7 normally locates to the spindle pole body during mitosis, and this appears to be unaffected in the cdc37ts mutant. Other evidence suggests that, in addition to mitosis and septum initiation, Cdc37 may also be required for septum cleavage. [Abstract/Link to Full Text]

Brown DW, Butchko RA, Busman M, Proctor RH
The Fusarium verticillioides FUM gene cluster encodes a Zn(II)2Cys6 protein that affects FUM gene expression and fumonisin production.
Eukaryot Cell. 2007 Jul;6(7):1210-8.
Fumonisins are mycotoxins produced by some Fusarium species and can contaminate maize or maize products. Ingestion of fumonisins is associated with diseases, including cancer and neural tube defects, in humans and animals. In fungi, genes involved in the synthesis of mycotoxins and other secondary metabolites are often located adjacent to each other in gene clusters. Such genes can encode structural enzymes, regulatory proteins, and/or proteins that provide self-protection. The fumonisin biosynthetic gene cluster includes 16 genes, none of which appear to play a role in regulation. In this study, we identified a previously undescribed gene (FUM21) located adjacent to the fumonisin polyketide synthase gene, FUM1. The presence of a Zn(II)2Cys6 DNA-binding domain in the predicted protein suggested that FUM21 was involved in transcriptional regulation. FUM21 deletion (Deltafum21) mutants produce little to no fumonisin in cracked maize cultures but some FUM1 and FUM8 transcripts in a liquid GYAM medium. Complementation of a Deltafum21 mutant with a wild-type copy of the gene restored fumonisin production. Analysis of FUM21 cDNAs identified four alternative splice forms (ASFs), and microarray analysis indicated the ASFs were differentially expressed. Based on these data, we present a model for how FUM21 ASFs may regulate fumonisin biosynthesis. [Abstract/Link to Full Text]

Judelson HS, Tani S
Transgene-induced silencing of the zoosporogenesis-specific NIFC gene cluster of Phytophthora infestans involves chromatin alterations.
Eukaryot Cell. 2007 Jul;6(7):1200-9.
Clustered within the genome of the oomycete phytopathogen Phytophthora infestans are four genes encoding spore-specific nuclear LIM interactor-interacting factors (NIF proteins, a type of transcriptional regulator) that are moderately conserved in DNA sequence. NIFC1, NIFC2, and NIFC3 are zoosporogenesis-induced and grouped within 4 kb, and 20 kb away resides a sporulation-induced form, NIFS. To test the function of the NIFC family, plasmids expressing full-length hairpin constructs of NIFC1 or NIFC2 were stably transformed into P. infestans. This triggered silencing of the cognate gene in about one-third of transformants, and all three NIFC genes were usually cosilenced. However, NIFS escaped silencing despite its high sequence similarity to the NIFC genes. Silencing of the three NIFC genes impaired zoospore cyst germination by 60% but did not affect other aspects of the life cycle. Silencing was transcriptional based on nuclear run-on assays and associated with tighter chromatin packing based on nuclease accessibility experiments. The chromatin alterations extended a few hundred nucleotides beyond the boundaries of the transcribed region of the NIFC cluster and were not associated with increased DNA methylation. A plasmid expressing a short hairpin RNA having sequence similarity only to NIFC1 silenced both that gene and an adjacent member of the gene cluster, likely due to the expansion of a heterochromatic domain from the targeted locus. These data help illuminate the mechanism of silencing in Phytophthora and suggest that caution should be used when interpreting silencing experiments involving closely spaced genes. [Abstract/Link to Full Text]

Rodgers MJ, Albanesi JP, Phillips MA
Phosphatidylinositol 4-kinase III-beta is required for Golgi maintenance and cytokinesis in Trypanosoma brucei.
Eukaryot Cell. 2007 Jul;6(7):1108-18.
The parasitic protozoan Trypanosoma brucei contains two type III phosphatidylinositol 4-kinases (alpha and beta). We have cloned the gene encoding the T. brucei type III phosphatidylinositol 4-kinase beta (TbPI4KIII-beta), expressed the protein in COS-7 cells, and confirmed that the protein catalyzes the phosphorylation of phosphatidylinositol. Depletion of TbPI4KIII-beta in procyclic T. brucei by RNA interference (RNAi) resulted in inhibition of cell growth and a distorted cellular morphology. RNAi cells had a distorted Golgi apparatus, and lysosomal and flagellar pocket proteins were mislocalized. Ultrastructural analysis revealed the internal accumulation of a heterogeneous population of vesicles, abnormal positioning of organelles, and a loss of cell polarity. Scanning electron microcopy revealed a twisted phenotype, and dividing cells often exhibited a detached daughter flagellum and lacked a cleavage furrow. Cell cycle analysis confirmed that cells depleted of TbPI4KIII-beta have a postmitotic cytokinesis block that occurs after a single round of mitosis, suggestive of a specific cell cycle block. In summary, TbPI4KIII-beta is an essential protein in procyclic T. brucei, required for maintenance of Golgi structure, protein trafficking, normal cellular shape, and cytokinesis. [Abstract/Link to Full Text]

McBride AE, Zurita-Lopez C, Regis A, Blum E, Conboy A, Elf S, Clarke S
Protein arginine methylation in Candida albicans: role in nuclear transport.
Eukaryot Cell. 2007 Jul;6(7):1119-29.
Protein arginine methylation plays a key role in numerous eukaryotic processes, such as protein transport and signal transduction. In Candida albicans, two candidate protein arginine methyltransferases (PRMTs) have been identified from the genome sequencing project. Based on sequence comparison, C. albicans candidate PRMTs display similarity to Saccharomyces cerevisiae Hmt1 and Rmt2. Here we demonstrate functional homology of Hmt1 between C. albicans and S. cerevisiae: CaHmt1 supports growth of S. cerevisiae strains that require Hmt1, and CaHmt1 methylates Npl3, a major Hmt1 substrate, in S. cerevisiae. In C. albicans strains lacking CaHmt1, asymmetric dimethylarginine and omega-monomethylarginine levels are significantly decreased, indicating that Hmt1 is the major C. albicans type I PRMT1. Given the known effects of type I PRMTs on nuclear transport of RNA-binding proteins, we tested whether Hmt1 affects nuclear transport of a putative Npl3 ortholog in C. albicans. CaNpl3 allows partial growth of S. cerevisiae npl3Delta strains, but its arginine-glycine-rich C terminus can fully substitute for that of ScNpl3 and also directs methylation-sensitive association with ScNpl3. Expression of green fluorescent protein-tagged CaNpl3 proteins in C. albicans strains with and without CaHmt1 provides evidence for CaHmt1 facilitating export of CaNpl3 in this fungus. We have also identified the C. albicans Rmt2, a type IV fungus- and plant-specific PRMT, by amino acid analysis of an rmt2Delta/rmt2Delta strain, as well as biochemical evidence for additional cryptic PRMTs. [Abstract/Link to Full Text]

Davis CA, Brown MP, Singh U
Functional characterization of spliceosomal introns and identification of U2, U4, and U5 snRNAs in the deep-branching eukaryote Entamoeba histolytica.
Eukaryot Cell. 2007 Jun;6(6):940-8.
Pre-mRNA splicing is essential to ensure accurate expression of many genes in eukaryotic organisms. In Entamoeba histolytica, a deep-branching eukaryote, approximately 30% of the annotated genes are predicted to contain introns; however, the accuracy of these predictions has not been tested. In this study, we mined an expressed sequence tag (EST) library representing 7% of amoebic genes and found evidence supporting splicing of 60% of the testable intron predictions, the majority of which contain a GUUUGU 5' splice site and a UAG 3' splice site. Additionally, we identified several splice site misannotations, evidence for the existence of 30 novel introns in previously annotated genes, and identified novel genes through uncovering their spliced ESTs. Finally, we provided molecular evidence for the E. histolytica U2, U4, and U5 snRNAs. These data lay the foundation for further dissection of the role of RNA processing in E. histolytica gene expression. [Abstract/Link to Full Text]

Bachand F
Protein arginine methyltransferases: from unicellular eukaryotes to humans.
Eukaryot Cell. 2007 Jun;6(6):889-98. [Abstract/Link to Full Text]

Georg RC, Gomes SL
Transcriptome analysis in response to heat shock and cadmium in the aquatic fungus Blastocladiella emersonii.
Eukaryot Cell. 2007 Jun;6(6):1053-62.
The global transcriptional response of the chytridiomycete Blastocladiella emersonii to environmental stress conditions was explored by sequencing a large number of expressed sequence tags (ESTs) from three distinct cDNA libraries, constructed with mRNA extracted from cells exposed to heat shock and different concentrations of cadmium chloride. A total of 6,350 high-quality EST sequences were obtained and assembled into 2,326 putative unigenes, 51% of them not previously described in B. emersonii. To approximately 59% of the unigenes it was possible to assign an orthologue in another organism, whereas 41% of them remained without a putative identification, with transcripts related to protein folding and antioxidant activity being highly enriched in the stress libraries. A microarray chip was constructed encompassing 3,773 distinct ESTs from the B. emersonii transcriptome presently available, which correspond to a wide range of biological processes. Global gene expression analysis of B. emersonii cells exposed to stress conditions revealed a large number of differentially expressed genes: 122 up- and 60 downregulated genes during heat shock and 189 up- and 110 downregulated genes during exposure to cadmium. The main functional categories represented among the upregulated genes were protein folding and proteolysis, proteins with antioxidant properties, and cellular transport. Interestingly, in response to cadmium stress, B. emersonii cells induced genes encoding six different glutathione S-transferases and six distinct metacaspases, as well as genes coding for several proteins of sulfur amino acid metabolism, indicating that cadmium causes oxidative stress and apoptosis in this fungus. All sequences described in this study have been submitted to the GenBank EST section with the accession numbers EE 730389 to EE 736848. [Abstract/Link to Full Text]

Nielsen K, De Obaldia AL, Heitman J
Cryptococcus neoformans mates on pigeon guano: implications for the realized ecological niche and globalization.
Eukaryot Cell. 2007 Jun;6(6):949-59.
The ecological niche that a species can occupy is determined by its resource requirements and the physical conditions necessary for survival. The niche to which an organism is most highly adapted is the realized niche, whereas the complete range of habitats that an organism can occupy represents the fundamental niche. The growth and development of Cryptococcus neoformans and Cryptococcus gattii on pigeon guano were examined to determine whether these two species occupy the same or different ecological niches. C. neoformans is a cosmopolitan pathogenic yeast that infects predominantly immunocompromised individuals, exists in two varieties (grubii [serotype A] and neoformans [serotype D]), and is commonly isolated from pigeon guano worldwide. By contrast, C. gattii often infects immunocompetent individuals and is associated with geographically restricted environments, most notably, eucalyptus trees. Pigeon guano supported the growth of both species, and a brown pigment related to melanin, a key virulence factor, was produced. C. neoformans exhibited prolific mating on pigeon guano, whereas C. gattii did not. The observations that C. neoformans completes the life cycle on pigeon guano but that C. gattii does not indicates that pigeon guano could represent the realized ecological niche for C. neoformans. Because C. gattii grows on pigeon guano but cannot sexually reproduce, pigeon guano represents a fundamental but not a realized niche for C. gattii. Based on these studies, we hypothesize that an ancestral Cryptococcus strain gained the ability to sexually reproduce in pigeon guano and then swept the globe. [Abstract/Link to Full Text]

Cui L, Miao J, Furuya T, Li X, Su XZ, Cui L
PfGCN5-mediated histone H3 acetylation plays a key role in gene expression in Plasmodium falciparum.
Eukaryot Cell. 2007 Jul;6(7):1219-27.
Histone acetylation, regulated by the opposing actions of histone acetyltransferases (HATs) and deacetylases, is an important epigenetic mechanism in eukaryotic transcription. Although an acetyltransferase (PfGCN5) has been shown to preferentially acetylate histone H3 at K9 and K14 in Plasmodium falciparum, the scale of histone acetylation in the parasite genome and its role in transcriptional activation are essentially unknown. Using chromatin immunoprecipitation (ChIP) and DNA microarray, we mapped the global distribution of PfGCN5, histone H3K9 acetylation (H3K9ac) and trimethylation (H3K9m3) in the P. falciparum genome. While the chromosomal distributions of H3K9ac and PfGCN5 were similar, they are radically different from that of H3K9m3. In addition, there was a positive, though weak correlation between relative occupancy of H3K9ac on individual genes and the levels of gene expression, which was inversely proportional to the distance of array elements from the putative translational start codons. In contrast, H3K9m3 was negatively correlated with gene expression. Furthermore, detailed mapping of H3K9ac for selected genes using ChIP and real-time PCR in three erythrocytic stages detected stage-specific peak H3K9ac enrichment at the putative transcriptional initiation sites, corresponding to stage-specific expression of these genes. These data are consistent with H3K9ac and H3K9m3 as epigenetic markers of active and silent genes, respectively. We also showed that treatment with a PfGCN5 inhibitor led to reduced promoter H3K9ac and gene expression. Collectively, these results suggest that PfGCN5 is recruited to the promoter regions of genes to mediate histone acetylation and activate gene expression in P. falciparum. [Abstract/Link to Full Text]

Tian C, Kasuga T, Sachs MS, Glass NL
Transcriptional profiling of cross pathway control in Neurospora crassa and comparative analysis of the Gcn4 and CPC1 regulons.
Eukaryot Cell. 2007 Jun;6(6):1018-29.
Identifying and characterizing transcriptional regulatory networks is important for guiding experimental tests on gene function. The characterization of regulatory networks allows comparisons among both closely and distantly related species, providing insight into network evolution, which is predicted to correlate with the adaptation of different species to particular environmental niches. One of the most intensely studied regulatory factors in the yeast Saccharomyces cerevisiae is the bZIP transcription factor Gcn4p. Gcn4p is essential for a global transcriptional response when S. cerevisiae experiences amino acid starvation. In the filamentous ascomycete Neurospora crassa, the ortholog of GCN4 is called the cross pathway control-1 (cpc-1) gene; it is required for the ability of N. crassa to induce a number of amino acid biosynthetic genes in response to amino acid starvation. Here, we deciphered the CPC1 regulon by profiling transcription in wild-type and cpc-1 mutant strains with full-genome N. crassa 70-mer oligonucleotide microarrays. We observed that at least 443 genes were direct or indirect CPC1 targets; these included 67 amino acid biosynthetic genes, 16 tRNA synthetase genes, and 13 vitamin-related genes. Comparison among the N. crassa CPC1 transcriptional profiling data set and the Gcn4/CaGcn4 data sets from S. cerevisiae and Candida albicans revealed a conserved regulon of 32 genes, 10 of which are predicted to be directly regulated by Gcn4p/CPC1. The 32-gene conserved regulon comprises mostly amino acid biosynthetic genes. The comparison of regulatory networks in species with clear orthology among genes sheds light on how gene interaction networks evolve. [Abstract/Link to Full Text]

Fleige T, Fischer K, Ferguson DJ, Gross U, Bohne W
Carbohydrate metabolism in the Toxoplasma gondii apicoplast: localization of three glycolytic isoenzymes, the single pyruvate dehydrogenase complex, and a plastid phosphate translocator.
Eukaryot Cell. 2007 Jun;6(6):984-96.
Many apicomplexan parasites, such as Toxoplasma gondii and Plasmodium species, possess a nonphotosynthetic plastid, referred to as the apicoplast, which is essential for the parasites' viability and displays characteristics similar to those of nongreen plastids in plants. In this study, we localized several key enzymes of the carbohydrate metabolism of T. gondii to either the apicoplast or the cytosol by engineering parasites which express epitope-tagged fusion proteins. The cytosol contains a complete set of enzymes for glycolysis, which should enable the parasite to metabolize imported glucose into pyruvate. All the glycolytic enzymes, from phosphofructokinase up to pyruvate kinase, are present in the T. gondii genome, as duplicates and isoforms of triose phosphate isomerase, phosphoglycerate kinase, and pyruvate kinase were found to localize to the apicoplast. The mRNA expression levels of all genes with glycolytic products were compared between tachyzoites and bradyzoites; however, a strict bradyzoite-specific expression pattern was observed only for enolase I. The T. gondii genome encodes a single pyruvate dehydrogenase complex, which was located in the apicoplast and absent in the mitochondrion, as shown by targeting of epitope-tagged fusion proteins and by immunolocalization of the native pyruvate dehydrogenase complex. The exchange of metabolites between the cytosol and the apicoplast is likely to be mediated by a phosphate translocator which was localized to the apicoplast. Based on these localization studies, a model is proposed that explains the supply of the apicoplast with ATP and the reduction power, as well as the exchange of metabolites between the cytosol and the apicoplast. [Abstract/Link to Full Text]

Ohtaka A, Okuzaki D, Saito TT, Nojima H
Mcp4, a meiotic coiled-coil protein, plays a role in F-actin positioning during Schizosaccharomyces pombe meiosis.
Eukaryot Cell. 2007 Jun;6(6):971-83.
Some meiosis-specific proteins of Schizosaccharomyces pombe harbor coiled-coil motifs and play essential roles in meiotic progression. Here we describe Mcp4, a novel meiosis-specific protein whose expression is abruptly induced at the horsetail phase and which remains expressed until sporulation is finished. Fluorescence microscopic analysis revealed that Mcp4 alters its subcellular localization during meiosis in a manner that partially resembles the movement of F-actin during meiosis. Mcp4 and F-actin never colocalize; rather, they are located in a side-by-side manner. When forespore membrane formation begins at metaphase II, the Mcp4 signals assemble at the lagging face of the dividing nuclei. At this stage, they are sandwiched between F-actin and the nucleus. Mcp4, in turn, appears to sandwich F-actin with Meu14. In mcp4Delta cells at anaphase II, the F-actin, which is normally dumbbell-shaped, adopts an abnormal balloon shape. Spores of mcp4Delta cells were sensitive to NaCl, although their shape and viability were normal. Taken together, we conclude that Mcp4 plays a role in the accurate positioning of F-actin during S. pombe meiosis. [Abstract/Link to Full Text]

Shimada N, Kawata T
Evidence that noncoding RNA dutA is a multicopy suppressor of Dictyostelium discoideum STAT protein Dd-STATa.
Eukaryot Cell. 2007 Jun;6(6):1030-40.
Dd-STATa, a Dictyostelium discoideum homologue of metazoan STAT transcription factors, is necessary for culmination. We created a mutant strain with partial Dd-STATa activity and used it to screen for unlinked suppressor genes. We screened approximately 450,000 clones from a slug-stage cDNA library for their ability to rescue the culmination defect when overexpressed. There were 12 multicopy suppressors of Dd-STATa, of which 4 encoded segments of a known noncoding RNA, dutA. Expression of dutA is specific to the pstA zone, the region where Dd-STATa is activated. In suppressed strains the expression patterns of several putative Dd-STATa target genes become similar to the wild-type strain. In addition, the amount of the tyrosine-phosphorylated form of Dd-STATa is significantly increased in the suppressed strain. These results indicate that partial copies of dutA may act upstream of Dd-STATa to regulate tyrosine phosphorylation by an unknown mechanism. [Abstract/Link to Full Text]

Ledford HK, Chin BL, Niyogi KK
Acclimation to singlet oxygen stress in Chlamydomonas reinhardtii.
Eukaryot Cell. 2007 Jun;6(6):919-30.
In an aerobic environment, responding to oxidative cues is critical for physiological adaptation (acclimation) to changing environmental conditions. The unicellular alga Chlamydomonas reinhardtii was tested for the ability to acclimate to specific forms of oxidative stress. Acclimation was defined as the ability of a sublethal pretreatment with a reactive oxygen species to activate defense responses that subsequently enhance survival of that stress. C. reinhardtii exhibited a strong acclimation response to rose bengal, a photosensitizing dye that produces singlet oxygen. This acclimation was dependent upon photosensitization and occurred only when pretreatment was administered in the light. Shifting cells from low light to high light also enhanced resistance to singlet oxygen, suggesting an overlap in high-light and singlet oxygen response pathways. Microarray analysis of RNA levels indicated that a relatively small number of genes respond to sublethal levels of singlet oxygen. Constitutive overexpression of either of two such genes, a glutathione peroxidase gene and a glutathione S-transferase gene, was sufficient to enhance singlet oxygen resistance. Escherichia coli and Saccharomyces cerevisiae exhibit well-defined responses to reactive oxygen but did not acclimate to singlet oxygen, possibly reflecting the relative importance of singlet oxygen stress for photosynthetic organisms. [Abstract/Link to Full Text]

Xu X, Müller-Taubenberger A, Adley KE, Pawolleck N, Lee VW, Wiedemann C, Sihra TS, Maniak M, Jin T, Williams RS
Attenuation of phospholipid signaling provides a novel mechanism for the action of valproic acid.
Eukaryot Cell. 2007 Jun;6(6):899-906.
Valproic acid (VPA) is used to treat epilepsy and bipolar disorder and to prevent migraine. It is also undergoing trials for cancer therapy. However, the biochemical and molecular biological actions of VPA are poorly understood. Using the social amoeba Dictyostelium discoideum, we show that an acute effect of VPA is the inhibition of chemotactic cell movement, a process partially dependent upon phospholipid signaling. Analysis of this process shows that VPA attenuates the signal-induced translocation of PH(Crac)-green fluorescent protein from cytosol to membrane, suggesting the inhibition of phosphatidylinositol-(3,4,5)-trisphosphate (PIP(3)) production. Direct labeling of lipids in vivo also shows a reduction in PIP and PIP(2) phosphorylation following VPA treatment. We further show that VPA acutely reduces endocytosis and exocytosis-processes previously shown to be dependent upon PIP(3) production. These results suggest that in Dictyostelium, VPA rapidly attenuates phospholipid signaling to reduce endocytic trafficking. To examine this effect in a mammalian model, we also tested depolarization-dependent neurotransmitter release in rat nerve terminals, and we show that this process is also suppressed upon application of VPA and an inhibitor of phosphatidylinositol 3-kinase. Although a more comprehensive analysis of the effect of VPA on lipid signaling will be necessary in mammalian systems, these results suggest that VPA may function to reduce phospholipid signaling processes and thus may provide a novel therapeutic effect for this drug. [Abstract/Link to Full Text]

Haarer BK, Helfant AH, Nelson SA, Cooper JA, Amberg DC
Stable preanaphase spindle positioning requires Bud6p and an apparent interaction between the spindle pole bodies and the neck.
Eukaryot Cell. 2007 May;6(5):797-807.
Faithful partitioning of genetic material during cell division requires accurate spatial and temporal positioning of nuclei within dividing cells. In Saccharomyces cerevisiae, nuclear positioning is regulated by an elegant interplay between components of the actin and microtubule cytoskeletons. Regulators of this process include Bud6p (also referred to as the actin-interacting protein Aip3p) and Kar9p, which function to promote contacts between cytoplasmic microtubule ends and actin-delimited cortical attachment points. Here, we present the previously undetected association of Bud6p with the cytoplasmic face of yeast spindle pole bodies, the functional equivalent of metazoan centrosomes. Cells lacking Bud6p show exaggerated movements of the nucleus between mother and daughter cells and display reduced amounts of time a given spindle pole body spends in close association with the neck region of budding cells. Furthermore, overexpression of BUD6 greatly enhances interactions between the spindle pole body and mother-bud neck in a spindle alignment-defective dynactin mutant. These results suggest that association of either spindle pole body with neck components, rather than simply entry of a spindle pole body into the daughter cell, provides a positive signal for the progression of mitosis. We propose that Bud6p, through its localization at both spindle pole bodies and at the mother-bud neck, supports this positive signal and provides a regulatory mechanism to prevent excessive oscillations of preanaphase nuclei, thus reducing the likelihood of mitotic delays and nuclear missegregation. [Abstract/Link to Full Text]

Odds FC, Bougnoux ME, Shaw DJ, Bain JM, Davidson AD, Diogo D, Jacobsen MD, Lecomte M, Li SY, Tavanti A, Maiden MC, Gow NA, d'Enfert C
Molecular phylogenetics of Candida albicans.
Eukaryot Cell. 2007 Jun;6(6):1041-52.
We analyzed data on multilocus sequence typing (MLST), ABC typing, mating type-like locus (MAT) status, and antifungal susceptibility for a panel of 1,391 Candida albicans isolates. Almost all (96.7%) of the isolates could be assigned by MLST to one of 17 clades. eBURST analysis revealed 53 clonal clusters. Diploid sequence type 69 was the most common MLST strain type and the founder of the largest clonal cluster, and examples were found among isolates from all parts of the world. ABC types and geographical origins showed statistically significant variations among clades by univariate analysis of variance, but anatomical source and antifungal susceptibility data were not significantly associated. A separate analysis limited to European isolates, thereby minimizing geographical effects, showed significant differences in the proportions of isolates from blood, commensal carriage, and superficial infections among the five most populous clades. The proportion of isolates with low antifungal susceptibility was highest for MAT homozygous a/a types and then alpha/alpha types and was lowest for heterozygous a/alpha types. The tree of clades defined by MLST was not congruent with trees generated from the individual gene fragments sequenced, implying a separate evolutionary history for each fragment. Analysis of nucleic acid variation among loci and within loci supported recombination. Computational haplotype analysis showed a high frequency of recombination events, suggesting that isolates had mixed evolutionary histories resembling those of a sexually reproducing species. [Abstract/Link to Full Text]

Li F, Svarovsky MJ, Karlsson AJ, Wagner JP, Marchillo K, Oshel P, Andes D, Palecek SP
Eap1p, an adhesin that mediates Candida albicans biofilm formation in vitro and in vivo.
Eukaryot Cell. 2007 Jun;6(6):931-9.
Candida albicans is the leading cause of systemic fungal infections in immunocompromised humans. The ability to form biofilms on surfaces in the host or on implanted medical devices enhances C. albicans virulence, leading to antimicrobial resistance and providing a reservoir for infection. Biofilm formation is a complex multicellular process consisting of cell adhesion, cell growth, morphogenic switching between yeast form and filamentous states, and quorum sensing. Here we describe the role of the C. albicans EAP1 gene, which encodes a glycosylphosphatidylinositol-anchored, glucan-cross-linked cell wall protein, in adhesion and biofilm formation in vitro and in vivo. Deleting EAP1 reduced cell adhesion to polystyrene and epithelial cells in a gene dosage-dependent manner. Furthermore, EAP1 expression was required for C. albicans biofilm formation in an in vitro parallel plate flow chamber model and in an in vivo rat central venous catheter model. EAP1 expression was upregulated in biofilm-associated cells in vitro and in vivo. Our results illustrate an association between Eap1p-mediated adhesion and biofilm formation in vitro and in vivo. [Abstract/Link to Full Text]

Sato N, Moriyama T
Genomic and biochemical analysis of lipid biosynthesis in the unicellular rhodophyte Cyanidioschyzon merolae: lack of a plastidic desaturation pathway results in the coupled pathway of galactolipid synthesis.
Eukaryot Cell. 2007 Jun;6(6):1006-17.
The acyl lipids making up the plastid membranes in plants and algae are highly enriched in polyunsaturated fatty acids and are synthesized by two distinct pathways, known as the prokaryotic and eukaryotic pathways, which are located within the plastids and the endoplasmic reticulum, respectively. Here we report the results of biochemical as well as genomic analyses of lipids and fatty acids in the unicellular rhodophyte Cyanidioschyzon merolae. All of the glycerolipids usually found in photosynthetic algae were found, such as mono- and digalactosyl diacylglycerol, sulfolipid, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. However, the fatty acid composition was extremely simple. Only palmitic, stearic, oleic, and linoleic acids were found as major acids. In addition, 3-trans-hexadecanoic acid was found as a very minor component in phosphatidylglycerol. Unlike the case for most other photosynthetic eukaryotes, polyenoic fatty acids having three or more double bonds were not detected. These results suggest that polyunsaturated fatty acids are not necessary for photosynthesis in eukaryotes. Genomic analysis suggested that C. merolae lacks acyl lipid desaturases of cyanobacterial origin as well as stearoyl acyl carrier protein desaturase, both of which are major desaturases in plants and green algae. The results of labeling experiments with radioactive acetate showed that the desaturation leading to linoleic acid synthesis occurs on phosphatidylcholine located outside the plastids. Monogalactosyl diacylglycerol is therefore synthesized by the coupled pathway, using plastid-derived palmitic acid and endoplasmic reticulum-derived linoleic acid. These results highlight essential differences in lipid biosynthetic pathways between the red algae and the green lineage, which includes plants and green algae. [Abstract/Link to Full Text]

Liu XH, Lu JP, Zhang L, Dong B, Min H, Lin FC
Involvement of a Magnaporthe grisea serine/threonine kinase gene, MgATG1, in appressorium turgor and pathogenesis.
Eukaryot Cell. 2007 Jun;6(6):997-1005.
We isolated an MgATG1 gene encoding a serine/threonine protein kinase from the rice blast fungus Magnaporthe grisea. In the DeltaMgatg1 mutant, in which the MgATG1 gene had been deleted, autophagy was blocked; the mutant also showed fewer lipid droplets in its conidia, lower turgor pressure of the appressorium, and such defects in morphogenesis as delayed initiation and slower germination of conidia. As a result of lower turgor pressure of the appressorium, the DeltaMgatg1 mutant lost its ability to penetrate and infect the two host plants, namely, rice and barley. However, normal values of the parameters and infective abilities were restored on reintroducing an intact copy of the MgATG1 gene into the mutant. Autophagy is thus necessary for turnover of organic matter during the formation of conidia and appressoria and for normal development and pathogenicity in M. grisea. [Abstract/Link to Full Text]

Schaefer D, Côte P, Whiteway M, Bennett RJ
Barrier activity in Candida albicans mediates pheromone degradation and promotes mating.
Eukaryot Cell. 2007 Jun;6(6):907-18.
Mating in Candida albicans and Saccharomyces cerevisiae is regulated by the secretion of peptide pheromones that initiate the mating process. An important regulator of pheromone activity in S. cerevisiae is barrier activity, involving an extracellular aspartyl protease encoded by the BAR1 gene that degrades the alpha pheromone. We have characterized an equivalent barrier activity in C. albicans and demonstrate that the loss of C. albicans BAR1 activity results in opaque a cells exhibiting hypersensitivity to alpha pheromone. Hypersensitivity to pheromone is clearly seen in halo assays; in response to alpha pheromone, a lawn of C. albicans Deltabar1 mutant cells produces a marked zone in which cell growth is inhibited, whereas wild-type strains fail to show halo formation. C. albicans mutants lacking BAR1 also exhibit a striking mating defect in a cells, but not in alpha cells, due to overstimulation of the response to alpha pheromone. The block to mating occurs prior to cell fusion, as very few mating zygotes were observed in mixes of Deltabar1 a and alpha cells. Finally, in a barrier assay using a highly pheromone-sensitive strain, we were able to demonstrate that barrier activity in C. albicans is dependent on Bar1p. These studies reveal that a barrier activity to alpha pheromone exists in C. albicans and that the activity is analogous to that caused by Bar1p in S. cerevisiae. [Abstract/Link to Full Text]

Llamas A, Tejada-Jimenez M, González-Ballester D, Higuera JJ, Schwarz G, Galván A, Fernández E
Chlamydomonas reinhardtii CNX1E reconstitutes molybdenum cofactor biosynthesis in Escherichia coli mutants.
Eukaryot Cell. 2007 Jun;6(6):1063-7.
We have isolated and characterized the Chlamydomonas reinhardtii genes for molybdenum cofactor biosynthesis, namely, CNX1G and CNX1E, and expressed them and their chimeric fusions in Chlamydomonas and Escherichia coli. In all cases, the wild-type phenotype was restored in individual mutants as well as in a CNX1G CNX1E double mutant. Therefore, CrCNX1E is the first eukaryotic protein able to complement an E. coli moeA mutant. [Abstract/Link to Full Text]

Peńas MM, Hervás-Aguilar A, Múnera-Huertas T, Reoyo E, Peńalva MA, Arst HN, Tilburn J
Further characterization of the signaling proteolysis step in the Aspergillus nidulans pH signal transduction pathway.
Eukaryot Cell. 2007 Jun;6(6):960-70.
The Aspergillus nidulans pH-responsive transcription factor PacC is modulated by limited, two-step proteolysis. The first, pH-regulated cleavage occurs in the 24-residue highly conserved "signaling protease box" in response to the alkaline pH signal. This is transduced by the Pal signaling pathway, containing the predicted calpain-like cysteine protease and likely signaling protease, PalB. In this work, we carried out classical mutational analysis of the putative signaling protease PalB, and we describe 9 missense and 18 truncating loss-of-function (including null) mutations. Mutations in the region of and affecting directly the predicted catalytic cysteine strongly support the deduction that PalB is a cysteine protease. Truncating and missense mutations affecting the C terminus highlight the importance of this region. Analysis of three-hemagglutinin-tagged PalB in Western blots demonstrates that PalB levels are independent of pH and Pal signal transduction. We have followed the processing of MYC(3)-tagged PacC in Western blots. We show unequivocally that PalB is essential for signaling proteolysis and is definitely not the processing protease. In addition, we have replaced 15 residues of the signaling protease box of MYC(3)-tagged PacC (pacC900) with alanine. The majority of these substitutions are silent. Leu481Ala, Tyr493Ala, and Gln499Ala result in delayed PacC processing in response to shifting from acidic to alkaline medium, as determined by Western blot analysis. Leu498Ala reduces function much more markedly, as determined by plate tests and processing recalcitrance. Excepting Leu498, this demonstrates that PacC signaling proteolysis is largely independent of sequence in the cleavage region. [Abstract/Link to Full Text]

---

Recent Articles in The Journal of Biological Chemistry

---

Jamieson KV, Wu J, Hubbard SR, Meruelo D
Crystal structure of the human laminin receptor precursor.
J Biol Chem. 2007 Dec 6; .
The human laminin receptor (LamR) interacts with many ligands, including laminin, prions, Sindbis virus, and the polyphenol EGCG, and has been implicated in a number of diseases. LamR is overexpressed on tumor cells, and targeting LamR elicits anti-cancer effects. Here, we report the crystal structure of human LamR, which provides insights into its function and should facilitate the design of novel therapeutics targeting LamR. [Abstract/Link to Full Text]

Chen K, Tu Y, Zhang Y, Blair HC, Zhang L, Wu C
Pinch-1 regulates the ERK-Bim pathway and contributes to apoptosis resistance in cancer cells.
J Biol Chem. 2007 Dec 6;
Resistance to apoptosis is a hallmark of cancer cells. We report here that PINCH-1, a cytoplasmic component of cell-extracellular matrix (ECM) adhesions, is required for protection of multiple types of cancer cells from apoptosis. Furthermore, using HT-1080 fibrosarcoma cells as a model system, we have investigated the signaling pathway through which PINCH-1 contributes to apoptosis resistance. Loss of PINCH-1 markedly increases the level of Bim and promotes Bim translocation to mitochondria, resulting in activation of the intrinsic apoptosis pathway. Depletion of Bim completely blocked apoptosis induced by the loss of PINCH-1. Thus, PINCH-1 contributes to apoptosis resistance through suppression of Bim. Mechanistically, PINCH-1 suppresses Bim not only transcriptionally but also post-transcriptionally. PINCH-1 promotes activating phosphorylation of Src family kinase (SFK) and extracellular signal-regulated kinase1/2 (ERK1/2). Consistent with this, ERK1/2-mediated Ser69 phosphorylation of Bim, a key signal for turnover of Bim, is suppressed by the removal of PINCH-1. Our results demonstrate a strong dependence of multiple types of apoptosis resistant cancer cells on PINCH-1 and provide new insights into the molecular mechanism by which cancer cells are protected from apoptosis. [Abstract/Link to Full Text]

Miyamoto T, Oshiro N, Yoshino KI, Nakashima A, Eguchi S, Takahashi M, Ono Y, Kikkawa U, Yonezawa K
AMP-activated protein kinase phosphorylates Golgi-specific brefeldin A resistance factor 1 at Thr1337 to induce disassembly of Golgi apparatus.
J Biol Chem. 2007 Dec 6;
Sufficiency and depletion of nutrients regulate the cellular activities through the protein phosphorylation reaction, however, many protein substrates remain to be clarified. Golgi-specific brefeldin A resistance factor 1 (GBF1), a guanine nucleotide exchange factor for the ADP-ribosylation factor family associated with the Golgi apparatus, was isolated as a phosphoprotein from the glucose-depleted cells by using the phospho-Akt-substrate antibody, which recognizes the substrate proteins of several protein kinases. The phosphorylation of GBF1 was induced by 2-deoxyglucose (2-DG), which blocks glucose utilization and increases the intracellular AMP concentration, and by AICAR, an AMP-activated protein kinase (AMPK) activator. This phosphorylation was observed in the cells expressing the constitutively active AMPK. The 2-DG-induced phosphorylation of GBF1 was suppressed by Compound C, an AMPK-specific inhibitor, and by the overexpression of the kinase-negative AMPK. Analysis using the deletion and point mutants identified Thr1337 as the 2-DG induced phosphorylation site in GBF1, which is phosphorylated by AMPK in vitro. ATP depletion is known to provoke the Golgi apparatus disassembly. Immunofluorescent microscopic analysis with the Golgi markers indicated that GBF1 associates with the fragmented Golgi apparatus in the cells treated with 2-DG and AICAR. The expression of the kinase-negative AMPK and the GBF1 mutant replacing Thr1337 by Ala prevented the 2-DG-induced Golgi disassembly. These results indicate that GBF1 is a novel AMPK substrate, and that the AMPK-mediated phosphorylation of GBF1 at Thr1337 has a critical role, presumably by attenuating the function of GBF1, in the disassembly of the Golgi apparatus induced under stress conditions that lower the intracellular ATP concentration. [Abstract/Link to Full Text]

Yamano K, Yatsukawa YI, Esaki M, Hobbs AE, Jensen RE, Endo T
TOM20 and TOM22 share the common signal recognition pathway in mitochondrial protein import.
J Biol Chem. 2007 Dec 6;
Precise targeting of mitochondrial precursor proteins to mitochondria requires receptor functions of Tom20, Tom22, and Tom70 on the mitochondrial surface. Tom20 is a major import receptor that recognizes preferentially mitochondrial presequences, and Tom70 is a specialized receptor that recognizes presequence-less inner membrane proteins. The cytosolic domain of Tom22 appears to function as a receptor in cooperation with Tom20, but how its substrate specificity differs from that of Tom20 remains unclear. To reveal possible difference, if any, in substrate specificities between Tom20 and Tom22, we deleted the receptor domain of Tom20 or Tom22 of mitochondria in vitro by introducing cleavage sites for TEV protease between the receptor domains and transmembrane segments of Tom20 and Tom22. Then mitochondria without the receptor domain of Tom20 or Tom22 were analyzed for their abilities to import various mitochondrial precursor proteins targeted to different mitochondrial subcompartments in vitro. The effects of deletion of the receptor domains on the import of different mitochondrial proteins for different import pathways were quite similar between Tom20 and Tom22. Therefore Tom20 and Tom22 are apparently involved in the same step or sequential steps along same pathway of targeting signal recognition in import. [Abstract/Link to Full Text]

Zdebik AA, Zifarelli G, Bergsdorf EY, Soliani P, Scheel O, Jentsch TJ, Pusch M
Determinants of anion-proton coupling in mammalian endosomal CLC proteins.
J Biol Chem. 2007 Dec 6;
Many proteins of the CLC gene family are Cl(-) channels, whereas others, like the bacterial ecClC-1 or mammalian ClC-4 and ClC 5, mediate Cl(-)/H(+) exchange. Mutating a 'gating glutamate' (E224 in ClC-4; E211 in ClC-5) converted these exchangers into anion conductances, as did the neutralization of another, intracellular 'proton glutamate' in ecClC-1. We show here that neutralizing the 'proton glutamate' of ClC 4 (E281) and ClC-5 (E268), but not its replacement by aspartate, histidine or tyrosine, rather abolished Cl(-) and H(+) transport. Surface expression was unchanged by these mutations. Uncoupled Cl(-) transport could be restored in the ClC- 4E281A and ClC-5E268A proton glutamate mutations by additionally neutralizing the gating glutamates, suggesting that WT proteins transport anions only when protons are supplied through a cytoplasmic H(+) donor. Each monomeric unit of the dimeric protein was found to be able to carry out Cl(-)/H(+) exchange independently from the transport activity of the neighboring subunit. NO(3)(-) or SCN(-) transport was partially uncoupled from H(+) but still depended on the 'proton glutamate'. Inserting 'proton glutamates' into CLC channels altered their gating but failed to convert them into Cl(-)/H(+) exchangers. Noise analysis indicated that ClC-5 switches between silent and transporting states with an apparent unitary conductance of 0.5 pS. Our results are consistent with the idea that Cl(-)/H(+) exchange of the endosomal ClC-4 and ClC-5 proteins relies on proton delivery from an intracellular titratable residue at position 268 (numbering of ClC-5), and that the strong rectification of currents arises from the voltage-dependent proton transfer from E268 to E211. [Abstract/Link to Full Text]

Bogenhagen DF, Rousseau D, Burke S
The layered structure of human mtDNA nucleoids.
J Biol Chem. 2007 Dec 6;
Mitochondrial DNA (mtDNA) occurs in cells in nucleoids containing several copies of the genome. Previous studies have identified proteins associated with these large DNA structures when they are biochemically purified by sedimentation and immunoaffinity chromatography. In this study, formaldehyde crosslinking was performed to determine which nucleoid proteins are in close contact with the mtDNA. A set of core nucleoid proteins is found in both native and crosslinked nucleoids, including 13 proteins with known roles in mtDNA transactions. Several other metabolic proteins and chaperones identified in native nucleoids including ATAD3 were not observed to crosslink to mtDNA. Additional immunofluorescence and protease susceptibility studies showed that an N-terminal domain of ATAD3 previously proposed to bind to the mtDNA D-loop is directed away from the mitochondrial matrix, so that it is unlikely to interact with mtDNA in vivo. These results are discussed in relation to a model for a layered structure of mtDNA nucleoids in which replication and transcription occur in the central core while translation and complex assembly may occur in the peripheral region. [Abstract/Link to Full Text]

Curaba J, Chen X
Biochemical activities of Arabidopsis RNA-dependent RNA polymerase 6.
J Biol Chem. 2007 Dec 6;
In Arabidopsis, genetic evidence demonstrates that RNA-dependent RNA polymerase6 (RDR6) plays a fundamental role in at least four RNA silencing pathways whose functions range from defense against transgenes or viruses to endogene regulation in development and in stress responses. Despite its critical role in RNA silencing, the biochemical activities of RDR6 have yet to be characterized. In this study, we transiently expressed Arabidopsis RDR6 in Nicotiana benthamiana and investigated the biochemical activities of immunopurified RDR6 in vitro. We showed that RDR6 possesses terminal nucleotidyl transferase activity as well as primer-independent RNA polymerase activity on single-stranded RNAs. We found that RDR6 cannot distinguish RNAs with or without a cap or polyA tail. We also demonstrated that RDR6 has strong polymerasae activity on single-stranded DNA. All these activities require the conserved catalytic Asp867 residue. Our findings have important implications on the processes involving RDR6 in vivo and provide new biochemical insights into the mechanisms of RNA silencing in Arabidopsis. [Abstract/Link to Full Text]

Raven JF, Baltzis D, Wang S, Mounir Z, Papadakis AI, Gao HQ, Koromilas AE
PKR and PERK induce the proteasome-dependent degradation of cyclin D1 via a mechanism requiring eIF2alpha phosphorylation.
J Biol Chem. 2007 Dec 6;
The cell cycle protein cyclin D1 plays a critical role in controlling the G1/S transition via its ability to regulate the activity of multiple cyclin-dependent kinases (CDKs). The transcriptional regulation of the cyclin D1 gene is well understood, and several studies have indicated that cyclin D1 translation is decreased upon activation of the eIF2a kinases. We examined the effect of activation of the eIF2a kinases PKR and PERK on cyclin D1 protein levels and translation, and determined that cyclin D1 protein levels decrease upon the induction of PKR and PERK catalytic activity, but that this decrease is not due to translation. Inhibition of the 26S proteasome with MG132 rescued cyclin D1 protein levels, indicating that rather than inhibiting translation, PKR and PERK are acting to increase cyclin D1 degradation. Interestingly, this effect still requires eIF2a phosphorylation at serine 51, as cyclin D1 remains unaffected in cells containing a non-phosphorylatable form of the protein. This proteasome-dependent degradation of cyclin D1 requires an intact ubiquitination pathway, although the ubiquitination of cyclin D1 is not itself affected. Furthermore, this degradation is independent of phosphorylation of cyclin D1 at threonine 286, which is mediated by the glycogen synthase kinase 3ss (GSK-3ss) and mitogen activated protein kinase (MAPK) pathways as described in previous studies. Our study reveals a novel functional cross-talk between the eIF2a phosphorylation and the proteasomal degradation of cyclin D1, and that this degradation is dependent upon eIF2a phosphorylation during short, but not prolonged periods of stress. [Abstract/Link to Full Text]

Dong WJ, Xing J, Ouyang Y, An J, Cheung HC
Structural kinetics of cardiac troponin C mutants linked to familial hypertrophic and dilated cardiomyopathy in troponin complexes.
J Biol Chem. 2007 Dec 5;
The key events in regulating cardiac muscle contraction involve Ca2+ binding to and release from cTnC (troponin C) and structural changes in cTnC and other thin filament proteins triggered by Ca2+ movement. Single mutations L29Q and G159D in human cTnC have been reported to associate with familial hypertrophic and dilated cardiomyopathy, respectively. We have examined the effects of these individual mutations on structural transitions in the regulatory N-domain of cTnC triggered by Ca2+ binding and dissociation. This study was carried out with a double mutant or triple mutants of cTnC, reconstituted into troponin with tryptophanless cTnI and cTnT. The double mutant, cTnC(L12W/N51C) labeled with 1,5-IAEDANS at Cys51, served as a control to monitor Ca2+-induced opening and closing of the N-domain by Förster resonance energy transfer (FRET). The triple mutants contained both L12W and N51C labeled with 1,5-IAEDANS, and either L29Q or G159D. Both mutations had minimal effects on the equilibrium distance between Trp12 and Cys51-AEDANS in the absence or presence of bound Ca2+. L29Q had no effect on the closing rate of the N-domain triggered by release of Ca2+, but reduced the Ca2+-induced opening rate. G159D reduced both the closing and opening rates. Previous results showed that the closing rate of cTnC N-domain triggered by Ca2+ dissociation was substantially enhanced by PKA phosphorylation of cTnI. This rate enhancement was abolished by L29Q or G159D. These mutations alter the kinetics of structural transitions in the regulatory N-domain of cTnC that are involved in either activation (L29Q) or deactivation (G159D). Both mutations appear to be antagonistic toward phosphorylation signaling between cTnI and cTnC. [Abstract/Link to Full Text]

Flashman E, Bagg EA, Chowdhury R, Mecinovic J, Loenarz C, McDonough MA, Hewitson KS, Schofield CJ
Kinetic rationale for selectivity towards N- and C-terminal oxygen dependent degradation domain substrates mediated by a loop region of HIF prolyl hydroxylases.
J Biol Chem. 2007 Dec 5;
Hydroxylation of two conserved prolyl residues in the N- and C-terminal oxygen dependent degradation domains (NODD and CODD) of the a-subunit of hypoxia-inducible factor (HIF) signals for its degradation via the ubiquitin-proteasome pathway. In human cells, three prolyl hydroxylases (PHDs 1-3) belonging to the Fe(II) and 2-oxoglutarate (2OG) dependent oxygenase family catalyze prolyl hydroxylation with differing selectivity for CODD and NODD. Sequence analysis of the catalytic domains of the PHDs in the light of crystal structures for PHD2, and results for other 2OG oxygenases, suggested that either the C-terminal region or a loop linking two ss-strands (ss2 and ss3 in human PHD2) are important in determining substrate selectivity. Mutation analyses on PHD2 revealed that the ss2ss3 loop is a major determinant in conferring selectivity for CODD over NODD peptides. A chimeric PHD in which the ss2ss3 loop of PHD2 was replaced with that of PHD3 displayed an almost complete selectivity for CODD, as observed for wild type PHD3. Kinetic studies suggest this is effected by both changes in strength of binding and catalytic efficiency. [Abstract/Link to Full Text]

Severi E, Muller A, Potts JR, Leech A, Williamson D, Wilson KS, Thomas GH
Sialic acid mutarotation is catalysed by the Escherichia coli beta -propeller protein YJHT.
J Biol Chem. 2007 Dec 5;
The acquisition of host-derived sialic acid is an important virulence factor for some bacterial pathogens but in vivo this sugar acid is sequestered in sialoconjugates as the a-anomer. In solution, however, sialic acid is present mainly as the ss-anomer, formed by a slow spontaneous mutarotation. We studied the Escherichia coli protein YjhT as a member of a family of uncharacterised proteins present in many sialic acid-utilising pathogens. This protein is able to accelerate the equilibration of the a- and ss-anomers of the sialic acid N-acetylneuraminic acid, thus describing a novel sialic acid mutarotase activity. The structure of this periplasmic protein, solved to 1.5 A resolution, reveals a dimeric 6-bladed unclosed ss-propeller, the first of a bacterial Kelch-domain protein. Mutagenesis of conserved residues in YjhT demonstrated an important role for Glu209 and Arg215 in mutarotase activity. We also present data suggesting that the ability to utilise a-N-acetylneuraminic acid released from complex sialoconjugates in vivo provides a physiological advantage to bacteria containing YjhT. [Abstract/Link to Full Text]

Wu J, Bohanan CS, Neumann JC, Lingrel JB
KLF2 transcription factor modulates blood vessel maturation through smooth muscle cell migration.
J Biol Chem. 2007 Dec 5;
Vasculogenesis, angiogenesis and maturation are three major phases of the development of blood vessels. Although many receptors required for blood vessel formation have been defined, the intracellular signal transduction pathways involved in vascular maturation remain unclear. KLF2-/- embryos fail to develop beyond 13.5 days because of a lack of blood vessel stabilization. The molecular mechanism of KLF2 function in embryonic vascular vessels is still largely unknown. Here we show a normal development pattern of endothelial cells in KLF2-/- embryos but a defect of smooth muscle cells at the dorsal side of the aorta. This phenotype results from arrested vascular maturation characterized by the failure of mural cells to migrate around endothelial cells. This migration defect is also observed when platelet-derived growth factor-B (PDGF) controlled migration is studied in MEF cells from KLF2-/- animals. In addition, KLF2-/- MEFs exhibit a significant growth defect, indicating that KLF2 is required to maintain the viability of MEF cells. The PDGF signal is mediated through the Src signaling pathway and a downstream target of KLF2 is sphingosine 1-phosphate receptor 1. These studies demonstrate that KLF2 is required for smooth muscle cell migration and elucidate a novel mechanism involving communication between PDGF and KLF2 in vascular maturation. [Abstract/Link to Full Text]

Jiang L, Fan J, Bai L, Wang Y, Chen T, Yang L, Chen L, Xu T
Direct quantification of fusion rate reveals a distal role for AS160 in insulin-stimulated fusion of GLUT4 storage vesicles.
J Biol Chem. 2007 Dec 6;
Insulin-stimulated GLUT4 translocation to the plasma membrane constitutes a key process for blood glucose control. However, convenient and robust assays to monitor this dynamic process in real time are lacking, which hinders current progress towards elucidation of the underlying molecular events as well as screens for drugs targeting this particular pathway. Here, we have developed a novel dual-colored probe to monitor the translocation process of GLUT4 based on dual color fluorescence measurement. We demonstrate that this probe is more than an order of magnitude more sensitive than the current technology for detecting fusion events from single GLUT4 storage vesicles (GSVs). A small fraction of fusion events were found to be of the "kiss-and-run" type. For the first time, we show that insulin stimulation evokes a ~40 folds increase in the fusion of GSVs in 3T3-L1 adipocytes, compared to basal conditions. The probe can also be used to monitor the pre-fusion behavior of GSVs. By quantifying both the docking and fusion rates simultaneously, we demonstrate a proportional inhibition in both docking and fusion of GSVs by a dominant negative mutant of AS160, indicating a role for AS160 in the docking of GSVs, but not in the regulation of GSV fusion after docking. [Abstract/Link to Full Text]

Shen Y, Hirsch DS, Sasiela CA, Wu WJ
CDC42 regulates E-cadherin ubiquitination and degradation through an EGF receptor to SRC-mediated pathway.
J Biol Chem. 2007 Dec 5;
E-cadherins play an essential role in maintaining epithelial polarity by forming calcium dependent adherens junctions between epithelial cells. Here, we report that calcium depletion induces E-cadherin ubiquitination and lysosomal degradation, and that Cdc42 plays an important role in regulating this process. We demonstrate that calcium depletion induces activation of Cdc42. This in turn upregulates EGFR signaling to mediate Src activation, leading to E-cadherin ubiquitination and lysosomal degradation. Silencing Cdc42 blocks activation of EGFR and Src induced by calcium depletion, resulting in a reduction in E-cadherin degradation. The role of Cdc42 in regulating E-cadherin ubiquitination and degradation is underscored by the fact that constitutively active Cdc42(F28L) increases the activity of EGFR and Src, and significantly enhances E-cadherin ubiquitination and lysosomal degradation. Furthermore, we found that GTP-dependent binding of Cdc42 to E-cadherin is critical for Cdc42 to induce the dissolution of adherens junctions. Our data support a model that activation of Cdc42 contributes to mesenchyme-like phenotype by targeting of E-cadherin for lysosomal degradation. [Abstract/Link to Full Text]

Connell CM, Colnaghi R, Wheatley SP
Nuclear survivin has reduced stability and is not cytoprotective.
J Biol Chem. 2007 Dec 5;
Survivin is an essential mitotic protein that is over expressed in many cancers and its presence is correlated with increased resistance to radiation and chemotherapy. Here we demonstrate that sending survivin into the nucleus accelerates its degradation in a cdh1 dependent manner, abolishes the radio resistance normally conferred to cells by its over expression, and prevents survivin from inhibiting apoptosis without affecting its mitotic localisation. Our data suggest that targeting survivin to the nucleus provides an efficient means of eliminating it from the cell and may prove a novel strategy in cancer treatment, particularly in combination with radiotherapy. [Abstract/Link to Full Text]

Ohgaki R, Fukura N, Matsushita M, Mitsui K, Kanazawa H
Cell surface levels of organellar Na+/H+ exchanger isoform 6 are regulated by interaction with the receptor for activated C-kinase 1.
J Biol Chem. 2007 Dec 5;
In mammalian cells, four Na+/H+ exchangers (NHE6-9) are localized to intracellular compartments. NHE6 and NHE9 are predominantly localized to sorting and recycling endosomes, NHE7 to the trans-Golgi network (TGN), and NHE8 to the mid-trans Golgi stacks. The unique localization of NHEs may contribute to establishing organelle-specific pHs and ion homeostasis in cells. Mechanisms underlying the regulation and targeting of organellar NHEs are largely unknown. We identified an interaction between NHE9 and receptor for activated C-kinase 1 (RACK1), a cytoplasmic scaffold protein, by yeast two-hybrid screening using the NHE9 C-terminus as bait. The NHE9 C-terminus is exposed to the cytoplasm, verifying that the interaction is topologically possible. The binding region was further delineated to the central region of the NHE9 C-terminus. RACK1 also bound NHE6 and NHE7, but not NHE8, in vitro. Endogenous association between NHE6 and RACK1 was confirmed by co-immunoprecipitation and co-localization in HeLa cells. The luminal pH of the recycling endosome was elevated in RACK1 knockdown cells, accompanied by a decrease in the amount of NHE6 on the cell surface, although the total level of NHE6 was not significantly altered. These results indicate that RACK1 plays a role in regulating the distribution of NHE6 between endosomes and the plasma membrane and contributes to maintaining luminal pH of the endocytic-recycling compartments. [Abstract/Link to Full Text]

Kainov DE, Mancini EJ, Telenius J, Lisal J, Grimes JM, Bamford DH, Stuart DI, Tuma R
Structural basis of mechano-chemical coupling in a hexameric molecular motor.
J Biol Chem. 2007 Dec 5;
The P4 protein of bacteriophage phi12 is a hexameric molecular motor closely related to super family 4 (SF4) helicases. P4 converts chemical energy from ATP hydrolysis into mechanical work, to translocate single stranded RNA into a viral capsid. The molecular basis of mechano-chemical coupling, i.e. how small ~1A changes in the ATP binding site are amplified into nanometer scale motion along the nucleic acid, is not understood at atomic level. Here we study in atomic detail the mechano-chemical coupling using structural and biochemical analyses of P4 mutants. We show that a conserved region, comprising SF4 helicase motifs H3 and H4 and loop L2, constitutes the moving lever of the motor. The lever tip encompasses an RNA binding site which moves along the mechanical reaction coordinate. The lever is flanked by gamma-phosphate sensors (Asn234 and Ser252) which report the nucleotide state of neighboring subunits and control the lever position. Insertion of an arginine finger (Arg279) into the neighboring catalytic site is concomitant with lever movement and commences ATP hydrolysis. This assures cooperative sequential hydrolysis which is tightly coupled to mechanical motion. Given the structural conservation the mutated residues may play similar roles in other hexameric helicases and related molecular motors. [Abstract/Link to Full Text]

Aguiar RS, Lovsin N, Tanuri A, Peterlin BM
VPR.A3A chimera inhibits HIV replication.
J Biol Chem. 2007 Dec 5;
Several APOBEC3 proteins (A3F and A3G), which are cytidine deaminases, restrict HIV replication in the absence of the viral Vif protein. However, Vif leads to their degradation and counteracts their effects. Another member, A3A restricts some retrotransposons and another virus but not HIV. We reasoned that this failure was due to the lack of appropriate targeting. Thus, we fused A3A to another viral protein, Vpr, which binds p6 in Gag and is incorporated into viral cores. Indeed, our Vpr.A3A chimera but not A3A was found abundantly in the viral core. It also restricted potently the replication of HIV and SIV in the presence and absence of Vif. Since we identified a high frequency of G to A mutations in viral cDNAs, this antiviral activity was mediated by DNA editing. Interestingly, our fusion protein did not restrict MLV, which does not incorporate Vpr. Thus, by targeting appropriately a potent single domain cytidine deaminase, we rendered HIV and SIV restriction resistant to Vif. [Abstract/Link to Full Text]

Yang PC, Yang CH, Huang CC, Hsu KS
Phosphatidylinositol 3 kinase activation is required for stress protocol-induced modification of hippocampal synaptic plasticity.
J Biol Chem. 2007 Dec 5;
Stress dramatically affects the induction of hippocampal synaptic plasticity; however, the molecular details of how it dose so remain unclear. Phosphatidylinositol 3 kinase (PI3K) signaling plays a crucial role in promoting neuronal survival and neuroplasticity, but its role, if any, in stress-induced alterations of long-term potentiation (LTP) and long-term depression (LTD) is unknown. We found here that inhibitors of PI3K signaling blocked the effects of acute restraint-tailshock stress protocol on LTP and LTD. Therefore the purpose of the present study is to explore the signaling events involving PI3K in terms of its role in mediating stress protocol-induced alterations of LTP and LTD. We found that stress protocol-induced PI3K activation can be blocked by various inhibitors including RU38486 for glucocorticoid receptors, LY294002 for PI3K, DL-2-amino-5- phosphonopentanoic acid for NMDA receptors or BDNF antisense oligonucleotides. Also, immunoblotting analyses revealed that stress protocol induced a profound and prolonged phosphorylation of numbers of PI3K downstream effectors, including 3-phosphoinositide-dependent protein kinase-1, protein kinase B, mammalian target of rapamycin (mTOR), p70S6 kinase and eukaryotic initiation factor 4B in hippocampal CA1 homogenate, which were prevented by the PI3K inhibitor pretreatment. More importantly, we found that stress protocol significantly increased the protein expression of dendritic scaffolding protein postsynaptic density-95 (PSD-95), which is known to involve in LTP and LTD, in an mTOR-dependent manner. These results identify a key role of PI3K signaling in mediating the stress protocol-induced modification of hippocampal synaptic plasticity and further suggest that PI3K may do so by invoking the protein expression of PSD-95. [Abstract/Link to Full Text]

Johnson AC, Li X, Pearlman E
MyD88 functions as a negative regulator of TLR3/TRIF induced corneal inflammation by inhibiting activation of c-jun N-terminal kinase (JNK).
J Biol Chem. 2007 Dec 5;
The adaptor molecule MyD88 is necessary for responses to all TLRs except TLR3 and a subset of TLR4 signaling events, which are mediated by the adaptor molecule TRIF. To determine the role of TRIF in host inflammatory responses, corneal epithelium of C57BL/6, TLR3-/-, TRIF-/-, and MyD88-/- mice was abraded and stimulated with the synthetic TLR3 ligand Poly(I:C). We found that Poly(I:C) induced a pronounced cellular infiltration into the corneal stroma, which was TLR3- and TRIF- dependent. Unexpectedly, the inflammatory response was exacerbated in MyD88-/- mice with enhanced neutrophil and F4/80+ cell infiltration into the corneal stroma, elevated corneal haze, which is an indicator of loss of corneal transparency. To determine if MyD88 - dependent inhibition of TLR3/TRIF responses is a general phenomenon, we examined cytokine production by MyD88-/- bone marrow-derived macrophages; however, no significant difference was observed between MyD88+/+ or MyD88-/- macrophages. In contrast, human corneal epithelial cells (HCECs) transfected with MyD88 siRNA, had significantly increased (2.5-fold) CCL5/RANTES production compared with control HCECs, demonstrating a negative regulatory role for MyD88 on TLR3/TRIF responses in these cells. Finally, knockdown of MyD88 in HCECs resulted in increased phosphorylation of JNK, but not p38, IRF-3, or NF-B. Consistent with this finding, the JNK inhibitor SP600125, but not p38 inhibitor SB203580, ablated this response. Taken together, these findings demonstrate a novel regulatory role for MyD88 on the TLR3/TRIF activation pathway by inhibition of JNK. [Abstract/Link to Full Text]

Wang J, Shiozawa Y, Wang J, Wang Y, Jung Y, Pienta KJ, Mehra R, Lober R, Taichman RS
The role of CXCR7/RDC1 as a chemokine receptor for CXCL12/ SDF-1 in prostate cancer.
J Biol Chem. 2007 Dec 5;
Several reports have recently documented that CXCR7/RDC1 functions as a chemokine receptor for SDF-1/CXCL12, which regulates a spectrum of normal and pathological processes. In this investigation, the role of CXCR7/RDC1 in prostate cancer (PCa) was explored. Staining of high-density tissue microarrays demonstrate that the levels of CXCR7/RDC1 expression increases as the tumors become more aggressive. In vitro and in vivo studies with PCa cell lines suggest that alterations in CXCR7/RDC1 expression are associated with enhanced adhesive and invasive activities in addition to a survival advantage. In addition, it was observed that CXCR7/RDC1 levels are regulated by CXCR4. Among the potential down stream targets of CXCR7/RDC1 are CD44 and cadherin-11, which are likely to contribute to the invasiveness of PCa cells. CXCR7/RDC1 also regulates the expression of the proangeogenic factors IL-8 or VEGF, which are likely to participate in the regulation of tumor angiogenesis. Finally, we found that signaling by CXCR7/RDC1 activates AKT pathways. Together, these data demonstrate a role for CXCR7/RDC1 in PCa metastasis and progression, and suggest potential targets therapeutic intervention. [Abstract/Link to Full Text]

Meng J, Parroche P, Golenbock DT, McKnight CJ
The differential impact of disulfide bonds and N-linked glycosylation on the stability and function of CD14.
J Biol Chem. 2007 Dec 5;
Innate immunity is the first-line defense against invading pathogens. During Gram-negative bacterial infection, the Toll-like receptor 4 and MD-2 complex recognize lipopolysaccharide (LPS) present in the bacterial cell wall. This recognition can be enhanced 100-1000 fold by CD14. However, the beneficial role provided by CD14 becomes detrimental in the context of sepsis and septic shock. An understanding of how CD14 functions will therefore benefit treatments targeted at both immune suppression and immune enhancement. In the present study, we use site-directed mutagenesis to address the role of disulfide bonds and N-linked glycosylation on CD14. A differential impact is observed for the five disulfide bonds on CD14 folding, with the first two (C6-C17, C15-C32) being indispensable, the third and fourth (C168-C198, C222-C253) being important, and the last (C287-C333) being dispensable. A functional role is observed for the first disulfide bond as the C6A substitution severely reduces the ability of CD14 to confer LPS responsiveness to U373 cells. Two of the four predicted glycosylation sites, asparagines 132 and 263, are actually involved in N-linked glycosylation, resulting in heterogeneity in CD14 molecular weight. Furthermore, glycosylation at N132 plays a role in CD14 trafficking and upstream and/or downstream ligand interactions. When mapped onto the crystal structure of mouse CD14, the first two disulfide bonds and N132 are in close proximity to the initial ss strands of the leucine rich repeat domain. Thus, disulfide bonds and N-linked glycosylation in the initial ss sheets of the inner concave surface of CD14 are crucial for structure and function. [Abstract/Link to Full Text]

Bundgaard JR, Rehfeld JF
Distinct linkage between posttranslational processing and differential secretion of progastrin derivatives in endocrine cells.
J Biol Chem. 2007 Dec 5;
Prohormones often undergo extensive cellular processing prior to secretion. These posttranslational processing events occur in organelles of the constitutive or regulated secretory pathway. The aim of this study was to examine the relationship between post-translational modifications and the secretory pathways taken by peptides derived from progastrin, the prohormone of gastrin, which in vivo is secreted by cells of the pyloric glands and stimulates the release of gastric acid. Targeting progastrin to compartments of the early secretory pathway shows that endoproteolytic processing is initiated in a pre-trans Golgi network compartment of endocrine but not non-endocrine cells. The resulting N-terminal fragments of progastrin are secreted via the constitutive pathway, whereas endoproteolytically processed C-terminal fragments are secreted via the regulated or constitutive-like pathways. C-terminal fragments derived from progastrin differ in characteristic manners in levels and patterns of carboxyamidation and tyrosine sulfation in accordance with the secretory pathway taken. Point mutations introduced into a sorting motif disrupt these patterns, suggesting that differences in post-translational modifications are attributable to differential intracellular sorting of precursors. The results suggest a two-step sorting mechanism for progastrin leading to differential secretion of processed fragments via different secretory pathways. [Abstract/Link to Full Text]

Li L, Kobayashi M, Kaneko H, Nakajima-Takagi Y, Nakayama Y, Yamamoto M
Molecular evolution of Keap1: Two Keap1 molecules with distinctive IVR structures are conserved among fish.
J Biol Chem. 2007 Dec 5;
Keap1 is a BTB-Kelch type substrate adaptor protein of the Cul3-dependent ubiquitin ligase complex. Keap1 facilitates the degradation of Nrf2, a transcription factor regulating the inducible expression of many cytoprotective genes. Through comparative genome analyses, we found that amino acid residues comprising the pocket of Keap1 that interacts with Nrf2 are highly conserved among Keap1 orthologs and related proteins in all vertebrates and in certain invertebrates, including flies and mosquitoes. The interaction between Nrf2 and Keap1 appears to be widely preserved in vertebrates. Similarly, cysteine residues corresponding to Cys273 and Cys288 in the intervening region of mouse Keap1, which are essential for the repression of Nrf2 activity in cultured cells, are conserved among Keap1 orthologs in vertebrates and invertebrates, except fish. We found that fish have two types of Keap1, Keap1a and Keap1b. To our surprise, Keap1a and Keap1b contain the cysteine residue corresponding to Cys288 and Cys273, respectively. In our analysis of zebrafish Keap1a and Keap1b activities, both Keap1a and Keap1b were able to facilitate the degradation of Nrf2 protein and repress Nrf2-mediated target gene activation. Individual mutation of either residual cysteine residue in Keap1a and Keap1b disrupted the ability of Keap1 to repress Nrf2, indicating that the presence of either Cys273 or Cys288 is sufficient for fish Keap1 molecules to fully function. These results provide an important insight into the means by which Keap1 cysteines act as sensors of electrophiles and oxidants. [Abstract/Link to Full Text]

Xi JH, Bai F, Gross J, Townsend RR, Menko AS, Andley UP
Mechanism of small heat shock protein function in vivo: A knockin mouse model demonstrates that the R49C mutation in alpha A-crystallin enhances protein insolubility and cell death.
J Biol Chem. 2007 Dec 5;
alphaA-crystallin (cryaa/HSPB4) is a small heat shock protein and molecular chaperone that prevents non-specific aggregation of denaturing proteins. Several point mutations in alphaA-crystallin gene cause congenital human cataracts by unknown mechanisms. We took a novel approach to investigate the molecular mechanism of cataract formation in vivo by creating gene knockin mice expressing the arginine 49 to cysteine mutation (R49C) in alphaA-crystallin (alphaA-R49C). This mutation has been linked with autosomal dominant hereditary cataracts in a four generation Caucasian family. Homologous recombination in embryonic stem cells was performed using a plasmid containing the C to T transition in exon 1 of the cryaa gene. alphaA-R49C heterozygosity led to early cataracts characterized by nuclear opacities. Unexpectedly, alphaA-R49C homozygosity led to small eye phenotype and severe cataracts at birth. Wild type littermates did not show these abnormalities. Lens fiber cells of alphaA-R49C homozygous mice displayed an increase in cell death by apoptosis mediated by a five-fold decrease in phosphorylated Bad, an anti-apoptotic protein, but an increase in Bcl-2 expression. However, proliferation measured by in vivo BrdU labeling did not decline. The alphaA-R49C heterozygous and homozygous knockin lenses demonstrated an increase in insoluble alphaA-crystallin and alphaB-crystallin and a surprising increase in expression of cytoplasmic -crystallin, while no changes in ss-crystallin were observed. Co-immunoprecipitation analysis showed an increased interaction between alphaA-crystallin and lens substrate proteins in the heterozygous knockin lenses. To our knowledge this is the first knockin mouse model for a crystallin mutation causing hereditary human cataract and establishes that alphaA-R49C promotes protein insolubility and cell death in vivo. [Abstract/Link to Full Text]

Kainu V, Hermansson M, Somerharju P
Electrospray ionization mass spectrometry and exogenous heavy isotope-labeled lipid species provide detailed information on aminophospholipid acyl chain remodeling.
J Biol Chem. 2007 Dec 4;
Mammalian cells maintain the phospholipid compositions of their different membranes remarkably constant. Beside de novo synthesis, degradation and intracellular trafficking, acyl chain remodeling plays an important role in phospholipid homeostasis. However, many key details of this process remain unresolved, largely due to limitations of existing methodologies. Here we describe a novel approach that allows one to study metabolism of individual phospholipid species in unprecedented detail. Forty different phosphatidylethanolamine (PE) or -serine (PS) species with a deuterium-labeled head group were synthesized and introduced to BHK21 or HeLa cells using cyclodextrin-mediated transfer. Their metabolism was then monitored in detail by electrospray ionization mass spectrometry. Atypical PE and PS species were rapidly remodeled at both sn1 and sn2 position, yielding a molecular species profile similar to that the endogenous PE and PS. In contrast, remodeling of exogenous species identical or similar to major endogenous ones was more limited and much slower. Major differences in remodeling pathways and kinetics were observed between species within a class, as well as between corresponding PE and PS species. These data along with those obtained with pharmacological inhibitors strongly suggest that multiple lipid class specific A-type phospholipases and acyl transferases are involved in aminophospholipid remodeling. In conclusion, the approach described here provides highly detailed information on remodeling of exogenously added (amino)glycerophospholipids and should thus be very helpful when elucidating the proteins and processes maintaining molecular species homeostasis. [Abstract/Link to Full Text]

Metcalfe CL, Macdonald IK, Murphy EJ, Brown KA, Raven EL, Moody PC
The tuberculosis prodrug isoniazid bound to activating peroxidases.
J Biol Chem. 2007 Dec 5;
Isoniazid (INH, isonicotinic acid hydrazine) is one of only two therapeutic agents effective in treating tuberculosis. This prodrug is activated by the heme enzyme catalase-peroxidase (KatG) endogenous to Mycobacterium tuberculosis, but the mechanism of activation is poorly understood, in part because the binding interaction has not been properly established. The class I peroxidases ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP) have very similar active site structures to KatG, and are also capable of activating isoniazid. We report here the first crystal structures of complexes of isoniazid bound to APX and CcP. These are the first structures of isoniazid bound to any activating enzymes. The structures show that isoniazid binds close to the d-heme edge in both APX and CcP, although the precise binding orientation varies slightly in the two cases. A second binding site for INH is found in APX at the g-heme edge close to the established ascorbate binding site, indicating that the g-heme edge can also support the binding of aromatic substrates. We also show that in an active site mutant of sAPX (W41A) INH can bind directly to the heme iron to become an inhibitor, and in a different mode when the distal histidine is replaced by alanine(H42A). These structures provide the first unambiguous evidence for the location of the isoniazid binding site in the class I peroxidases, and provide rationalisation of isoniazid resistance in naturally occurring KatG mutant strains of M. tuberculosis. [Abstract/Link to Full Text]

Rossi F, Garavaglia S, Montalbano V, Walsh MA, Rizzi M
Crystal structure of human kynurenine aminotransferase II, a drug target for the treatment of Schizophrenia.
J Biol Chem. 2007 Dec 7;
Kynurenic acid is an endogenous neuroactive compound whose unbalancing is involved in the pathogenesis and progression of several neurological diseases. Kynurenic acid synthesis in the human brain is sustained by the catalytic activity of two kynurenine aminotransferases, hKAT I and hKAT II. A wealth of pharmacological data highlight hKAT II as a sensible target for the treatment of neuropathological conditions characterized by a kynurenic acid excess, such as schizophrenia and cognitive impairment. We have solved the structure of human KAT II by means of the single-wavelength anomalous dispersion method at 2.3 A resolution. Although closely resembling the classical aminotransferase fold, the hKAT II architecture displays unique features. Structural comparison with a prototypical aspartate aminotranferase reveals a novel antiparallel strand-loop-strand motif that forms an unprecedented inter-subunit ss-sheet in the functional hKAT II dimer. Moreover, the N-terminal regions of hKAT II and aspartate aminotransferase appear to have converged to highly similar although two-fold symmetry-related conformations, that fulfill the same functional role. A detailed structural comparison of hKAT I and hKAT II reveals a larger and more aliphatic character to the active site of hKAT II due to the absence of the aromatic cage involved in ligand binding in hKAT I. The observed structural differences could be exploited for the rational design of highly selective hKAT II inhibitors. [Abstract/Link to Full Text]

Han Q, Robinson H, Li J
Crystal structure of human kynurenine aminotransferase II.
J Biol Chem. 2007 Dec 5;
Human kynurenine aminotransferase II (hKAT-II) efficiently catalyzes the transamination of knunrenine to kynurenic acid (KYNA). KYNA is the only known endogenous antagonist of N-methyl-D-aspartate (NMDA) receptors and is also an antagonist of 7-nicotinic acetylcholine receptors. Abnormal concentrations of brain KYNA have been implicated in the pathogenesis and development of several neurological and psychiatric diseases in humans. Consequently, enzymes involved in the production of brain KYNA have been considered potential regulatory targets. In this article, we report a 2.16 Angstrom crystal structure of hKAT-II and a 1.95 Angstrom structure of its complex with kynurenine. The protein architecture of hKAT-II reveals that it belongs to the fold-type I pyridoxal 5-phosphate (PLP) dependent enzymes. In comparison with all subclasses of fold-type I-PLP-dependent enzymes, we propose that hKAT-II represents a novel subclass in the fold-type I enzymes because of the unique folding of its first 65 N-terminal residues. This study provides a molecular basis for future effort in maintaining physiological concentrations of KYNA through molecular and biochemical regulation of hKAT-II. [Abstract/Link to Full Text]

Collom SL, Laddusaw RM, Burch AM, Kuzmic P, Perry MD, Miller GP
CYP2E1 substrate inhibition: Mechanistic interpretation through an effector site for monocyclic compounds.
J Biol Chem. 2007 Dec 4;
In this study we offer a mechanistic interpretation of the previously known, but unexplained substrate inhibition observed for CYP2E1. At low substrate concentrations, p-nitrophenol (pNP) was rapidly turned over (47 min(1)) with relatively low Km (24 muM); nevertheless, at concentrations > 100 muM, the rate of pNP oxidation gradually decreased as a second molecule bound to CYP2E1 through an effector site (Kss 260 muM), which inhibited activity at the catalytic site. 4-Methlylpyrazole (4MP) was a potent inhibitor for both sites through a mixed inhibition mechanism. The Ki for the catalytic site was 2.0 muM. Although we unable to discriminate whether an EIS or ESI complex formed, the respective inhibition constants were far lower than Kss. Bicyclic indazole (IND) inhibited catalysis through a single CYP2E1 site (Ki 0.12 muM). Similarly, 4MP and IND yielded Type II binding spectra that reflected the association of either two 4MP or one IND molecule(s) to CYP2E1, respectively. Based on computational docking studies with a homology model for CYP2E1, the two sites for monocyclic molecules, pNP and 4MP, exist within a narrow channel connecting the active site to the surface of the enzyme. Due to the presence of the heme iron, one site supports catalysis, while the other, more distal effector site binds molecules that can influence the binding orientation and egress of molecules for the catalytic site. Although IND did not bind these sites simultaneously, the presence of IND at the catalytic site blocked binding at the effector site. [Abstract/Link to Full Text]

---

Recent Articles in Bioscience, Biotechnology, and Biochemistry

---

Mega T
Plant-Type N-Glycans Containing Fucose and Xylose in Bryophyta (Mosses) and Tracheophyta (Ferns).
Biosci Biotechnol Biochem. 2007 Dec 7;
The presence of typical plant-type N-glycans (eg, M3FX, Gn2M3FX, and Le(a)2M3FX) in mosses, ferns, and other organisms was examined to determine which plant initially acquired glycosyltransferases to produce plant-type N-glycans during organic evolution. No M3FX-type N-glycan was detected in lichens (Cladonia humilis) or in any one of the three preland plants Enteromorpha prolifera, Ulva pertusa Kjellman, and Chara braunii Gmelin. In Bryophyta, M3FX-type N-glycan was detected at trace amounts in Anthocerotopsida (hornworts) and at certain amounts in Bryopsida (mosses), but not in Hepaticopsida (liverworts). Le(a)2M3FX was detected in some Bryopsida of relatively high M3FX content. Most Tracheophyta (ferns and higher plants) contained the three typical M3FX-type glycans as the main N-glycans in different ratios. These results suggest that organisms acquired xylosyltransferase and fucosyltransferase during the development of mosses from liverworts, and that later all plants retained both enzymes. Bryopsida have also obtained galactosyltransferase and fucosyltransferase to synthesize the Le(a) antigen. [Abstract/Link to Full Text]

Tamura T, Tomimatsu K, Katakura Y, Yamashita M, Matsumoto SE, Aiba Y, Jung YS, Abe Y, Fujiki T, Teruya K, Shirahata S
Anti-Peptide Antibody Production Elicited by in Vitro Immunization of Human Peripheral Blood Mononuclear Cells.
Biosci Biotechnol Biochem. 2007 Dec 7;
Human monoclonal antibodies have great potential for use in the treatment of various diseases. We have established an in vitro immunization protocol for inducing antigen-specific antibody production from human peripheral blood mononuclear cells (PBMCs). In the in vitro immunization protocol, PBMCs are pretreated with L-leucyl-L-leucine methyl ester (LLME) to remove suppressive cells, and are sensitized and cultured with a soluble antigen in the presence of IL-2, IL-4 and muramyl dipeptide for 8 d, and then an antigen-specific antibody is produced. In this study, we examined the novel possibility of an in vitro immunization protocol, specifically, whether LLME-treated PBMCs can be sensitized with a peptide antigen to produce an anti-peptide antibody. The results indicate that antigen-specific immune responses were elicited by a peptide antigen derived from rice allergen, a cholera toxin B subunit, and TNF-alpha as a sensitizing antigen in in vitro immunization. These results suggest that the in vitro immunization protocol is applicable in the generation of an anti-peptide antibody against various antigens, including food allergens, foreign antigens, and self-antigens. [Abstract/Link to Full Text]

Ohno T, Tomatsu M, Toeda K, Ohisa N
Texture of Cooked Rice Prepared from Aged Rice and Its Improvement by Reducing Agents.
Biosci Biotechnol Biochem. 2007 Dec 7;
The textures of cooked rice prepared from aged rice grains and their improvement by reducing agents were investigated. For aged rice that was stored for 5 months without air by the operation of a vacuum packing machine, the stickiness/hardness ratio of cooked rice was as low as that of aged rice stored in air. The results of electrophoresis showed that oxidation of proteins in the former was advanced to the same degree as in the latter. The stickiness/hardness ratios of the aged rice were increased by the addition of sodium sulfite, cysteine, and dithiothreitol to the cooking water. Sodium sulfite, cysteine, and dithiothreitol cleave disulfide bonds to sulfhydryl groups. Therefore, cleaving disulfide bonds to sulfhydryl groups improved the texture. The addition of them to the cooking water also increased the extractable solids at the time of heating. Hence cleaving disulfide bonds to sulfhydryl groups must increase extractable solids. Consequently, the gelatinized paste layer thickened and the thick paste layer softened the cooked rice. [Abstract/Link to Full Text]

Ito Y, Watanabe T, Nagatomo S, Seki T, Niimi S, Ariga T
Annexin A3-Expressing Cellular Phenotypes Emerge from Necrotic Lesion in the Pericentral Area in 2-Acetylaminofluoren/Carbon Tetrachloride-Treated Rat Livers.
Biosci Biotechnol Biochem. 2007 Dec 7;
Recently we found a small hepatocyte-specific protein, annexin A3 (AnxA3), in fractionated adult rat hepatocytes. Here we describe the results of an in vivo demonstration of AnxA3-expressing cellular phenotypes in the liver with 2-acetylaminofluoren (2-AAF)/carbon tetrachloride (CCl(4))-injury. In association with an elevation of alanine amino transferase (ALT) and aspartic acid amino transferase (AST) activities, hepatic AnxA3 mRNA increased markedly. AnxA3-positive cells were detected in clustered cells present in or emerging from the pericentral region. These albumin-expressed cells were histologically similar to cells expressing CD34, a hematopoietic cell marker protein. The number of clusters decreased in the days following CCl(4) treatment, and annexin-negative, but albumin-positive, oval cells appeared. We concluded that the agent-induced liver defect initially recruits bone marrow-derived cells, and that it promotes differentiation of these cells into AnxA3-positive cells, followed by emergence of the oval cells, which might have a role in the restitution of the damaged liver. [Abstract/Link to Full Text]

Nozaki H, Hayashi KI, Nishimura N, Kawaide H, Matsuo A, Takaoka D
Momilactone A and B as Allelochemicals from Moss Hypnum plumaeforme: First Occurrence in Bryophytes.
Biosci Biotechnol Biochem. 2007 Dec 7;
Momilactones A (1) and B (2), which have been identified as phytoalexins in rice, were isolated from extracts of the moss Hypnum plumaeforme. This is the first isolation and identification of momilactones as allelochemicals from a bryophyte. H. plumaeforme produces considerable amounts of momilactones (isolated yield: 8.4 mg/Kg plant for 1; 4.2 mg/Kg for 2). EtOAc extracts from H. plumaeforme and 2 showed growth inhibitory activity against angiosperms, moss, and liverwort plants. On the other hand, the growth of H. plumaeforme was insensitive to its extract and 2. Our finding suggests that momilactones play an important role as allelochemicals in this moss. [Abstract/Link to Full Text]

Kasahara Y, Takayanagi Y, Kawada T, Itoi K, Nishimori K
Impaired Thermoregulatory Ability of Oxytocin-Deficient Mice during Cold-Exposure.
Biosci Biotechnol Biochem. 2007 Dec 7;
We analyzed temperature homeostasis in oxytocin-deficient (Oxt(-/-)) mice and found that Oxt(-/-) mice exhibited lower body temperatures than wild-type animals when they were exposed to cold. Oxt(-/-) mice also showed slightly more weight gain, but there were no obvious differences in the morphology of white and brown adipose tissues as between wild-type and Oxt(-/-) mice. In cold-exposed conditions, oxytocin neurons containing c-Fos immunoreactivity existed in the paraventricular nucleus of the hypothalamus.These results suggest that the central oxytocin neurons constitute part of the thermoregulatory system involved in maintaining body temperature in cold environments. [Abstract/Link to Full Text]

Tanabe S, Hase M, Yano T, Sato M, Fujimura T, Akiyama H
A Real-Time Quantitative PCR Detection Method for Pork, Chicken, Beef, Mutton, and Horseflesh in Foods.
Biosci Biotechnol Biochem. 2007 Dec 7;
A rapid real-time quantitative PCR method to detect trace amounts of pork, beef, chicken, mutton, and horseflesh in foods was developed. The primers and TaqMan MGB (minor groove binder) probes were designed on the gene encoding cytochrome b for the specific detection of each species. The limit of quantification of this method was found to be 100 fg/mul of each mitochondrial DNA in 10 ng/mul of the wheat mitochondrial DNA matrix. The calculated R(2) values of the standard curves for the five species ranged between 0.994 and 0.999. This method would be particularly useful in the detection of hidden meat mince in processed foods, which would verify food labeling and gain consumers' trust. [Abstract/Link to Full Text]

Li JB, Hashimoto F, Shimizu K, Sakata Y
Anthocyanins from Red Flowers of Camellia reticulata LINDL.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2833-6.
Ten anthocyanins, cyanidin 3-sambubioside, 3-glucoside and their acylated derivatives, cyanidin 3-lathyroside and cyanidin 3-galactoside, were isolated from red flowers of Camellia reticulata. Their structures were determined on the basis of spectroscopic analyses, and the chemotaxonomic distribution of the accumulated anthocyanins in the petals of wild Camellia reticulata and C. pitardii var. yunnanica is discussed. [Abstract/Link to Full Text]

Sugio T, Taha TM, Kanao T, Takeuchi F
Increase in Fe(2+)-Producing Activity during Growth of Acidithiobacillus ferrooxidans ATCC23270 on Sulfur.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2663-9.
When Acidithiobacillus ferrooxidans ATCC23270 cells, grown for many generations on sulfur were grown in sulfur medium with and without Fe(3+), the bacterium markedly increased not only in iron oxidase activity but also in Fe(2+)-producing sulfide:ferric ion oxidoreductase (SFORase) activity during the early log phase, and retained part of these activities during the late log phase. The activity of SFORase, which catalyzes the production of Fe(2+) from Fe(3+) and sulfur, of sulfur-grown cells was approximately 10-20 fold higher than that of iron-grown cells. aa(3) type cytochrome c oxidase, an important component of iron oxidase in A. ferrooxidans, was partially purified from sulfur-grown cells. A. ferrooxidans ATCC23270 cells grown for many generations on sulfur had the ability to grow on iron as rapidly as that did iron-grown cells. These results suggest that both iron oxidase and Fe(2+)-producing SFORase have a role in the energy generation of A. ferrooxidans ATCC23270 from sulfur. [Abstract/Link to Full Text]

Katayama K, Shimazaki K, Tazaki H, Hasa Y, Miyoshi M, Koshino H, Furuki T, Nabeta K
Parvitexins A-E, Clerodane-Type Diterpenes Isolated from the in Vitro-Cultured Liverwort, Scapania parvitexta.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2751-8.
New clerodane-type diterpenes, designated as parvitexins A (1)-E (5), were isolated from the in vitro-cultured liverwort, Scapania parvitexta. These compounds were determined to be monoacetylated clerodane-type diterpenes based on spectroscopic evidence. [Abstract/Link to Full Text]

Kondo Y, Tadokoro E, Matsuura M, Iwasaki K, Sugimoto Y, Miyake H, Takikawa H, Sasaki M
Synthesis and seed germination stimulating activity of some imino analogs of strigolactones.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2781-6.
Strigolactones are germination stimulants for seeds of the root parasitic weeds, Striga and Orobanche spp. The imino analog of GR24 showed moderate germination stimulating activity against the seeds of S. hermonthica. The seed germination stimulating activity of some phenyliminoacetates and phenyliminoacetonitriles was also examined. The degree of activity of the phenyliminoacetate was less than that of the phenylacrylates. On the other hand, the degree of activity of the phenyliminoacetonitrile was comparable to that of the phenylacrylonitriles. Among the tested compounds, the 3-pyridyliminoacetonitrile showed higher activity against the seeds of O. crenata than GR24. These findings demonstrate that it is not always essential to have the Michael acceptor of the C-D ring junction moiety which has been proposed to react with nucleophilic species presented at the target site to enhance the activity. [Abstract/Link to Full Text]

Yajima A, Yamamoto M, Nukada T, Yabuta G
Efficient and Stereo-Selective Syntheses of Three Stereoisomers of the Sex Pheromone of the Cowpea Weevil, Callosobruchus maculatus.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2720-4.
An efficient and stereoselective synthesis of three stereoisomers of the sex pheromone of the cowpea weevil, Callosobruchus maculatus, is reported. [Abstract/Link to Full Text]

Morimura K, Yamazaki C, Hattori Y, Makabe H, Kamo T, Hirota M
A Tyrosinase Inhibitor, Daedalin A, from Mycelial Culture of Daedalea dickinsii.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2837-40.
A chromene-type compound, daedalin A (1), was isolated from mycelial culture broth of Daedalea dickinsii. Based on spectroscopic data, the structure of 1 was found to be (2R)-6-hydroxy-2-hydroxymethyl-2-methyl-2H-chromene. Daedalin A (1) strongly inhibited the activity of tyrosinase (IC(50): 194 muM). In addition, 1 also showed 1,1-diphenyl-2-picrylhydrazyl scavenging activity (IC(50): 16.9 muM) and superoxide anion scavenging activity (IC(50): 28.5 muM). [Abstract/Link to Full Text]

Chen J, Bai G, Cao Y, Gao Z, Zhang Q, Zhu Y, Yang W
One-Step Purification of a Fusion Protein of Glucagon-Like Peptide-1 and Human Serum Albumin Expressed in Pichia pastoris by an Immunomagnetic Separation Technique.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2655-62.
Glucagon-like peptide-1 (GLP-1) has great therapeutic potential to treat diabetes type 2, mainly due to its unique glucose-dependent stimulation of insulin secretion profiles, but its clinical application is limited by its short half-life in vivo, which resultes from degradation by dipeptidyl peptidase IV and/or renal clearance. Developing long-acting GLP-1 analogs is therefore an important step toward using them therapeutically. In this study, the GLP-1/human serum albumin (HSA) fusion protein gene was cloned into the secretor type expression vector pPIC9K and subsequently expressed in Pichia pastoris. The expression quantity reached 58.5 mg/l in small-scale incubation. After optimization and characterization, the GLP-1/HSA fusion protein was successfully purified from the supernatant of the broth using immunomagnetic cellulose microspheres. HPLC showed that the purified GLP-1/HSA had an overall purity of 93.9%, and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) confirmed the fusion protein exhibited the expected molecular mass of 70 kDa. Furthermore, that analysis of in vivo activity indicated that GLP-1/HSA reduced the blood glucose level after intraperitoneal administration to Chinese Kunming mice in a dose-dependent manner, and the effects held significantly 4 h after administration. Overall, this study illustrates the development of a long-acting GLP-1/HSA fusion protein expressed in Pichia pastoris. [Abstract/Link to Full Text]

Wakai S, Tsujita M, Kikumoto M, Manchur MA, Kanao T, Kamimura K
Purification and Characterization of Sulfide:Quinone Oxidoreductase from an Acidophilic Iron-Oxidizing Bacterium, Acidithiobacillus ferrooxidans.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2735-42.
Sulfide:quinone oxidoreductase (SQR) was purified from membrane of acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans NASF-1 cells grown on sulfur medium. It was composed of a single polypeptide with an apparent molecular mass of 47 kDa. The apparent K(m) values for sulfide and ubiquinone were 42 and 14 muM respectively. The apparent optimum pH for the SQR activity was about 7.0. A gene encoding a putative SQR of A. ferrooxidans NASF-1 was cloned and sequenced. The gene was expressed in Escherichia coli as a thioredoxin-fusion protein in inclusion bodies in an inactive form. A polyclonal antibody prepared against the recombinant protein reacted immunologically with the purified SQR. Western blotting analysis using the antibody revealed an increased level of SQR synthesis in sulfur-grown A. ferrooxidans NASF-1 cells, implying the involvement of SQR in elemental sulfur oxidation in sulfur-grown A. ferrooxidans NASF-1 cells. [Abstract/Link to Full Text]

Kadokura K, Sakamoto Y, Saito K, Ikegami T, Hirano T, Hakamata W, Oku T, Nishio T
Production and Secretion of a Recombinant Vibrio parahaemolyticus Chitinase by Escherichia coli and Its Purification from the Culture Medium.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2848-51.
An open reading frame encoding the chitinase gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome. An expression plasmid containing the gene was introduced into Escherichia coli cells, and recombinant chitinase (Pa-rChi) was produced and secreted into the culture medium with the aid of the signal peptide. Pa-rChi was purified and its substrate specificity was determined. [Abstract/Link to Full Text]

Lee YH, Hong SW, Jun W, Cho HY, Kim HC, Jung MG, Wong J, Kim HI, Kim CH, Yoon HG
Anti-histone acetyltransferase activity from allspice extracts inhibits androgen receptor-dependent prostate cancer cell growth.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2712-9.
Histone acetylation depends on the activity of two enzyme families, histone acetyltransferase (HAT) and deacetylase (HDAC). In this study, we screened various plant extracts to find potent HAT inhibitors. Hot water extracts of allspice inhibited HAT activity, especially p300 and CBP (40% at 100 mug/ml). The mRNA levels of two androgen receptor (AR) regulated genes, PSA and TSC22, decreased with allspice treatment (100 mug/ml). Importantly, in IP western analysis, AR acetylation was dramatically decreased by allspice treatment.Furthermore, chromatin immunoprecipitation indicated that the acetylation of histone H3 in the PSA and B2M promoter regions was also repressed. Finally, allspice treatment reduced the growth of human prostate cancer cells, LNCaP (50% growth inhibition at 200 mug/ml). Taken together, our data indicate that the potent HAT inhibitory activity of allspice reduced AR and histone acetylation and led to decreased transcription of AR target genes, resulting in inhibition of prostate cancer cell growth. [Abstract/Link to Full Text]

Izawa S, Kita T, Ikeda K, Miki T, Inoue Y
Formation of Cytoplasmic P-Bodies in Sake Yeast during Japanese Sake Brewing and Wine Making.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2800-7.
Recent studies have revealed that cytoplasmic processing bodies (P-bodies) play important roles in the control of eukaryotic gene expression in response to stress. Since the formation of P-bodies is in dynamic competition with translation, the status of translation is reflected in the assembly and disassembly of P-bodies in eukaryotic cells. During the brewing of Japanese sake and the making of wine, yeast cells are exposed to stress caused by increases in the concentration of ethanol. Here we found that ethanol stress enhances the formation of P-bodies in yeast cells in SD medium. In the wine-making process, P-body formation was also enhanced as alcoholic fermentation proceeded, but the formation of P-bodies was not simply affected by the ethanol concentration in the sake mash. These findings suggest differences in the rate of translation and the cytoplasmic mRNA flux during the sake brewing and wine making processes. [Abstract/Link to Full Text]

Taniguchi Y, Mizote A, Kohno K, Iwaki K, Oku K, Chaen H, Fukuda S
Effects of Dietary Lactosucrose (4(G)-beta-D-galactosylsucrose) on the IgE Response in Mice.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2766-73.
In this study, we examined the effects of dietary lactosucrose (LS, a non-digestible oligosaccharide) on the IgE response in mice immunized with ovalbumin (OVA)/alum. In addition to IgG1 and IgG2a responses, the anti-OVA IgE response in mice fed LS diets was dose-dependently suppressed, as compared with the control mice, while the serum total IgG levels were comparable. Moreover, dietary LS feeding inhibited antigen-specific IgE and IgG1 productions even after a second immunization. Regarding with cytokine production, when stimulated in vitro with OVA, splenocytes obtained from LS-fed mice produced a similar level of IFN-gamma, and lower levels of IL-4 and IL-5, as compared with the control mice. But IL-10 production by OVA-stimulated splenocytes was augmented in LS-fed mice, suggesting that IL-10 producing cells are responsible for the immunoregulatory effect of LS. Our findings indicate the further possibility that dietary LS supplementation can be used to prevent IgE-mediated allergic diseases. [Abstract/Link to Full Text]

Takemoto D, Takekawa Y, Soest RW, Fusetani N, Matsunaga S
Poecillastrin D: A New Cytotoxin of the Chondropsin Class from Marine Sponge Jaspis serpentina.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2697-700.
Poecillastrin D (2) was isolated together with poecillastrin C (1) from the deep sea sponge, Japsis serpentina. Its structure was elucidated to be that of a macrolide lactam by spectroscopic methods. These compounds showed potent cytotoxicity against various tumor cell lines. [Abstract/Link to Full Text]

Tanabe S, Kinuta Y, Yasumatsu H, Takayanagi M, Kobayashi S, Takido N, Sugiyama M
Effects of Citrus unshiu Powder on the Cytokine Balance in Peripheral Blood Mononuclear Cells of Patients with Seasonal Allergic Rhinitis to Pollen.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2852-5.
We evaluated the effects of a 50% methanol extract of Citrus unshiu powder (MEC) on cytokines in peripheral blood mononuclear cells (PBMCs) obtained from patients with seasonal allergic rhinitis to cedar pollen. The levels of cytokines, such as TNF-alpha, IFN-gamma, IL-2, IL-4, IL-5, IL-10, IL-12 (p70), IL-13, and GM-CSF, produced by pollen-stimulated PBMC were measured. We found that MEC suppressed pollen-induced TNF-alpha release and increased IFN-gamma release from PBMCs. The results suggest that Citrus unshiu powder has an immunomodulatory effect in vitro and that its use could improve seasonal allergic rhinitis symptoms. [Abstract/Link to Full Text]

Watanabe S, Yamashita K, Ochiai N, Fukumori F, Ichiishi A, Kimura M, Fujimura M
OS-2 Mitogen Activated Protein Kinase Regulates the Clock-Controlled Gene ccg-1 in Neurospora crassa.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2856-9.
OS-2 MAP kinase is involved in osmoadaptation in Neurospora crassa. Clock-controlled genes ccg-1, bli-3, and con-10 were induced by osmotic stress in an OS-2 dependent manner. In contrast, osmotic stress did not affect the expression of clock genes frq, wc-1, and wc-2 or of clock-controlled genes ccg-2 and bli-4. These results suggest that OS-2 participates in the regulation of certain circadian-clock output genes. [Abstract/Link to Full Text]

Watanabe T, Matsuo I, Maruyama J, Kitamoto K, Ito Y
Identification and Characterization of an Intracellular Lectin, Calnexin, from Aspergillus oryzae Using N-Glycan-Conjugated Beads.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2688-96.
Recently, asparagine-linked oligosaccharides (N-glycans) have been found to play a pivotal role in glycoprotein quality control in the endoplasmic reticulum (ER). In order to screen proteins interacting with N-glycans, we developed affinity chromatography by conjugating synthetic N-glycans on sepharose beads. Using the affinity beads with the dodecasaccharide Glc(1)Man(9)GlcNAc(2), one structure of the N-glycans, a 75-kDa protein, was isolated from the membranous fraction including the ER in Aspergillus oryzae. By LC-MS/MS analysis using the A. oryzae genome database, the protein was identified as one (AO090009000313) sharing similarities with calnexin. Further affinity chromatographic experiments suggested that the protein specifically bound to Glc(1)Man(9)GlcNAc(2), similarly to mammalian calnexins. We designated the gene AoclxA and expressed it as a fusion gene with egfp, revealing the ER localization of the AoClxA protein. Our results suggest that our affinity chromatography with synthetic N-glycans might help in biological analysis of glycoprotein quality control in the ER. [Abstract/Link to Full Text]

Tomita K, Nagura T, Okuhara Y, Nakajima-Adachi H, Shigematsu N, Aritsuka T, Kaminogawa S, Hachimura S
Dietary melibiose regulates th cell response and enhances the induction of oral tolerance.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2774-80.
We examined how dietary melibiose affected the T-helper (Th) cell responses induced by an orally fed antigen in ovalbumin (OVA)-specific T cell receptor transgenic mice (OVA 23-3). Dietary melibiose markedly decreased the Th2 type responses as shown by a significant decrease in the interleukin (IL)-4 production and T cell proliferative response induced by sensitization from the 7-d oral administration of OVA. It was additionally observed that the Th1 type responses tended to decrease. We therefore examined the effect of melibiose feeding on the induction of immunological tolerance induced by the oral administration of an antigen (oral tolerance). The Th cell responses induced in BALB/c mice by subcutaneous immunization with OVA were suppressed by the prior oral administration of OVA. Such responses in the OVA-fed and immunized mice were further diminished by dietary melibiose. These results suggest that dietary melibiose strongly affected the Th cell responses to an ingested antigen, and further demonstrate the potential of melibiose to enhance the induction of oral tolerance. [Abstract/Link to Full Text]

Kajitani N, Kobuchi H, Fujita H, Yano H, Fujiwara T, Yasuda T, Utsumi K
Mechanism of A23187-Induced Apoptosis in HL-60 Cells: Dependency on Mitochondrial Permeability Transition but Not on NADPH Oxidase.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2701-11.
Calcium ions (Ca(2+)) are involved in a number of physiological cellular functions including apoptosis. An elevation in intracellular levels of Ca(2+) in A23187-treated HL-60 cells was associated with the generation of both intracellular and extracellular reactive oxygen species (ROS) and induction of apoptotic cell death. A23187-induced apoptosis was prevented by cyclosporin A, a potent inhibitor of mitochondrial permeability transition (MPT). The generation of extracellular ROS was suppressed by the NADPH oxidase inhibitor diphenylene iodonium, and by superoxide dismutase, but these agents had no effect on A23187-induced apoptosis. In contrast, the blocking of intracellular ROS by a cell-permeant antioxidant diminished completely the induction of MPT and apoptosis. In isolated mitochondria, the addition of Ca(2+) induced a typical MPT concomitant with the generation of ROS, which leads to augmentation of intracellular ROS levels. These results indicate that intracellular not extracellular ROS generated by A23187 is associated with the opening of MPT pores that leads to apoptotic cell death. [Abstract/Link to Full Text]

Ichinomiya M, Uchida H, Koshi Y, Ohta A, Horiuchi H
A Protein Kinase C-Encoding Gene, pkcA, Is Essential to the Viability of the Filamentous Fungus Aspergillus nidulans.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2787-99.
A protein kinase C (PKC)-encoding gene (pkcA) was isolated from the filamentous fungus Aspergillus nidulans. Although we attempted to isolate pkcA deletion mutants, we obtained only heterokaryons that had both DeltapkcA and pkcA(+) nuclei. Conidia produced by the heterokaryon germinated. The germ tubes, however, lysed frequently and no colony formation was observed, indicating that the pkcA gene is essential to the viability of A. nidulans. We constructed conditional mutants (alcA(p)-pkcA mutants) that expressed pkcA under the control of the alcA promoter (alcA(p)). Under alcA(p)-repressing conditions, their colonies were smaller than those of the wild-type strains and their hyphae lysed frequently. These phenotypes were not remedied under moderate- or high-osmolarity conditions; the growth defect deteriorated further under the latter. Under alcA(p)-inducing conditions, the alcA(p)-pkcA mutants also showed growth-sensitivity to cell wall destabilizing agents. These results indicate that pkcA plays an important role in the maintenance of cell integrity. [Abstract/Link to Full Text]

Fukuda T, Isogawa D, Takagi M, Kato-Murai M, Kimoto H, Kusaoke H, Ueda M, Suye S
Yeast Cell-Surface Expression of Chitosanase from Paenibacillus fukuinensis.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2845-7.
To produce chitoorigosaccharides using chitosan, we attempted to construct Paenibacillus fukuinensis chitosanase-displaying yeast cells as a whole-cell biocatalyst through yeast cell-surface engineering. The localization of the chitosanase on the yeast cell surface was confirmed by immunofluorescence labeling of cells. The chitosanase activity of the constructed yeast was investigated by halo assay and the dinitrosalicylic acid method. [Abstract/Link to Full Text]

Sezutsu H, Kajiwara H, Kojima K, Mita K, Tamura T, Tamada Y, Kameda T
Identification of Four Major Hornet Silk Genes with a Complex of Alanine-Rich and Serine-Rich Sequences in Vespa simillima xanthoptera Cameron.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2725-34.
Hornet silk, a fibrous protein in the cocoon produced by the larva of the vespa, is composed of four major proteins. In this study, we constructed silk-gland cDNA libraries from larvae of the hornet Vespa simillima xanthoptera Cameron and deduced the full amino acid sequences of the four hornet silk proteins, which were named Vssilk 1-4 in increasing order of molecular size. Portions of the amino acid sequences of the four proteins were confirmed by Matrix-assisted laser desorption/ionization-time of flight/mass spectrometry (MALDI-TOF/MS) and N-terminal protein sequencing. The primary sequences of the four Vssilk proteins (1-4) were highly divergent, but the four proteins had some common properties: (i) the amino acid compositions of all four proteins were similar to each other in that the well-defined and characteristic repetitive patterns present in most of the known silk proteins were absent; and (ii) the characteristics of the amino acid sequences of the four proteins were also similar in that Ser-rich structures such as sericin were localized at both ends of the chains and Ala-rich structures such as fibroin were found in the center. These characteristic primary structures might be responsible for the coexisting alpha-helix and beta-sheet conformations that make up the unique secondary structure of hornet silk proteins in the native state. Because heptad repeat sequences of hydrophobic residue are present in the Ala-rich region, we believe that the Ala-rich region of hornet silk predominantly forms a coiled coil with an alpha-helix conformation. [Abstract/Link to Full Text]

Tsuruoka N, Yamato R, Sakai Y, Yoshitake Y, Yonekura H
Promotion by collagen tripeptide of type I collagen gene expression in human osteoblastic cells and fracture healing of rat femur.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2680-7.
Peptides produced by the enzymatic degradation of collagens are reported to have various activities of biological and medical interest. The mechanisms underlying their actions are, however, poorly understood. We have produced, by collagenase digestion of type I collagen, a highly purified, non-antigenic, and low allergenic tripeptide fraction (collagen tripeptide, Ctp). We report here the effects of Ctp on the in vivo bone fracture healing and in vitro calcification of osteoblastic cells. An oral administration of Ctp to rats with a femur fracture accelerated the fracture healing. Ctp apparently stimulated the calcification of human osteoblastic cells in culture. This osteotrophic effect was accompanied by a significant increase in type I collagen protein production and its mRNA levels. DNA microarray and quantitative RT-PCR analyses demonstrated that Ctp upregulated the bone-specific transcription factor, Osterix, suggesting that the induction of type I collagen gene expression by Ctp was mediated by upregulation of this factor. [Abstract/Link to Full Text]

Miura D, Miura Y, Yagasaki K
Effect of apple polyphenol extract on hepatoma proliferation and invasion in culture and on tumor growth, metastasis, and abnormal lipoprotein profiles in hepatoma-bearing rats.
Biosci Biotechnol Biochem. 2007 Nov;71(11):2743-50.
The effect of apple polyphenol extract (APE) on the proliferation and invasion of a rat ascites hepatoma cell line of AH109A was examined in vitro. APE suppressed both the hepatoma proliferation and invasion in a dose-dependent manner up to 200 mug/ml. Serum obtained from rats orally given APE also inhibited hepatoma proliferation and invasion when added to the culture medium. Subsequently, the effect of dietary APE on growth and the metastasis of AH109A hepatomas were investigated in vivo. APE reduced the growth and metastasis of solid hepatomas and significantly suppressed the serum lipid peroxide level in rats transplanted with AH109A. APE also suppressed the serum very-low-density lipoprotein + low-density lipoprotein (VLDL + LDL)-cholesterol level. These in vitro and in vivo findings suggest that APE has anti-hepatoma activities. [Abstract/Link to Full Text]

---

Recent Articles in Experimental & Molecular Medicine

---

Oh KS, Kim EY, Yoon M, Lee CM
Swim training improves leptin receptor deficiency-induced obesity and lipid disorder by activating uncoupling proteins.
Exp Mol Med. 2007 Jun 30;39(3):385-94.
Leptin receptor deficiency causes morbid obesity and hyperlipidemia in mice. Since physical exercise enhances energy expenditure, it is an important part of successful weight-control regimens. We investigated the mechanism by which swim training regulates leptin receptor deficiency-induced obesity and lipid disorder in a mouse model of obesity (obese db/db mouse). Swim training for 6 weeks significantly decreased body weight gain and adipose tissue mass in both sexes of obese and lean mice, compared to their respective sedentary controls. These effects were particularly evident in obese mice. Swim training also caused significant decreases in serum levels of triglycerides, free fatty acids and total cholesterol in both obese and lean mice. In obese mice, swim training increased the levels of mRNAs and proteins encoding uncoupling protein 1 (UCP1), UCP2 and UCP3 in brown adipose tissue, white adipose tissue and skeletal muscle, respectively. In conclusion, these findings suggest that, in mice, swim training can effectively prevent body weight gain, adiposity and lipid disorders caused by leptin receptor deficiency, in part through activation of UCPs in adipose tissue and skeletal muscle, which may contribute to alleviating metabolic syndromes, such as obesity, hyperlipidemia and type 2 diabetes. [Abstract/Link to Full Text]

Lee MK, Kang SJ, Poncz M, Song KJ, Park KS
Resveratrol protects SH-SY5Y neuroblastoma cells from apoptosis induced by dopamine.
Exp Mol Med. 2007 Jun 30;39(3):376-84.
Dopamine (DA) is an oxidant that may contribute to the degeneration of dopaminergic neurons. The present study demonstrates that DA-induced cytotoxicity in human-derived neurotypic cells, SH-SY5Y, is prevented by resveratrol, one of the major antioxidative constituents found in the skin of grapes. SH-SY5Y cells, a neuroblastoma cell line, treated with DA at 300 and 500 microM for 24 h underwent apoptotic death as determined by characteristic morphological features, including nuclear condensation, and loss of mitochondrial membrane potential (MMP). Flow cytometric analysis using Annexin V showed that DA can induce significant and severe apoptosis. Exposure to resveratrol (5 microM) for 1 h prior to the DA treatment attenuated DA-induced cytotoxicity, and rescued the loss of MMP. To investigate the apoptotic signaling pathways relevant to the restoration of DA-induced apoptosis by resveratrol, we carried out quantitative analysis of Bcl-2, caspase-3, and cleaved poly ADP-ribose polymerase (PARP) by immunoblot analysis. Resveratrol pretreatment led to a decrease in cleavage of PARP, an increase in the Bcl-2 protein, and activation of caspase-3. These results suggest that DA may be a potential oxidant of neuronal cells at biologically relevant concentrations. Resveratrol may protect SH-SY5Y cells against this cytotoxicity, reducing intracellular oxidative stress through canonical signal pathways of apoptosis and may be of biological importance in the prevention of a dopaminergic neurodegenerative disorder such as Parkinson disease. [Abstract/Link to Full Text]

Hur GY, Lee SY, Lee SH, Kim SJ, Lee KJ, Jung JY, Lee EJ, Kang EH, Jung KH, Lee SY, Kim JH, Shin C, Shim JJ, In KH, Kang KH, Yoo SH
Potential use of an anticancer drug gefinitib, an EGFR inhibitor, on allergic airway inflammation.
Exp Mol Med. 2007 Jun 30;39(3):367-75.
The EGFR plays an essential role in goblet cell hyperplasia and mucus hypersecretion. EGFR has an intrinsic tyrosine kinase activity that, when activated, induces the production of MUC5AC through the signaling kinase cascade in the airway epithelium. We have investigated the effects of an EGFR tyrosine kinase inhibitor, gefitinib, on ovalbumin (OVA)-induced, allergic inflammation in airway epithelia of mice. OVA-sensitized mice were pretreated with gefitinib at two different doses (12.5 and 50 mg/kg) and then challenged with OVA. The OVA challenge increased the total cell count and eosinophil count in bronchoalveolar lavage fluid (BALF), as well as the concentrations of T-helper2 (Th2) cytokines, such as IL-4 and IL-13, overall eosinophil recruitment in the lung tissue and airway hyperresponsiveness (AHR). Pretreatment with gefitinib reduced the inflammatory cell counts and released cytokine concentrations (IL-4 and IL-13) in BALF, as well as eosinophil recruitment in the lungs and AHR, in a dose-dependent manner. This was associated with decreased EGFR and Akt phosphorylation. We showed that gefinitib inhibits EGFR and phosphoinositol 3'-kinase (PI3K)/Akt activation which were activated in OVA sensitized mice. These findings suggest that inhibitors of the EGFR cascade may have a role in the treatment of asthma. [Abstract/Link to Full Text]

Shin A, Kang D, Choi JY, Lee KM, Park SK, Noh DY, Ahn SH, Yoo KY
Cytochrome P450 1A1 (CYP1A1) polymorphisms and breast cancer risk in Korean women.
Exp Mol Med. 2007 Jun 30;39(3):361-6.
Cytochrome P450 1A1 (CYP1A1) is involved in the 2-hydroxylation of estrogen, the hormone that plays a critical role in the etiology of breast carcinoma. We evaluated the associations between two CYP1A1 polymorphisms [MspI (rs4646903); Ile462Val (rs1048943)] and breast cancer in a multicenter case-control study of 513 breast cancer cases and 447 controls in Korea. Women carrying the T allele of the CYP1A1 MspI polymorphism were found to have a 1.72-fold (95% CI 1.11-2.68) greater risk of developing breast cancer. No association was found between any CYP1A1 Ile462Val polymorphism and breast cancer. Haplotype analysis of the two loci showed that the CA haplotype was associated with the lowest risk of breast cancer, and CA/CA diplotypes were associated with a lower risk of breast cancer [OR=0.28 (0.13-0.61)] than others/others diplotypes. Moreover, this reduced risk was more pronounced among women with a lower body mass index (BMI) [OR=0.18 (0.06-0.58)] or with a shorter lifetime exposure to estrogen [OR=0.23 (0.07-0.81)]. The results obtained suggest that the CYP1A1 MspI polymorphisms could affect susceptibility to breast cancer. [Abstract/Link to Full Text]

Roh MS, Seo MS, Kim Y, Kim SH, Jeon WJ, Ahn YM, Kang UG, Juhnn YS, Kim YS
Haloperidol and clozapine differentially regulate signals upstream of glycogen synthase kinase 3 in the rat frontal cortex.
Exp Mol Med. 2007 Jun 30;39(3):353-60.
Glycogen synthase kinase 3 (GSK3) was recently suggested to be a potential target of psychotropics used in psychiatric illnesses such as schizophrenia and bipolar disorder. Relevant studies have found that antipsychotic drugs regulate GSK3 activity via an increase in either inhibitory serine phosphorylation or amount of GSK3 after acute or subchronic treatment. Recent evidence shows that GSK3 is regulated by dopaminergic or serotonergic systems implicated in the pathophysiology and treatment mechanisms of schizophrenia and bipolar disorder. Therefore, antipsychotics may regulate GSK3 via antagonizing dopaminergic or serotonergic activity. However, the signaling pathway that is involved in GSK3 regulation by dopaminergic or serotonergic systems has not been well established. Haloperidol is a typical antipsychotic with potent dopamine D(2) receptor antagonism. Clozapine is an atypical antipsychotic with potent serotonin 5HT(2) receptor antagonism. We injected rats with haloperidol or clozapine and examined the phosphorylation and amount of GSK3alpha/beta and its well-known upstream regulators Akt and Dvl in the rat frontal cortex by Western blotting. Both haloperidol and clozapine induced Ser21/9 phosphorylation of GSK3GSK3alpha/beta. Haloperidol increased the Ser473 phosphorylation of Akt transiently, whereas clozapine maintained the increase for 1 h. Haloperidol did not affect the phosphorylation and amount of Dvl, whereas clozapine increased both phosphorylation and the amount of Dvl. Our results suggest that GSK3 activity may be regulated by both typical and atypical antipsychotics and that Akt or Dvl, depending on the D(2)- or 5HT(2)- receptor antagonism properties of typical and atypical antipsychotics, mediate the regulation differently. [Abstract/Link to Full Text]

Moon EY, Ryu SK
TACI:Fc scavenging B cell activating factor (BAFF) alleviates ovalbumin-induced bronchial asthma in mice.
Exp Mol Med. 2007 Jun 30;39(3):343-52.
Asthma was induced by the sensitization and challenge with ovalbumin (OVA) in mice. B-cell activating factor (BAFF) plays a role in mature B cell generation and maintenance. Here, we investigated whether, BAFF expression was changed in OVA-induced mice and whether the control of BAFF expression level alleviates the symptom of bronchial asthma. BAFF expression was detected in alveolar-associated cells surrounding bronchi of OVA-induced mouse lung tissues. BAFF protein was also increased in OVA-induced mouse serum. The increased BAFF transcripts was detected in OVA-induced mouse splenocytes. OVA-induced asthma was associated with the increased number of eosinophils in bronchoalveolar lavage fluid (BALF). When TACI:Fc scavenging soluble BAFF was injected to OVA-induced mice, a significant inhibition was detected in the thickness of airway smooth muscle and glycol-containing cellular elements in airway that were visualized by hematoxylin/eosin Y and periodic acid-Schiff staining, respectively. In addition, when mice were treated with TACI:Fc protein, BAFF protein level was decreased in alveolar-associated cells surrounding bronchi of OVA-induced mouse lung tissues compared to control mice. When compared to OVA-induced control, TACI:Fc treatment reduced the percentage of non-lymphoid cells and no changes were detected in lymphoid cell population. Hypodiploid cell formation in BALF was decreased by OVA-challenge but it was recovered by TACI:Fc treatment. Collectively, data suggest that asthmatic symptom could be alleviated by scavenging BAFF and then BAFF could be a novel target for the development of anti-asthmatic agents. [Abstract/Link to Full Text]

Choi YH, Kang H, Lee WK, Kim T, Rhim H, Yu YG
An inhibitory compound against the interaction between G alpha(s) and the third intracellular loop region of serotonin receptor subtype 6 (5-HT6) disrupts the signaling pathway of 5-HT6.
Exp Mol Med. 2007 Jun 30;39(3):335-42.
Serotonin receptor subtype 6 (5-HT(6)) is a neurotransmitter receptor, which is involved in various brain functions such as memory and mood. It mediates signaling via the interaction with a stimulatory G-protein. Especially, the third intracellular loop (iL3) of 5-HT(6) and the alpha subunit of stimulatory G protein (G alpha(s)) are responsible for the signaling process of 5-HT(6). Chemical compounds that could inhibit the interaction between the iL3 region of 5-HT(6) and G alpha(s) were screened from a chemical library consisted of 5,600 synthetic compounds. One of the identified compounds bound to G alpha(s) and effectively blocked the interaction between G alpha(s) and the iL3 region of 5-HT(6). The identified compound was further shown to reduce the serotonin-induced accumulation of cAMP in 293T cells transformed with 5-HT(6) cDNA. It also lowered the Ca(2+) efflux induced by serotonin in cells expressing 5-HT(6) and chimeric G alpha(s5/q). These results indicate that the interaction between the iL3 of 5-HT(6) and G alpha(s) can be exploited for screening of regulatory compounds against the signaling pathway of 5-HT(6). [Abstract/Link to Full Text]

Alvarez-Aguilar C, Enríquez-Ramírez ML, Figueroa-Nuńez B, Gómez-García A, Rodríguez-Ayala E, Morán-Moguel C, Farías-Rodríguez VM, Mino-León D, López-Meza JE
Association between angiotensin-1 converting enzyme gene polymorphism and the metabolic syndrome in a Mexican population.
Exp Mol Med. 2007 Jun 30;39(3):327-34.
Metabolic Syndrome (MS) is recognized as a cluster of cardiovascular risk factors. All components of MS have a genetic base. Genes of the renin angiotensin system are potential candidate genes for MS. We investigated whether angiotensin converting enzyme (ACE) gene polymorphism increases susceptibility to MS as an entity in a Mexican population. In a cross-sectional study, 514 individuals were studied including 245 patients with MS and 269 subjects without MS criteria. ACE gene polymorphism was detected using PCR. MS was defined according to The National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) criteria, except that the raised fasting plasma glucose <or=100 mg/dl criterion for identification of intolerance fasting glucose was modified in accordance with the suggestion of the American Diabetes Association. Patients with MS were significantly different from subjects without MS in relation to mean body mass index (BMI), waist circumference (WC), systolic blood pressure, diastolic blood pressure, glucose, total cholesterol (C), triglycerides, HDL-C, and LDL-C (P<0.0001). The differences in the mean BMI, WC, glucose, total cholesterol, triglycerides, LDL-C, and HDL-C were maintained in patients with the MS and DD genotypes (P<0.01). The DD genotype was strongly associated with MS (adjusted OR=5.48, 95% CI 3.20-9.38, P<0.0001). We concluded that the DD genotype increases susceptibility to MS in a Mexican population. These results indicate that pharmacological and non-pharmacological treatment and a reduction in body fat will have important therapeutic implications in this disease. [Abstract/Link to Full Text]

Park BC, Lee YS, Park HJ, Kwak MK, Yoo BK, Kim JY, Kim JA
Protective effects of fustin, a flavonoid from Rhus verniciflua Stokes, on 6-hydroxydopamine-induced neuronal cell death.
Exp Mol Med. 2007 Jun 30;39(3):316-26.
6-Hydroxydopamine (6-OHDA) is a neurotoxin and is commonly used to generate experimental models of Parkinson's disease (PD). In this study, we investigated the signaling molecules involved in the 6-OHDA-induced cell death using a neuronal catecholaminergic cell line (SK-N-SH cells), and the protective effect of fustin, a flavonoid from Rhus verniciflua Stokes, on 6-OHDA-induced neuronal death. 6-OHDA significantly increased levels of reactive oxygen species (ROS), intracellular Ca(2+) ([Ca(2+)](i)), and p38 phosphorylation. In addition, this ROS increase by 6-OHDA was reduced by pretreatment with N-acetylcysteine (NAC), a free radical scavenger, but not by bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid (BAPTA), a Ca(2+) chelator. However, the [Ca(2+)](i) increase induced by 6-OHDA was suppressed by NAC. Moreover, pretreatment with NAC or BAPTA significantly prevented the 6-OHDA-induced increases in p38 phosphorylation, Bax/Bcl-2 ratio, and caspase-3 activity. Although 6-OHDA-increased phosphorylation of p38 was prevented by NAC or BAPTA, inhibition of p38 by SB203580 did not suppress ROS, Bax/Bcl-2 ratio, or caspase-3 activity increases, and only partially prevented 6-OHDA-induced cell death, thus demonstrating that p38 activation is a component of a signaling pathway leading to the initiation of 6-OHDA-induced cell death, which acts in parallel with an ROS-Ca(2+)-Bcl-2-caspase-3 pathway. Moreover, fustin not only suppressed 6-OHDA-induced cell death in a concentration-dependent manner but also blocked 6-OHDA-induced increases in ROS, [Ca(2+)](i), Bax/Bcl-2 ratio, caspase-3 activity, and p38 phosphorylation. These results suggest that fustin exerts neuroprotection against 6-OHDA-induced cell death. [Abstract/Link to Full Text]

Jung DS, Baek SY, Park KH, Chung YI, Kim HJ, Kim CD, Cho MK, Han ME, Park KP, Kim BS, Kim JB, Oh SO
Effects of retinoic acid on ischemic brain injury-induced neurogenesis.
Exp Mol Med. 2007 Jun 30;39(3):304-15.
Neurogenesis can be induced by pathological conditions such as cerebral ischemia. However the molecular mechanisms or modulating reagents of the reactive neurogenesis after the cerebral ischemia are poorly characterized. Retinoic acid (RA) has been shown to increase neurogenesis by enhancing the proliferation and neuronal differentiation of forebrain neuroblasts. Here, we examined whether RA can modulate the reactive neurogenesis after the cerebral ischemia. In contrast to our expectation, RA treatment decreased the reactive neurogenesis in subventricular zone (SVZ), subgranular zone (SGZ) and penumbral region. Furthermore, RA treatment also decreased the angiogenesis and gliosis in penumbral region. [Abstract/Link to Full Text]

Choi JW, Kim JT, Park JH, Park EK, Kim SY, Kwon TG, Kim EC, Shin HI
gp130 is important for the normal morphogenesis of Meckel's cartilage and subsequent mandibular development.
Exp Mol Med. 2007 Jun 30;39(3):295-303.
gp130-mediated signaling is involved in both chondrogenesis and osteogenesis, but its direct role in the formation of embryonic Meckel's cartilage and associated mandibular development has not yet been elucidated. In this study, we examined the influence of gp130 ablation on the developing mandibular Meckel's cartilage by evaluating the morphological and histological changes as well as the gene expression patterns in developing embryonic gp130-/- mice. The ablation of the gp130 gene showed no change in region-specific collagen mRNA expression except for a slight delay in its expression but caused shortened embryonic Meckel's cartilage, delayed hypertrophic chondrocyte maturation and subsequent bony replacement with characteristic bending of the intramandibular Meckel's cartilage. The bending of Meckel's cartilage led to a narrow mandibular arch at the rostral area with poor cortical plate formation. These findings indicate that gp130-mediated signaling is important for the normal morphogenesis of Meckel's cartilage and subsequent mandibular development. [Abstract/Link to Full Text]

Lee SA, Ryu YS, Choi HI, Han IS
Capsaicin promotes the development of burst-forming units--erythroid (BFU-E) from mouse bone marrow cells.
Exp Mol Med. 2007 Jun 30;39(3):278-83.
Capsaicin, the pungent component of chilli peppers, is known to induce mediators of hematopoiesis. We investigated the effect of capsaicin on hematopoiesis in mouse progenitor cells. Treatment of mouse bone marrow cells with capsaicin induced the formation of colony of burst-forming units-erythroid (BFU-E). We also found that the number of erythropoietin receptor (EpoR)-positive cells was increased by capsaicin. To clarify the effect of capsaicin on erythroid lineage, BFU-E colonies were separated from non-BFU-E colonies by colony-picking after in vitro culture of mouse bone marrow cells. Quantitative RT-PCR analysis revealed that capsaicin stimulated the expression of the erythroid-specific genes encoding EpoR, glycophorin A (GPA), beta-globin (Hbb-b1), GATA-1, PU.1, nuclear factor erythroid-derived 2 (NF-E2), and Krüppel-like factor 1 (KLF1) in the BFU-E colonies. Furthermore, capsaicin could effectively stimulate the transfected GATA-1 promoter in K562 cells. GATA-1 is known as an essential transcription factor for the development of erythroid cells. Our results show that development of the erythroid lineage from bone marrow cells can be induced by treatment with capsaicin, and that GATA-1 seems to play a role in this induced erythroid maturation. [Abstract/Link to Full Text]

Pae HO, Jeong GS, Jeong SO, Kim HS, Kim SA, Kim YC, Yoo SJ, Kim HD, Chung HT
Roles of heme oxygenase-1 in curcumin-induced growth inhibition in rat smooth muscle cells.
Exp Mol Med. 2007 Jun 30;39(3):267-77.
In vascular smooth muscle cells (VSMCs), induction of the heme oxygenase-1 (HO-1) confers vascular protection against cellular proliferation mainly via its up-regulation of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) that is involved in negative regulation of cellular proliferation. In the present study, we investigated whether the phytochemical curcumin and its metabolite tetrahydrocurcumin could induce HO-1 expression and growth inhibition in rat VSMCs and, if so, whether their antiproliferative effect could be mediated via HO-1 expression. At non-toxic concentrations, curcumin possessing two Michael-reaction acceptors induced HO-1 expression by activating antioxidant response element (ARE) through translocation of the nuclear transcription factor E2-related factor-2 (Nrf2) into the nucleus and also inhibited VSMC growth triggered by 5% FBS in a dose-dependent manner. In contrast, tetrahydrocurcumin lacking Michael-reaction acceptor showed no effect on HO-1 expression, ARE activation and VSMC growth inhibition. The antiproliferative effect of curcumin in VSMCs was accompanied by the increased expression of p21(WAF1/CIP1). Inhibition of VSMC growth and expression of p21(WAF1/CIP1) by curcumin were partially, but not completely, abolished when the cells were co- incubated with the HO inhibitor tin protoporphyrin. In human aortic smooth muscle cells (HASMCs), curcumin also inhibited growth triggered by TNF-alpha and increased p21(WAF1/CIP1) expression via HO-1-dependent manner. Our findings suggest that curcumin has an ability to induce HO-1 expression, presumably through Nrf2-dependent ARE activation, in rat VSMCs and HASMCs, and provide evidence that the antiproliferative effect of curcumin is considerably linked to its ability to induce HO-1 expression. [Abstract/Link to Full Text]

Liu T, Ma Z, Que H, Li X, Ni Y, Jing S, Liu S
Identification and characterization of scirr1, a novel gene up-regulated after spinal cord injury.
Exp Mol Med. 2007 Jun 30;39(3):255-66.
Spinal cord injury and regeneration involves transcriptional activity of many genes, of which many remain unknown. Using the rat spinal cord full- transection model, bioinformatics, cloning, expression assays, fusion proteins, and transfection techniques, we identified and characterized one such differentially expressed gene, termed scirr1 (spinal cord injury and/or regeneration related gene 1). Fourteen orthologs were found in 13 species from echinoderm to insect and human by Blast search of NCBI protein reference sequence database. However, no further information is available for these homologues. Using whole-mount in situ hybridization, mouse scirr1 mRNA was expressed temporally and spatially in accordance with the early development sequence of the central nervous system. In adult rat spinal cord, expression of scirr1 mRNA was localized to neurons of gray matter by in situ hybridization. Using immunohistochemistry, SCIRR1 protein was found to be up-regulated and expressed more highly in spinal cord neurons farther from the epicenter of injury. Although the precise function of SCIRR1 is unknown, its unique pattern of expression during CNS early development and up-regulation after spinal cord injury suggest that SCIRR1 should be involved in the succeeding injury and/or repair processes of the injured spinal cord. Also, the typical F-box and leucine-rich repeat (LRR) architecture of rat SCIRR1 indicated that it may play an important substrate recruiting role in the pleiotropic ubiquitin/proteasome pathway. All these make scirr1 a new interesting start to study the spinal cord injury and regeneration mechanism. [Abstract/Link to Full Text]

Lampiasi N, Azzolina A, Montalto G, Cervello M
Histamine and spontaneously released mast cell granules affect the cell growth of human hepatocellular carcinoma cells.
Exp Mol Med. 2007 Jun 30;39(3):284-94.
The role of mast cells in tumor growth is still controversial. In this study we analyzed the effects of both histamine and pre-formed mediators spontaneously released by mast cells on the growth of two human hepatocellular carcinoma cell lines, HA22T/VGH and HuH-6, with different characteristics of differentiation, biological behavior and genetic defects. We showed that total mast cell releasate, exocytosed granules (granule remnants) and histamine reduced cell viability and proliferation in HuH-6 cells. In contrast, in HA22T/VGH cells granule remnants and histamine induced a weak but significant increase in cell growth. We showed that both cell lines expressed histamine receptors H(1) and H(2) and that the selective H(1) antagonist terfenadine reverted the histamine-induced inhibition of HuH-6 cell growth, whereas the selective H(2) antagonist ranitidine inhibited the histamine-induced cell growth of HA22T/VGH cells. We demonstrated that histamine down-regulated the expression of beta-catenin, COX-2 and survivin in HuH-6 cells and that this was associated with caspase-3 activation and PARP cleavage. On the contrary, in HA22T/VGH cells expression of survivin and beta-catenin increased after treatment with granule remnants and histamine. Overall, our results suggest that mediators stored in mast cell granules and histamine may affect the growth of liver cancer cells. However, mast cells and histamine may play different roles depending on the tumor cell features. Finally, these data suggest that histamine and histamine receptor agonists/antagonists might be considered as "new therapeutic" drugs to inhibit liver tumor growth. [Abstract/Link to Full Text]

Cho EJ
RNA polymerase II carboxy-terminal domain with multiple connections.
Exp Mol Med. 2007 Jun 30;39(3):247-54.
The largest subunit of eukaryotic RNA polymerase II contains a unique domain at its carboxy-terminus, which is referred to as the carboxy-terminal domain (CTD). The CTD is made up of an evolutionarily conserved heptapeptide repeat (YSPTSPS). Over the past decade, there has been increasing attention on the role of the CTD in transcription regulation in the view of mRNA processing and chromatin remodeling. This paper provides a brief overview of the recent progress in the dynamic changes in CTD phosphorylation and its role in integrating multiple nuclear events. [Abstract/Link to Full Text]

Lim EJ, Lee SH, Lee JG, Kim JR, Yun SS, Baek SH, Lee C
Toll-like receptor 9 dependent activation of MAPK and NF-kB is required for the CpG ODN-induced matrix metalloproteinase-9 expression.
Exp Mol Med. 2007 Apr 30;39(2):239-45.
Unmethylated CpG oligodeoxynucleotides (CpG ODNs) activate immune cells to produce immune mediators. This study demonstrates that in murine macrophage RAW 264.7 cells, CpG ODN-mediated matrix metalloproteinase-9 (MMP-9) expression is regulated at transcriptional level and requires de novo protein synthesis. Inhibition of ERK and p38 MAPK, but not JNK, results in significant decrease of CpG ODN-induced MMP-9 expression. We found that endosomal maturation inhibitors, chloroquine and bafilomycin A, block CpG ODN-induced ERK and p38 MAPK activation and the subsequent MMP-9 expression. We also observed that CpG ODN induces NF-kappaB activation and NF-kappaB is a downstream target of p38 MAPK. Taken together, our data demonstrate that CpG ODN triggers MMP-9 expression via TLR-9 dependent ERK and p38 MAPK activation followed by NF-kappaB activation. [Abstract/Link to Full Text]

Lee SJ, Namkoong S, Ha KS, Nam WD, Kwon YG, Lee H, Yoon EY, Chang DJ, Kim SO, Kim YM
Colchicine-derived compound CT20126 promotes skin allograft survival by regulating the balance of Th1 and Th2 cytokine production.
Exp Mol Med. 2007 Apr 30;39(2):230-8.
Colchicine has been shown to regulate the expression of inflammatory gene, but this compound possesses much weaker anti-inflammatory activity. In this study, we synthesized a new colchicine derivative CT20126 and examined its immunomodulatory property. CT20126 was found to have immunosuppressive effects by inhibiting lymphocyte proliferation without cytotoxicity and effectively inhibit the transcriptional expression of the inflammatory genes, iNOS, TNF-alpha, and IL-1beta, in macrophages stimulated by LPS. This effect was nearly comparable to that of cyclosporine A. This compound also significantly suppressed the production of nitric oxide and Th1-related pro-inflammatory cytokines, IL-1beta, TNF-alpha, and IL-2, with minimal suppression of Th2-related anti-inflammatory cytokines IL-4 and IL-10 in the sponge matrix allograft model. Moreover, administration of CT20126 prolonged the survival of allograft skins from BALB/c mice (H-2(d)) to the dorsum of C57BL/6 (H-2(b)) mice. The in vivo immune suppressive effects of CT20126 were similar to that of cyclosporine A. These results indicate that this compound may have potential therapeutic value for transplantation rejection and other inflammatory diseases. [Abstract/Link to Full Text]

Park CE, Kim MJ, Lee JH, Min BI, Bae H, Choe W, Kim SS, Ha J
Resveratrol stimulates glucose transport in C2C12 myotubes by activating AMP-activated protein kinase.
Exp Mol Med. 2007 Apr 30;39(2):222-9.
trans-Resveratrol (t-RVT), a naturally occurring polyphenol found in Polygonum cuspidatum, grape, and red wine, has been reported to have anti-inflammatory, cardioprotective, and cancer chemopreventive properties. However antidiabetic effect of t-RVT has not yet been reported. In this study, we show that t-RVT increases glucose uptake in C2C12 myotubes by activating AMP-activated protein kinase (AMPK), uncovering an antidiabetic potential of t-RVT for the first time. AMPK plays a central role in the regulation of glucose and lipid metabolism, and hence it is considered a novel therapeutic target for metabolic syndrome such as type 2 diabetes. t-RVT significantly induced glucose uptake in C2C12 cells, via AMPK activation, but not a phosphatidylinositol-3 kinase (PI-3 kinase) signal pathway. The induced glucose uptake was attenuated by pretreatment with a pharmacological inhibitor for AMPK, indicating that the effect of t-RVT primarily depends on AMPK activation. However, in the presence of insulin, t-RVT also potentiated the effect of insulin on glucose uptake via AMPK activation, which led to further activation of PI-3 kinase/Akt signal pathway. [Abstract/Link to Full Text]

Yi T, Baek JH, Kim HJ, Choi MH, Seo SB, Ryoo HM, Kim GS, Woo KM
Trichostatin A-mediated upregulation of p21(WAF1) contributes to osteoclast apoptosis.
Exp Mol Med. 2007 Apr 30;39(2):213-21.
Histone deacetylase inhibitors (HDIs), a new class of anti-cancer agents, have been reported to suppress formation of osteoclast precursors and their fusion into multinucleated cells. However, little is known about the effect of HDIs on mature osteoclasts, which may have significance for their therapeutic use. Here, we demonstrate a novel action of HDIs on osteoclast apoptosis. Primary multinucleated mature osteoclasts were prepared from mouse bone marrow cells. Treatment of osteoclasts with the HDI trichostatin A (TSA) caused apoptosis, as confirmed by annexin V staining and caspase activation. TSA caused the upregulation of p21WAF1 in osteoclasts. To understand the role of p21(WAF1) upregulation in TSA-treated osteoclasts, shRNA against p21(WAF1)-containing lentivirus was introduced into osteoclasts. The suppression of p21(WAF1) decreased TSA-directed osteoclast apoptosis. Collectively, our results provide evidence that TSA causes osteoclast apoptosis, which involves, in part, TSA-induced upregulation of p21(WAF1), and strongly supports HDIs as potential therapeutic agents for excessive bone resorption. [Abstract/Link to Full Text]

Kim MA, Kim HJ, Brown AL, Lee MY, Bae YS, Park JI, Kwak JY, Chung JH, Yun J
Identification of novel substrates for human checkpoint kinase Chk1 and Chk2 through genome-wide screening using a consensus Chk phosphorylation motif.
Exp Mol Med. 2007 Apr 30;39(2):205-12.
Checkpoint kinase 1 (Chk1) and Chk2 are effector kinases in the cellular DNA damage response and impairment of their function is closely related to tumorigenesis. Previous studies revealed several substrate proteins of Chk1 and Chk2, but identification of additional targets is still important in order to understand their tumor suppressor functions. In this study, we screened novel substrates for Chk1 and Chk2 using substrate target motifs determined previously by an oriented peptide library approach. The potential candidates were selected by genome-wide peptide database searches and were examined by in vitro kinase assays. ST5, HDAC5, PGC-1alpha, PP2A PR130, FANCG, GATA3, cyclin G, Rad51D and MAD1a were newly identified as in vitro substrates for Chk1 and/or Chk2. Among these, HDAC5 and PGC-1a were further analyzed to substantiate the screening results. Immunoprecipitation kinase assay of full-length proteins and site-directed mutagenesis analysis of the target motifs demonstrated that HDAC5 and PGC-1alpha were specific targets for Chk1 and/or Chk2 at least in vitro. [Abstract/Link to Full Text]

Park HY, Jeon YK, Shin HJ, Kim IJ, Kang HC, Jeong SJ, Chung DH, Lee CW
Differential promoter methylation may be a key molecular mechanism in regulating BubR1 expression in cancer cells.
Exp Mol Med. 2007 Apr 30;39(2):195-204.
The BubR1 mitotic-checkpoint protein monitors proper attachment of microtubules to kinetochores, and links regulation of chromosome-spindle attachment to mitotic-checkpoint signaling. Thus, disruption of BubR1 activity results in a loss of checkpoint control, chromosomal instability caused by a premature anaphase, and/or the early onset of tumorigenesis. The mechanisms by which deregulation and/or abnormalities of BubR1 expression operate, however, remain to be elucidated. In this study, we demonstrate that levels of BubR1 expression are significantly increased by demethylation. Bisulfite sequencing analysis revealed that the methylation status of two CpG sites in the essential BubR1 promoter appear to be associated with BubR1 expression levels. Associations of MBD2 and HDAC1 with the BubR1 promoter were significantly relieved by addition of 5-aza-2'-deoxycytidine, an irreversible DNA methyltransferase inhibitor. However, genomic DNA isolated from 31 patients with colorectal carcinomas exhibited a +84A/G polymorphic change in approximately 60% of patients, but this polymorphism had no effect on promoter activity. Our findings indicate that differential regulation of BubR1 expression is associated with changes in BubR1 promoter hypermethylation patterns, but not with promoter polymorphisms, thus providing a novel insight into the molecular regulation of BubR1 expression in human cancer cells. [Abstract/Link to Full Text]

Kim MK, Park KS, Lee H, Kim YD, Yun J, Bae YS
Phytosphingosine-1-phosphate stimulates chemotactic migration of L2071 mouse fibroblasts via pertussis toxin-sensitive G-proteins.
Exp Mol Med. 2007 Apr 30;39(2):185-94.
Phytosphingosine-1-phosphate (PhS1P) was found to stimulate an intracellular calcium increase via phospholipase C but not pertussis toxin (PTX)-sensitive G-proteins in L2071 mouse fibroblasts. PhS1P also activated ERK and p38 kinase, and these activations by PhS1P were inhibited by PTX. Moreover, PhS1P stimulated the chemotactic migration of L2071 cells via PTX-sensitive Gi protein(s). In addition, the PhS1P-induced chemotactic migration of L2071 cells was also dramatically inhibited by LY294002 and SB203580 (inhibitors of phosphoinositide 3-kinase and p38 kinase, respectively). L2071 cells are known to express four S1P receptors, i.e., S1P1, S1P2, S1P3, and S1P4, and pretreatment with an S1P1 and S1P3 antagonist (VPC 23019) did not affect on PhS1P-induced chemotaxis. This study demonstrates that PhS1P stimulates at least two different signaling cascades, one is a PTX-insensitive but phospholipase C dependent intracellular calcium increase, and the other is a PTX-sensitive chemotactic migration mediated by phosphoinositide 3-kinase and p38 kinase. [Abstract/Link to Full Text]

Oh KI, Kim BK, Ban YL, Choi EY, Jung KC, Lee IS, Park SH
CD99 activates T cells via a costimulatory function that promotes raft association of TCR complex and tyrosine phosphorylation of TCR zeta.
Exp Mol Med. 2007 Apr 30;39(2):176-84.
We investigated the co-stimulatory role of a cell-surface protein, CD99. Co-ligation of CD99 and suboptimal CD3 induced T-cell activation to a level comparable to that obtained with optimal CD3 or CD3+CD28. We also noted concomitant enhancement of the earliest T-cell receptor (TCR) signaling events. In addition, co-ligation of CD99 and CD3 led to translocation of TCR complexes into the lipid raft, without concomitant migration of CD99 to the raft, and consequent enhancement of TCR zeta-mediated signal 1. These data demonstrate the unique properties of CD99 co-stimulation that distinguish this molecule from CD28 and other raft-resident co-stimulatory factors. [Abstract/Link to Full Text]

Liu Y, Okada T, Nomoto T, Ke X, Kume A, Ozawa K, Xiao S
Promoter effects of adeno-associated viral vector for transgene expression in the cochlea in vivo.
Exp Mol Med. 2007 Apr 30;39(2):170-5.
The aims of this study were to evaluate the expression of enhanced green fluorescent protein (EGFP) driven by 6 different promoters, including cytomegalovirus IE enhancer and chicken beta-actin promoter (CAG), cytomegalovirus promoter (CMV), neuron-specific enolase promoter (NSE), myosin 7A promoter (Myo), elongation factor 1alpha promoter (EF-1alpha), and Rous sarcoma virus promoter (RSV), and assess the dose response of CAG promoter to transgene expression in the cochlea. Serotype 1 adeno-associated virus (AAV1) vectors with various constructs were transduced into the cochleae, and the level of EGFP expression was examined. We found the highest EGFP expression in the inner hair cells and other cochlear cells when CAG promoter was used. The CMV and NSE promoter drove the higher EGFP expression, but only a marginal activity was observed in EF-1alpha promoter driven constructs. RSV promoter failed to driven the EGFP expression. Myo promoter driven EGFP was exclusively expressed in the inner hair cells of the cochlea. When driven by CAG promoter, reporter gene expression was detected in inner hair cells at a dose as low as 3x10(7) genome copies, and continued to increase in a dose-dependent manner. Our data showed that individual promoter has different ability to drive reporter gene expression in the cochlear cells. Our results might provide important information with regard to the role of promoters in regulating transgene expression and for the proper design of vectors for gene expression and gene therapy. [Abstract/Link to Full Text]

Cho KH, Park SH, Han JM, Kim HC, Chung YJ, Choi I, Kim JR
A point mutant of apolipoprotein A-I, V156K, exhibited potent anti-oxidant and anti-atherosclerotic activity in hypercholesterolemic C57BL/6 mice.
Exp Mol Med. 2007 Apr 30;39(2):160-9.
In our previous study, two point mutants of apolipoprotein A-I, designated V156K and A158E, revealed peculiar characteristics in their lipid-free and lipid-bound states. In order to determine the putative therapeutic potential of these mutants, several in vitro and in vivo evaluations were conducted. In the lipid-free state, V156K showed more profound antioxidant activity against LDL oxidation than did the wildtype (WT) or A158E variants in an in vitro assay. In the lipid-bound state, V156K-rHDL showed an enhanced cholesterol delivery activity to HepG2 cells in a time-dependent manner, as compared to WT-rHDL, A158E-rHDL, and R173C-rHDL. We assessed the physiological activities of the mutants in circulation, using hypercholesterolemic mice (C57BL6/J). Palmitoyloleoyl phosphatidylcholine (POPC)-rHDL preparations containing each of the apoA-I variants were injected into the mice at a dosage of 30 mg of apoA-I/kg of body weight. Forty eight hours after injection, the sera of the V156K-rHDL injected group showed the most potent antioxidant abilities in the ferric acid removal assay. The V156K-rHDL- or R173C-rHDL-injected mice showed no atherosclerotic lesions and manifested striking increases in their serum apo-E levels, as compared to the mice injected with WT-rHDL or A158E-rHDL. In conclusion, V156K-rHDL exhibited the most pronounced antioxidant activity and anti-atherosclerotic activity, both in vitro and in vivo. These results support the notion that HDL-therapy may prove beneficial due to its capacity to induce accelerated cholesterol excretion, as well as its enhanced antioxidant and anti-inflammatory effects and lesion regression effect. [Abstract/Link to Full Text]

Kim EK, Kwon KB, Han MJ, Song MY, Lee JH, Lv N, Ka SO, Yeom SR, Kwon YD, Ryu DG, Kim KS, Park JW, Park R, Park BH
Coptidis rhizoma extract protects against cytokine-induced death of pancreatic beta-cells through suppression of NF-kappaB activation.
Exp Mol Med. 2007 Apr 30;39(2):149-59.
We demonstrated previously that Coptidis rhizoma extract (CRE) prevented S-nitroso-N-acetylpenicillamine-induced apoptotic cell death via the inhibition of mitochondrial membrane potential disruption and cytochrome c release in RINm5F (RIN) rat insulinoma cells. In this study, the preventive effects of CRE against cytokine-induced beta-cell death was assessed. Cytokines generated by immune cells infiltrating pancreatic islets are crucial mediators of beta-cell destruction in insulin-dependent diabetes mellitus. The treatment of RIN cells with IL-1beta and IFN-gamma resulted in a reduction of cell viability. CRE completely protected IL-1beta and IFN-gamma-mediated cell death in a concentration-dependent manner. Incubation with CRE induced a significant suppression of IL-1beta and IFN-gamma-induced nitric oxide (NO) production, a finding which correlated well with reduced levels of the iNOS mRNA and protein. The molecular mechanism by which CRE inhibited iNOS gene expression appeared to involve the inhibition of NF-kappaB activation. The IL-1beta and IFN-gamma-stimulated RIN cells showed increases in NF-kappaB binding activity and p65 subunit levels in nucleus, and IkappaB alpha degradation in cytosol compared to unstimulated cells. Furthermore, the protective effects of CRE were verified via the observation of reduced NO generation and iNOS expression, and normal insulin-secretion responses to glucose in IL-1beta and IFN-gamma-treated islets. [Abstract/Link to Full Text]

Abuarqoub H, Green CJ, Foresti R, Motterlini R
Curcumin reduces cold storage-induced damage in human cardiac myoblasts.
Exp Mol Med. 2007 Apr 30;39(2):139-48.
Curcumin is a polyphenolic compound possessing interesting anti-inflammatory and antioxidant properties and has the ability to induce the defensive protein heme oxygenase-1 (HO-1). The objective of this study was to investigate whether curcumin protects against cold storage-mediated damage of human adult atrial myoblast cells (Girardi cells) and to assess the potential involvement of HO-1 in this process. Girardi cells were exposed to either normothermic or hypothermic conditions in Celsior preservation solution in the presence or absence of curcumin. HO-1 protein expression and heme oxygenase activity as well as cellular damage were assessed after cold storage or cold storage followed by re-warming. In additional experiments, an inhibitor of heme oxygenase activity (tin protoporphyrin IX, 10 microM) or siRNA for HO-1 were used to investigate the participation of HO-1 as a mediator of curcumin-induced effects. Treatment with curcumin produced a marked induction of cardiac HO-1 in normothermic condition but cells were less responsive to the polyphenolic compound at low temperature. Cold storage-induced damage was markedly reduced in the presence of curcumin and HO-1 contributed to some extent to this effect. Thus, curcumin added to Celsior preservation solution effectively prevents the damage caused by cold-storage; this effect involves the protective enzyme HO-1 but also other not yet identified mechanisms. [Abstract/Link to Full Text]

Ko J, Yun CY, Lee JS, Kim JH, Kim IS
p38 MAPK and ERK activation by 9-cis-retinoic acid induces chemokine receptors CCR1 and CCR2 expression in human monocytic THP-1 cells.
Exp Mol Med. 2007 Apr 30;39(2):129-38.
9-cis-Retinoic acid (9CRA) plays an important role in the immune response; this includes cytokine production and cell migration. We have previously demonstrated that 9CRA increases expression of chemokine receptors CCR1 and CCR2 in human monocytes. To better understand how 9CRA induces CCR1 and CCR2 expression, we examined the contribution of signaling proteins in human monocytic THP-1 cells. The mRNA and surface protein up-regulation of CCR1 and CCR2 in 9CRA-stimulated cells were weakly blocked by the pretreatment of SB202190, a p38 MAPK inhibitor, and PD98059, an upstream ERK inhibitor. Activation of p38 MAPK and ERK1/2 was induced in both a time and dose-dependent manner after 9CRA stimulation. Both p38 MAPK and ERK1/2 phosphorylation peaked at 2 h after a 100 nM 9CRA treatment. 9CRA increased calcium influx and chemotactic activity in response to CCR1-dependent chemokines, Lkn-1/CCL15, MIP-1alpha/CCL3, and RANTES/CCL5, and the CCR2-specific chemokine, MCP-1/CCL2. Both SB202190 and PD98059 pretreatment diminished the increased calcium mobilization and chemotactic ability due to 9CRA. SB202190 inhibited the expression and functional activities of CCR1 and CCR2 more effectively than did PD98059. Therefore, our results demonstrate that 9CRA transduces the signal through p38 MAPK and ERK1/2 for CCR1 and CCR2 up-regulation, and may regulate the pro-inflammatory process through the p38 MAPK and ERK-dependent signaling pathways. [Abstract/Link to Full Text]

Lee YR, Yu HN, Noh EM, Youn HJ, Song EK, Han MK, Park CS, Kim BS, Park YS, Park BK, Lee SH, Kim JS
TNF-alpha upregulates PTEN via NF-kappaB signaling pathways in human leukemic cells.
Exp Mol Med. 2007 Feb 28;39(1):121-7.
TNF-alpha plays a variety of biological functions such as apoptosis, inflammation and immunity. PTEN also has various cellular function including cell growth, proliferation, migration and differentiation. Thus, possible relationships between the two molecules are suggested. TNF-alpha has been known to downregulate PTEN via NF-kappaB pathway in the human colon cell line, HT-29. However, here we show the opposite finding that TNF-alpha upregulates PTEN via activation of NF-kappaB in human leukemic cells. TNF-alpha increased PTEN expression at HL-60 cells in a time- and dose-dependent manner, but the response was abolished by disruption of NF-kappaB with p65 antisense phosphorothioate oligonucleotide or pyrrolidine dithiocarbamate. We found that TNF-alpha activated the NF-kappaB pathways, evidenced by the translocation of p65 to the nucleus in TNF-alpha-treated cells. We conclude that TNF-alpha induces upregulation of PTEN expression through NF-kappaB activation in human leukemic cells. [Abstract/Link to Full Text]

---

Recent Articles in BMC Biochemistry

---

Fraccascia P, Sniekers M, Casteels M, Van Veldhoven PP
Presence of thiamine pyrophosphate in mammalian peroxisomes.
BMC Biochem. 2007;810.
BACKGROUND: Thiamine pyrophosphate (TPP) is a cofactor for 2-hydroxyacyl-CoA lyase 1 (HACL1), a peroxisomal enzyme essential for the alpha-oxidation of phytanic acid and 2-hydroxy straight chain fatty acids. So far, HACL1 is the only known peroxisomal TPP-dependent enzyme in mammals. Little is known about the transport of metabolites and cofactors across the peroxisomal membrane and no peroxisomal thiamine or TPP carrier has been identified in mammals yet. This study was undertaken to get a better insight into these issues and to shed light on the role of TPP in peroxisomal metabolism. RESULTS: Because of the crucial role of the cofactor TPP, we reanalyzed its subcellular localization in rat liver. In addition to the known mitochondrial and cytosolic pools, we demonstrated, for the first time, that peroxisomes contain TPP (177 +/- 2 pmol/mg protein). Subsequently, we verified whether TPP could be synthesized from its precursor thiamine, in situ, by a peroxisomal thiamine pyrophosphokinase (TPK). However, TPK activity was exclusively recovered in the cytosol. CONCLUSION: Our results clearly indicate that mammalian peroxisomes do contain TPP but that no pyrophosphorylation of thiamine occurs in these organelles, implying that thiamine must enter the peroxisome already pyrophosphorylated. Consequently, TPP entry may depend on a specific transport system or, in a bound form, on HACL1 translocation. [Abstract/Link to Full Text]

Lin SC, Liu WT, Liu SH, Chou WI, Hsiung BK, Lin IP, Sheu CC, Dah-Tsyr Chang M
Role of the linker region in the expression of Rhizopus oryzae glucoamylase.
BMC Biochem. 2007;89.
BACKGROUND: Rhizopus oryzae glucoamylase (RoGA) consists of three domains: an amino (N)-terminal raw starch-binding domain (SBD), a glycosylated linker domain, and a carboxy (C)-terminal catalytic domain. The 36-amino-acid linker region (residues 132-167) connects the two functional domains, but its structural and functional roles are unclear. RESULTS: To characterize the linker sequences of RoGA and its involvement in protein expression, a number of RoGA variants containing deletions and mutations were constructed and expressed in Saccharomyces cerevisiae. Deletion analyses demonstrate that the linker region, especially within residues 161 to 167, is required for protein expression. In addition, site-directed mutagenesis and deglycosylation studies reveal that the linker region of RoGA contains both N- and O-linked carbohydrate moieties, and the N-linked oligosaccharides play a major role in the formation of active enzyme. Although the linker segment itself appears to have no ordered secondary structural conformation, the flexible region indeed contributes to the stabilization of functional N- and C-terminal domains. CONCLUSION: Our data provide direct evidence that the length, composition, and glycosylation of the interdomain linker play a central role in the structure and function of RoGA. [Abstract/Link to Full Text]

Kalra S, Jena G, Tikoo K, Mukhopadhyay AK
Preferential inhibition of xanthine oxidase by 2-amino-6-hydroxy-8-mercaptopurine and 2-amino-6-purine thiol.
BMC Biochem. 2007;88.
BACKGROUND: The anticancer drug, 6-mercaptopurine (6MP) is subjected to metabolic clearance through xanthine oxidase (XOD) mediated hydroxylation, producing 6-thiouric acid (6TUA), which is excreted in urine. This reduces the effective amount of drug available for therapeutic efficacy. Co-administration of allopurinol, a suicide inhibitor of XOD, which blocks the hydroxylation of 6MP inadvertently enhances the 6MP blood level, counters this reduction. However, allopurinol also blocks the hydroxylation of hypoxanthine, xanthine (released from dead cancer cells) leading to their accumulation in the body causing biochemical complications such as xanthine nephropathy. This necessitates the use of a preferential XOD inhibitor that selectively inhibits 6MP transformation, but leaves xanthine metabolism unaffected. RESULTS: Here, we have characterized two such unique inhibitors namely, 2-amino-6-hydroxy-8-mercaptopurine (AHMP) and 2-amino-6-purinethiol (APT) on the basis of IC50 values, residual activity in bi-substrate simulative reaction and the kinetic parameters like Km, Ki, kcat. The IC50 values of AHMP for xanthine and 6MP as substrate are 17.71 +/- 0.29 microM and 0.54 +/- 0.01 microM, respectively and the IC50 values of APT for xanthine and 6MP as substrates are 16.38 +/- 0.21 microM and 2.57 +/- 0.08 microM, respectively. The Ki values of XOD using AHMP as inhibitor with xanthine and 6MP as substrate are 5.78 +/- 0.48 microM and 0.96 +/- 0.01 microM, respectively. The Ki values of XOD using APT as inhibitor with xanthine and 6MP as substrate are 6.61 +/- 0.28 microM and 1.30 +/- 0.09 microM. The corresponding Km values of XOD using xanthine and 6MP as substrate are 2.65 +/- 0.02 microM and 6.01 +/- 0.03 microM, respectively. The results suggest that the efficiency of substrate binding to XOD and its subsequent catalytic hydroxylation is much superior for xanthine in comparison to 6MP. In addition, the efficiency of the inhibitor binding to XOD is much more superior when 6MP is the substrate instead of xanthine. We further undertook the toxicological evaluation of these inhibitors in a single dose acute toxicity study in mice and our preliminary experimental results suggested that the inhibitors were equally non-toxic in the tested doses. CONCLUSION: We conclude that administration of either APT or AHMP along with the major anti-leukemic drug 6MP might serve as a good combination cancer chemotherapy regimen. [Abstract/Link to Full Text]

Ostrow JD, Mukerjee P
Revalidation and rationale for high pKa values of unconjugated bilirubin.
BMC Biochem. 2007;87.
BACKGROUND: Our prior solvent partition analysis, published in 1992, yielded pKa values for unconjugated bilirubin of about 8.1 and 8.4, but these results have been challenged and studies by other methods have suggested pKa values below 5.0. METHODS: We repeated our published solvent partition studies, using 14C-unconjugated bilirubin highly purified by extraction of residual labeled impurities from CHCl3 into an aqueous buffer, pH 7.0. Partition ratios at six pH values from 5.0 to 9.0 were determined by radioassay and compared with our prior values obtained by diazo assay. RESULTS: At pH values ranging from 4.8 to 9.2, stable aqueous/chloroform 14C-partition ratios did not differ significantly from our published partition ratios based on diazo assay. CONCLUSION: These results support the high pKa values of unconjugated bilirubin, above 8.0, derived from our earlier solvent partition study. In both studies, our measurements were based on the rapid analysis of clearly under-saturated solutions of highly-purified bilirubin over a wide pH range, using properly purified and preserved solvents. No previous direct estimate of the aqueous pKa values of unconjugated bilirubin meets all these preconditions. Three theoretical factors acting in combination, each related to the unique, extensive internal H-bonding of the -COOH groups, are proposed to support high pKa values of unconjugated bilirubin in water: a) donation of an H-bond from the -OH moiety of the -COOH group, which is broken on ionization; b) hindered solvation of the -COO- group after ionization; and c) restricted rotation of the -COO- and -COOH groups. Our findings and rationale rebut methodological and theoretical criticisms leveled against our prior work. High pKa values for unconjugated bilirubin dictate that: a) bilirubin diacid, which readily diffuses across membranes and can cause neurotoxicity, is the dominant unbound bilirubin species of unconjugated bilirubin in plasma at physiological pH; b) at the near-neutral pH range of gallbladder bile, the monoanion is the major unconjugated bilirubin anion present, concordant with the finding that the calcium bilirubinate precipitated in gallstones is the monoanion salt. Our conclusions are thus relevant to understanding bilirubin-induced neurological disease in severely jaundiced neonates and the precipitation of calcium bilirubinate salts in gallstones. [Abstract/Link to Full Text]

Rigter A, Langeveld JP, Timmers-Parohi D, Jacobs JG, Moonen PL, Bossers A
Mapping of possible prion protein self-interaction domains using peptide arrays.
BMC Biochem. 2007;86.
BACKGROUND: The common event in transmissible spongiform encephalopathies (TSEs) or prion diseases is the conversion of host-encoded protease sensitive cellular prion protein (PrPC) into strain dependent isoforms of scrapie associated protease resistant isoform (PrPSc) of prion protein (PrP). These processes are determined by similarities as well as strain dependent variations in the PrP structure. Selective self-interaction between PrP molecules is the most probable basis for initiation of these processes, potentially influenced by chaperone molecules, however the mechanisms behind these processes are far from understood. We previously determined that polymorphisms do not affect initial PrPC to PrPSc binding but rather modulate a subsequent step in the conversion process. Determining possible sites of self-interaction could elucidate which amino acid(s) or amino acid sequences contribute to binding and further conversion into other isoforms. To this end, ovine - and bovine PrP peptide-arrays consisting of 15-mer overlapping peptides were probed with recombinant sheep PrPC fused to maltose binding protein (MBP-PrP). RESULTS: The peptide-arrays revealed two distinct high binding areas as well as some regions of lower affinity in PrPC resulting in total in 7 distinct amino acid sequences (AAs). The first high binding area comprises sheep-PrP peptides 43-102 (AA 43-116), including the N-terminal octarepeats. The second high binding area of sheep-PrP peptides 134-177 (AA 134-191), encompasses most of the scrapie susceptibility-associated polymorphisms in sheep. This concurs with previous studies showing that scrapie associated-polymorphisms do not modulate the initial binding of PrPC to PrPSc. Comparison of ovine - and bovine peptide-array binding patterns revealed that amino acid specific differences can influence the MBP-PrP binding pattern. PrP-specific antibodies were capable to completely block interaction between the peptide-array and MBP-PrP. MBP-PrP was also capable to specifically bind to PrP in a Western blot approach. The octarepeat region of PrP seems primarily important for this interaction because proteinase K pre-treatment of PrPSc completely abolished binding. CONCLUSION: Binding of MBP-PrP to PrP-specific sequences indicate that several specific self-interactions between individual PrP molecules can occur and suggest that an array of interactions between PrPC-PrPC as well as PrPC-PrPSc may be possible, which ultimately lead to variations in species barrier and strain differences. [Abstract/Link to Full Text]

Tsuchiya Y, Yamashita S
p42MAPK-mediated phosphorylation of xEIAP/XLX in Xenopus cytostatic factor-arrested egg extracts.
BMC Biochem. 2007;85.
BACKGROUND: BIR family proteins are evolutionarily conserved anti-apoptotic molecules. One member of Xenopus BIR family proteins, xEIAP/XLX, is a weak apoptosis inhibitor and rapidly degraded in a cell-free apoptotic execution system derived from interphase egg extracts. However, unfertilized eggs are naturally arrested at the metaphase of meiosis II by the concerted activities of Mos-MEK-p42MAPK-p90Rsk kinase cascade (cytostatic factor pathway) and many mitotic kinases. Previous studies suggest that cytostatic factor-arrested egg extracts are more resistant to spontaneous apoptosis than interphase egg extracts in a p42MAPK-dependent manner. We tested whether xEIAP/XLX might be phosphorylated in cytostatic factor-arrested egg extracts, and also examined whether xEIAP/XLX could be functionally regulated by phosphorylation. RESULTS: We found that p42MAPK was the major kinase phosphorylating xEIAP/XLX in cytostatic factor-arrested egg extracts, and three Ser residues (Ser 235/251/254) were identified as p42MAPK-mediated phosphorylation sites. We characterized the behaviors of various xEIAP/XLX mutants that could not be phosphorylated by p42MAPK. However, neither protein stability nor anti-apoptotic ability of xEIAP/XLX was significantly altered by the substitution of Ser with either Ala or Asp at these three sites. CONCLUSION: xEIAP/XLX is physiologically phosphorylated by p42MAPK in Xenopus unfertilized eggs. However, this protein may not serve as an essential mediator of p42MAPK-dependent anti-apoptotic activity. [Abstract/Link to Full Text]

Qu Q, Morizono H, Shi D, Tuchman M, Caldovic L
A novel bifunctional N-acetylglutamate synthase-kinase from Xanthomonas campestris that is closely related to mammalian N-acetylglutamate synthase.
BMC Biochem. 2007;84.
BACKGROUND: In microorganisms and plants, the first two reactions of arginine biosynthesis are catalyzed by N-acetylglutamate synthase (NAGS) and N-acetylglutamate kinase (NAGK). In mammals, NAGS produces an essential activator of carbamylphosphate synthetase I, the first enzyme of the urea cycle, and no functional NAGK homolog has been found. Unlike the other urea cycle enzymes, whose bacterial counterparts could be readily identified by their sequence conservation with arginine biosynthetic enzymes, mammalian NAGS gene was very divergent, making it the last urea cycle gene to be discovered. Limited sequence similarity between E. coli NAGS and fungal NAGK suggests that bacterial and eukaryotic NAGS, and fungal NAGK arose from the fusion of genes encoding an ancestral NAGK (argB) and an acetyltransferase. However, mammalian NAGS no longer retains any NAGK catalytic activity. RESULTS: We identified a novel bifunctional N-acetylglutamate synthase and kinase (NAGS-K) in the Xanthomonadales order of gamma-proteobacteria that appears to resemble this postulated primordial fusion protein. Phylogenetic analysis indicated that xanthomonad NAGS-K is more closely related to mammalian NAGS than to other bacterial NAGS. We cloned the NAGS-K gene from Xanthomonas campestis, and characterized the recombinant NAGS-K protein. Mammalian NAGS and its bacterial homolog have similar affinities for substrates acetyl coenzyme A and glutamate as well as for their allosteric regulator arginine. CONCLUSION: The close phylogenetic relationship and similar biochemical properties of xanthomonad NAGS-K and mammalian NAGS suggest that we have identified a close relative to the bacterial antecedent of mammalian NAGS and that the enzyme from X. campestris could become a good model for mammalian NAGS in structural, biochemical and biophysical studies. [Abstract/Link to Full Text]

Westwood IM, Sim E
Kinetic characterisation of arylamine N-acetyltransferase from Pseudomonas aeruginosa.
BMC Biochem. 2007;83.
BACKGROUND: Arylamine N-acetyltransferases (NATs) are important drug- and carcinogen-metabolising enzymes that catalyse the transfer of an acetyl group from a donor, such as acetyl coenzyme A, to an aromatic or heterocyclic amine, hydrazine, hydrazide or N-hydroxylamine acceptor substrate. NATs are found in eukaryotes and prokaryotes, and they may also have an endogenous function in addition to drug metabolism. For example, NAT from Mycobacterium tuberculosis has been proposed to have a role in cell wall lipid biosynthesis, and is therefore of interest as a potential drug target. To date there have been no studies investigating the kinetic mechanism of a bacterial NAT enzyme. RESULTS: We have determined that NAT from Pseudomonas aeruginosa, which has been described as a model for NAT from M. tuberculosis, follows a Ping Pong Bi Bi kinetic mechanism. We also describe substrate inhibition by 5-aminosalicylic acid, in which the substrate binds both to the free form of the enzyme and the acetyl coenzyme A-enzyme complex in non-productive reaction pathways. The true kinetic parameters for the NAT-catalysed acetylation of 5-aminosalicylic acid with acetyl coenzyme A as the co-factor have been established, validating earlier approximations. CONCLUSION: This is the first reported study investigating the kinetic mechanism of a bacterial NAT enzyme. Additionally, the methods used herein can be applied to investigations of the interactions of NAT enzymes with new chemical entities which are NAT ligands. This is likely to be useful in the design of novel potential anti-tubercular agents. [Abstract/Link to Full Text]

Liu H, Zheng S, Bellemare V, Pelletier G, Labrie F, Luu-The V
Expression and localization of estrogenic type 12 17beta-hydroxysteroid dehydrogenase in the cynomolgus monkey.
BMC Biochem. 2007;82.
BACKGROUND: We have recently discovered that human type 12 17beta-HSD (h17beta-HSD12), a homolog of type 3 17beta-HSD, is a new estrogen-specific 17beta-hydroxysteroid dehydrogenase involved in the production of estradiol (E2). To further characterize this estradiol-producing enzyme, we have isolated the corresponding cDNA in the cynomolgus monkey (Macaca fascicularis), characterized its enzymatic activities and performed cellular localization using in situ hybridization. RESULTS: Using HEK-293 cells stably expressing Macaca fascicularis type 12 17beta-HSD (mf17beta-HSD12), we have found that the mf17beta-HSD12 catalyzes efficiently and selectively the transformation of El into E2, in analogy with the h17beta-HSD12. We have also quantified the mf17beta-HSD12 mRNA expression levels in a series of Macaca fascicularis tissues using Quantitative RealTime PCR. The Macaca fascicularis 17beta-HSD12 mRNA is widely expressed with the highest levels tissues found in the cerebellum, spleen and adrenal with moderate level observed in all the other examined, namely the testis, ovary, cerebral cortex, liver, heart, prostate, mammary gland, myometrium, endometrium, skin, muscle and pancreas. To gain knowledge about the cellular localization of the mf17beta-HSD12 mRNA expression, we performed in situ hybridization using a 35S-labeled cRNA probe. Strong labeling was observed in epithelial cells and stromal cells of the mammary gland. In the uterus, the labeling is detected in epithelial cells and stromal cells of the endometrium. CONCLUSION: These results strongly suggest that the Macaca fascicularis 17beta-HSD12 is an essential partner of aromatase in the biosynthesis of estradiol (E2). It strongly suggests that in the estradiol biosynthesis pathway, the step of 17-ketoreduction comes after the step of the aromatization (the aromatization of 4-androstendione to estrone followed by the conversion of estrone into estradiol by estrogen specific l7beta-HSDs) which is in contrast with the hypothesis suggesting that 4-androstenedione is converted to testosterone followed by the aromatization of testosterone. [Abstract/Link to Full Text]

Filippou PS, Lioliou EE, Panagiotidis CA, Athanassopoulos CM, Garnelis T, Papaioannou D, Kyriakidis DA
Effect of polyamines and synthetic polyamine-analogues on the expression of antizyme (AtoC) and its regulatory genes.
BMC Biochem. 2007;81.
BACKGROUND: In bacteria, the biosynthesis of polyamines is modulated at the level of transcription as well as post-translationally. Antizyme (Az) has long been identified as a non-competitive protein inhibitor of polyamine biosynthesis in E. coli. Az was also revealed to be the product of the atoC gene. AtoC is the response regulator of the AtoS-AtoC two-component system and it functions as the positive transcriptional regulator of the atoDAEB operon genes, encoding enzymes involved in short chain fatty acid metabolism. The antizyme is referred to as AtoC/Az, to indicate its dual function as both a transcriptional and post-translational regulator. RESULTS: The roles of polyamines on the transcription of atoS and atoC genes as well as that of atoDAEB(ato) operon were studied. Polyamine-mediated induction was tested both in atoSC positive and negative E. coli backgrounds by using beta-galactosidase reporter constructs carrying the appropriate promoters patoDAEB, patoS, patoC. In addition, a selection of synthetic polyamine analogues have been synthesized and tested for their effectiveness in inducing the expression of atoC/Az, the product of which plays a pivotal role in the feedback inhibition of putrescine biosynthesis and the transcriptional regulation of the ato operon. The effects of these compounds were also determined on the ato operon expression. The polyamine analogues were also tested for their effect on the activity of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis and on the growth of polyamine-deficient E. coli. CONCLUSION: Polyamines, which have been reported to induce the protein levels of AtoC/Az in E. coli, act at the transcriptional level, since they cause activation of the atoC transcription. In addition, a series of polyamine analogues were studied on the transcription of atoC gene and ODC activity. [Abstract/Link to Full Text]

Weselake RJ, Madhavji M, Szarka SJ, Patterson NA, Wiehler WB, Nykiforuk CL, Burton TL, Boora PS, Mosimann SC, Foroud NA, Thibault BJ, Moloney MM, Laroche A, Furukawa-Stoffer TL
Acyl-CoA-binding and self-associating properties of a recombinant 13.3 kDa N-terminal fragment of diacylglycerol acyltransferase-1 from oilseed rape.
BMC Biochem. 2006;724.
BACKGROUND: Diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol to generate triacylglycerol and CoA. The deduced amino acid sequence of cDNAs encoding DGAT1 from plants and mammals exhibit a hydrophilic N-terminal region followed by a number of potential membrane-spanning segments, which is consistent with the membrane-bound nature of this enzyme family. In order to gain insight into the structure/function properties of DGAT1 from Brassica napus (BnDGAT1), we produced and partially characterized a recombinant polyHis-tagged N-terminal fragment of the enzyme, BnDGAT1(1-116)His6, with calculated molecular mass of 13,278 Da. RESULTS: BnDGAT1(1-116)His6 was highly purified from bacterial lysate and plate-like monoclinic crystals were grown using this preparation. Lipidex-1000 binding assays and gel electrophoresis indicated that BnDGAT1(1-116)His6 interacts with long chain acyl-CoA. The enzyme fragment displayed enhanced affinity for erucoyl (22:1cisDelta13)-CoA over oleoyl (18:1cisDelta9)-CoA, and the binding process displayed positive cooperativity. Gel filtration chromatography and cross-linking studies indicated that BnDGAT1(1-116)His6 self-associated to form a tetramer. Polyclonal antibodies raised against a peptide of 15 amino acid residues representing a segment of BnDGAT1(1-116)His6 failed to react with protein in microsomal vesicles following treatment with proteinase K, suggesting that the N-terminal fragment of BnDGAT1 was localized to the cytosolic side of the ER. CONCLUSION: Collectively, these results suggest that BnDGAT1 may be allosterically modulated by acyl-CoA through the N-terminal region and that the enzyme self-associates via interactions on the cytosolic side of the ER. [Abstract/Link to Full Text]

Celestino KR, Cunha RB, Felix CR
Characterization of a beta-glucanase produced by Rhizopus microsporus var. microsporus, and its potential for application in the brewing industry.
BMC Biochem. 2006;723.
BACKGROUND: In the barley malting process, partial hydrolysis of beta-glucans begins with seed germination. However, the endogenous 1,3-1,4-beta-glucanases are heat inactivated, and the remaining high molecular weight beta-glucans may cause severe problems such as increased brewer mash viscosity and turbidity. Increased viscosity impairs pumping and filtration, resulting in lower efficiency, reduced yields of extracts, and lower filtration rates, as well as the appearance of gelatinous precipitates in the finished beer. Therefore, the use of exogenous beta-glucanases to reduce the beta-glucans already present in the malt barley is highly desirable. RESULTS: The zygomycete microfungus Rhizopus microsporus var. microsporus secreted substantial amounts of beta-glucanase in liquid culture medium containing 0.5% chitin. An active protein was isolated by gel filtration and ion exchange chromatographies of the beta-glucanase activity-containing culture supernatant. This isolated protein hydrolyzed 1,3-1,4-beta-glucan (barley beta-glucan), but showed only residual activity against 1,3-beta-glucan (laminarin), or no activity at all against 1,4-beta-glucan (cellulose), indicating that the R. microsporus var. microsporus enzyme is a member of the EC 3.2.1.73 category. The purified protein had a molecular mass of 33.7 kDa, as determined by mass spectrometry. The optimal pH and temperature for hydrolysis of 1,3-1,4-beta-glucan were in the ranges of 4-5, and 50-60 degrees C, respectively. The Km and Vmax values for hydrolysis of beta-glucan at pH 5.0 and 50 degrees C were 22.39 mg.mL-1 and 16.46 mg.min-1, respectively. The purified enzyme was highly sensitive to Cu+2, but showed less or no sensitivity to other divalent ions, and was able to reduce both the viscosity and the filtration time of a sample of brewer mash. In comparison to the values determined for the mash treated with two commercial glucanases, the relative viscosity value for the mash treated with the 1,3-1,4-beta-glucanase produced by R. microsporus var. microsporus. was determined to be consistently lower. CONCLUSION: The zygomycete microfungus R. microsporus var. microsporus produced a 1,3-1,4-beta-D-glucan 4-glucanhydrolase (EC 3.2.1.73) which is able to hydrolyze beta-D-glucan that contains both the 1,3- and 1,4-bonds (barley beta-glucans). Its molecular mass was 33.7 kDa. Maximum activity was detected at pH values in the range of 4-5, and temperatures in the range of 50-60 degrees C. The enzyme was able to reduce both the viscosity of the brewer mash and the filtration time, indicating its potential value for the brewing industry. [Abstract/Link to Full Text]

Kamath N, Karwowska-Desaulniers P, Pflum MK
Limited proteolysis of human histone deacetylase 1.
BMC Biochem. 2006;722.
BACKGROUND: Histone deacetylase (HDAC) proteins are associated with cell proliferation, differentiation, apoptosis, and cancer. Specifically, HDAC1 is linked with cell growth, a hallmark of cancer formation. HDAC1 is a phosphoprotein and phosphorylation at S421 and S423 promotes HDAC1 enzymatic activity and protein association. While single and double point mutants of HDAC1 at S421 and S423 appear functionally similar, the evidence suggests that HDAC1 is phosphorylated simultaneously at both S421 and S423 in vivo. Additional experiments are necessary to probe the role of double phosphorylation of HDAC1 at S421 and S423. RESULTS: To characterize HDAC1 phosphorylation at S421 and S423, limited proteolysis of HDAC1 was performed for the first time. HDAC1 degraded without production of discrete fragments. By performing concentration-dependent proteolysis, HDAC1 double point mutants with disrupted phosphorylation at S421 and S423 displayed different trypsin sensitivities compared to wild type HDAC1. Unexpectedly, HDAC1 single point mutants with disrupted phosphorylation at either S421 or S423 demonstrated protease sensitivity similar to the wild type HDAC1. CONCLUSION: Concentration-dependent proteolysis experiments provide evidence that phosphorylation of S421 and S423 individually contribute to HDAC1 function. In addition, the limited proteolysis experiments support a model where associated proteins promote HDAC1 enzymatic activity, reinforcing the importance of protein interactions in HDAC1 structure and function. Finally, because HDAC1 does not display distinct regions of protease sensitivity, the proteolysis studies suggest that HDAC1 comprises inter-related structural regions. [Abstract/Link to Full Text]

Shikata K, Sasa-Masuda T, Okuno Y, Waga S, Sugino A
The DNA polymerase activity of Pol epsilon holoenzyme is required for rapid and efficient chromosomal DNA replication in Xenopus egg extracts.
BMC Biochem. 2006;721.
BACKGROUND: DNA polymerase epsilon (Pol epsilon) is involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the roles of replicative Pol alpha and Pol delta in chromosomal DNA replication are relatively well understood and well documented, the precise role of Pol epsilon in chromosomal DNA replication is not well understood. RESULTS: This study uses a Xenopus egg extract DNA replication system to further elucidate the replicative role(s) played by Pol epsilon. Previous studies show that the initiation timing and elongation of chromosomal DNA replication are markedly impaired in Pol epsilon-depleted Xenopus egg extracts, with reduced accumulation of replicative intermediates and products. This study shows that normal replication is restored by addition of Pol epsilon holoenzyme to Pol epsilon-depleted extracts, but not by addition of polymerase-deficient forms of Pol epsilon, including polymerase point or deletion mutants or incomplete enzyme complexes. Evidence is also provided that Pol epsilon holoenzyme interacts directly with GINS, Cdc45p and Cut5p, each of which plays an important role in initiation of chromosomal DNA replication in eukaryotic cells. CONCLUSION: These results indicate that the DNA polymerase activity of Pol epsilon holoenzyme plays an essential role in normal chromosomal DNA replication in Xenopus egg extracts. These are the first biochemical data to show the DNA polymerase activity of Pol epsilon holoenzyme is essential for chromosomal DNA replication in higher eukaryotes, unlike in yeasts. [Abstract/Link to Full Text]

Funderud A, Henanger HH, Hafte TT, Amieux PS, Orstavik S, Skĺlhegg BS
Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine Cbeta2.
BMC Biochem. 2006;720.
BACKGROUND: Two main genes encoding the catalytic subunits Calpha and Cbeta of cyclic AMP dependent protein kinase (PKA) have been identified in all vertebrates examined. The murine, bovine and human Cbeta genes encode several splice variants, including the splice variant Cbeta2. In mouse Cbeta2 has a relative molecular mass of 38 kDa and is only expressed in the brain. In human and bovine Cbeta2 has a relative molecular mass of 47 kDa and is mainly expressed in lymphoid tissues. RESULTS: We identified a novel 47 kDa splice variant encoded by the mouse Cbeta gene that is highly expressed in lymphoid cells. Cloning, expression, and production of a sequence-specific antiserum and characterization of PKA catalytic subunit activities demonstrated the 47 kDa protein to be a catalytically active murine homologue of human and bovine Cbeta2. Based on the present results and the existence of a human brain-specifically expressed Cbeta splice variant designated Cbeta4 that is identical to the former mouse Cbeta2 splice variant, the mouse splice variant has now been renamed mouse Cbeta4. CONCLUSION: Murine lymphoid tissues express a protein that is a homologue of human and bovine Cbeta2. The murine Cbeta gene encodes the splice variants Cbeta1, Cbeta2, Cbeta3 and Cbeta4, as is the case with the human Cbeta gene. [Abstract/Link to Full Text]

Higashibata A, Imamizo-Sato M, Seki M, Yamazaki T, Ishioka N
Influence of simulated microgravity on the activation of the small GTPase Rho involved in cytoskeletal formation--molecular cloning and sequencing of bovine leukemia-associated guanine nucleotide exchange factor.
BMC Biochem. 2006;719.
BACKGROUND: The irregular formation of cytoskeletal fibers in spaceflown experimental cells has been observed, but the disorganization process of fibers is still poorly understood. It is well known that the activation of the small GTPase Rho leads to actin stress fibers assembly. This study was performed to evaluate the effect of simulated microgravity on the activation of Rho that is involved in actin fiber remodeling in cells. RESULTS: Clinorotation influences actin fiber remodeling and its related signaling pathways that involve the small GTPase Rho. Actin stress fiber remodeling was significantly inhibited to a greater extent in cells cultured under clinorotation than in static cultured cells. From the gene and protein expression analyses, we found that the expression level of leukemia-associated Rho guanine nucleotide exchange factor (LARG), which activates Rho, was downregulated under clinorotation. Moreover, we identified the full-length LARG cDNA. The amount of GTP-bound RhoA, that is, the active form of RhoA, decreased under this condition. CONCLUSION: The activation of the small GTPase Rho was influenced by simulated microgravity generated by a three-dimensional (3D) clinostat. Furthermore, the full-length cDNA of bovine LARG, a member of the Rho guanine nucleotide exchange factor (GEF) family, was identified, and its gene expression was observed to be downregulated under clinorotation. This downregulation subsequently resulted in the repression of RhoA activation. These results indicated that the disorganization of the actin fibers was caused by the inhibition of Rho activation by 3D clinorotation. [Abstract/Link to Full Text]

Davies MN, Toseland CP, Moss DS, Flower DR
Benchmarking pK(a) prediction.
BMC Biochem. 2006;718.
BACKGROUND: pKa values are a measure of the protonation of ionizable groups in proteins. Ionizable groups are involved in intra-protein, protein-solvent and protein-ligand interactions as well as solubility, protein folding and catalytic activity. The pKa shift of a group from its intrinsic value is determined by the perturbation of the residue by the environment and can be calculated from three-dimensional structural data. RESULTS: Here we use a large dataset of experimentally-determined pKas to analyse the performance of different prediction techniques. Our work provides a benchmark of available software implementations: MCCE, MEAD, PROPKA and UHBD. Combinatorial and regression analysis is also used in an attempt to find a consensus approach towards pKa prediction. The tendency of individual programs to over- or underpredict the pKa value is related to the underlying methodology of the individual programs. CONCLUSION: Overall, PROPKA is more accurate than the other three programs. Key to developing accurate predictive software will be a complete sampling of conformations accessible to protein structures. [Abstract/Link to Full Text]

Badal S, Brown PD, Ragoobirsingh D
Nitric oxide agents impair insulin-mediated signal transduction in rat skeletal muscle.
BMC Biochem. 2006;717.
BACKGROUND: Evidence demonstrates that exogenously administered nitric oxide (NO) can induce insulin resistance in skeletal muscle. We have investigated the modulatory effects of two NO donors, S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and S-nitrosoglutathione (GSNO) on the early events in insulin signaling in rat skeletal myocytes. RESULTS: Skeletal muscle cells from 6-8 week old Sprague-Dawley rats were treated with SNAP or GSNO (25 ng/ml) in the presence or absence of glucose (25 mM) and insulin (100 nM). Cellular insulin receptor-beta levels and tyrosine phosphorylation in IRS-1 were significantly reduced, while serine phosphorylation in IRS-1 was significantly increased in these cells, when compared to the insulin-stimulated control. Reversal to near normal levels was achieved using the NO scavenger, 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO). CONCLUSION: These data suggest that NO is a potent modulator of insulin-mediated signal transduction and may play a significant role in the pathogenesis of type 2 diabetes mellitus. [Abstract/Link to Full Text]

Abdullah MA, Valaitis AP, Dean DH
Identification of a Bacillus thuringiensis Cry11Ba toxin-binding aminopeptidase from the mosquito, Anopheles quadrimaculatus.
BMC Biochem. 2006;716.
BACKGROUND: Aminopeptidase N (APN) type proteins isolated from several species of lepidopteran insects have been implicated as Bacillus thuringiensis (Bt) toxin-binding proteins (receptors) for Cry toxins. We examined brush border membrane vesicle (BBMV) proteins from the mosquito Anopheles quadrimaculatus to determine if APNs from this organism would bind mosquitocidal Cry toxins that are active to it. RESULTS: A 100-kDa protein with APN activity (APNAnq 100) was isolated from the brush border membrane of Anopheles quadrimaculatus. Native state binding analysis by surface plasmon resonance shows that APNAnq 100 forms tight binding to a mosquitocidal Bt toxin, Cry11Ba, but not to Cry2Aa, Cry4Ba or Cry11Aa. CONCLUSION: An aminopeptidase from Anopheles quadrimaculatus mosquitoes is a specific binding protein for Bacillus thuringiensis Cry11Ba. [Abstract/Link to Full Text]

Siadat OR, Lougarre A, Lamouroux L, Ladurantie C, Fournier D
The effect of engineered disulfide bonds on the stability of Drosophila melanogaster acetylcholinesterase.
BMC Biochem. 2006;712.
BACKGROUND: Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use in biosensors for detection of these insecticides. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, its stability has to be improved for extensive utilization. RESULTS: To create a disulfide bond that could increase the stability of the Drosophila melanogaster acetylcholinesterase, we selected seven positions taking into account first the distance between Cbeta of two residues, in which newly introduced cysteines will form the new disulfide bond and second the conservation of the residues in the cholinesterase family. Most disulfide bonds tested did not increase and even decreased the stability of the protein. However, one engineered disulfide bridge, I327C/D375C showed significant stability increase toward denaturation by temperature (170 fold at 50 degrees C), urea, organic solvent and provided resistance to protease degradation. The new disulfide bridge links the N-terminal domain (first 356 aa) to the C-terminal domain. The quantities produced by this mutant were the same as in wild-type flies. CONCLUSION: Addition of a disulfide bridge may either stabilize or unstabilize proteins. One bond out of the 7 tested provided significant stabilisation. [Abstract/Link to Full Text]

Hagge SO, Hammer MU, Wiese A, Seydel U, Gutsmann T
Calcium adsorption and displacement: characterization of lipid monolayers and their interaction with membrane-active peptides/proteins.
BMC Biochem. 2006;715.
BACKGROUND: The first target of antimicrobial peptides (AMPs) is the bacterial membrane. In the case of Gram-negative bacteria this is the outer membrane (OM), the lipid composition of which is extremely asymmetric: Whereas the inner leaflet is composed of a phospholipid mixture, the outer leaflet is made up solely from lipopolysaccharides (LPSs). LPS, therefore, represents the first target of AMPs. The binding and intercalation of polycationic AMPs is driven by the number and position of negatively charged groups of the LPS. Also, proteins other than cationic AMPs can interact with LPS, e.g. leading eventually to a neutralization of the endotoxic effects of LPS. We compared different biophysical techniques to gain insight into the properties of the electrical surface potentials of lipid monolayers and aggregates composed of LPSs and various phospholipids and their interaction with peptides and proteins. RESULTS: The net negative charge calculated from the chemical structure of the phospholipid and LPS molecules is linearly correlated with the adsorption of calcium to two-dimensional lipid monolayers composed of the respective lipids. However, the zeta-potentials determined by the electrophoretic mobility of LPS aggregates can only be interpreted by assuming a dependence of the plane of shear on the number of saccharides and charged groups. Various peptides and proteins were able to displace calcium adsorbed to monolayers. CONCLUSION: To characterize the electrical properties of negatively charged phospholipids and LPSs and their electrostatic interaction with various polycationic peptides/proteins, the adsorption of calcium to and displacement from lipid monolayers is a suitable parameter. Using the calcium displacement method, the binding of peptides to monolayers can be determined even if they do not intercalate. The interpretation of zeta-potential data is difficulty for LPS aggregates, because of the complex three-dimensional structure of the LPS molecules. However, the influence of peptides/proteins on the zeta-potential can be used to characterize the underlying interaction mechanisms. [Abstract/Link to Full Text]

Gorelick DA, Praetorius J, Tsunenari T, Nielsen S, Agre P
Aquaporin-11: a channel protein lacking apparent transport function expressed in brain.
BMC Biochem. 2006;714.
BACKGROUND: The aquaporins are a family of integral membrane proteins composed of two subfamilies: the orthodox aquaporins, which transport only water, and the aquaglyceroporins, which transport glycerol, urea, or other small solutes. Two recently described aquaporins, numbers 11 and 12, appear to be more distantly related to the other mammalian aquaporins and aquaglyceroporins. RESULTS: We report on the characterization of Aquaporin-11 (AQP11). AQP11 RNA and protein is found in multiple rat tissues, including kidney, liver, testes and brain. AQP11 has a unique distribution in brain, appearing in Purkinje cell dendrites, hippocampal neurons of CA1 and CA2, and cerebral cortical neurons. Immunofluorescent staining of Purkinje cells indicates that AQP11 is intracellular. Unlike other aquaporins, Xenopus oocytes expressing AQP11 in the plasma membrane failed to transport water, glycerol, urea, or ions. CONCLUSION: AQP11 is functionally distinct from other proteins of the aquaporin superfamily and could represent a new aquaporin subfamily. Further studies are necessary to elucidate the role of AQP11 in the brain. [Abstract/Link to Full Text]

Arnesen T, Betts MJ, Pendino F, Liberles DA, Anderson D, Caro J, Kong X, Varhaug JE, Lillehaug JR
Characterization of hARD2, a processed hARD1 gene duplicate, encoding a human protein N-alpha-acetyltransferase.
BMC Biochem. 2006;713.
BACKGROUND: Protein acetylation is increasingly recognized as an important mechanism regulating a variety of cellular functions. Several human protein acetyltransferases have been characterized, most of them catalyzing epsilon-acetylation of histones and transcription factors. We recently described the human protein acetyltransferase hARD1 (human Arrest Defective 1). hARD1 interacts with NATH (N-Acetyl Transferase Human) forming a complex expressing protein N-terminal alpha-acetylation activity. RESULTS: We here describe a human protein, hARD2, with 81 % sequence identity to hARD1. The gene encoding hARD2 most likely originates from a eutherian mammal specific retrotransposition event. hARD2 mRNA and protein are expressed in several human cell lines. Immunoprecipitation experiments show that hARD2 protein potentially interacts with NATH, suggesting that hARD2-NATH complexes may be responsible for protein N-alpha-acetylation in human cells. In NB4 cells undergoing retinoic acid mediated differentiation, the level of endogenous hARD1 and NATH protein decreases while the level of hARD2 protein is stable. CONCLUSION: A human protein N-alpha-acetyltransferase is herein described. ARD2 potentially complements the functions of ARD1, adding more flexibility and complexity to protein N-alpha-acetylation in human cells as compared to lower organisms which only have one ARD. [Abstract/Link to Full Text]

Jovine L, Janssen WG, Litscher ES, Wassarman PM
The PLAC1-homology region of the ZP domain is sufficient for protein polymerisation.
BMC Biochem. 2006;711.
BACKGROUND: Hundreds of extracellular proteins polymerise into filaments and matrices by using zona pellucida (ZP) domains. ZP domain proteins perform highly diverse functions, ranging from structural to receptorial, and mutations in their genes are responsible for a number of severe human diseases. Recently, PLAC1, Oosp1-3, Papillote and CG16798 proteins were identified that share sequence homology with the N-terminal half of the ZP domain (ZP-N), but not with its C-terminal half (ZP-C). The functional significance of this partial conservation is unknown. RESULTS: By exploiting a highly engineered bacterial strain, we expressed in soluble form the PLAC1-homology region of mammalian sperm receptor ZP3 as a fusion to maltose binding protein. Mass spectrometry showed that the 4 conserved Cys residues within the ZP-N moiety of the fusion protein adopt the same disulfide bond connectivity as in full-length native ZP3, indicating that it is correctly folded, and electron microscopy and biochemical analyses revealed that it assembles into filaments. CONCLUSION: These findings provide a function for PLAC1-like proteins and, by showing that ZP-N is a biologically active folding unit, prompt a re-evaluation of the architecture of the ZP domain and its polymers. Furthermore, they suggest that ZP-C might play a regulatory role in the assembly of ZP domain protein complexes. [Abstract/Link to Full Text]

Franklin RB, Zou J, Yu Z, Costello LC
EAAC1 is expressed in rat and human prostate epithelial cells; functions as a high-affinity L-aspartate transporter; and is regulated by prolactin and testosterone.
BMC Biochem. 2006;710.
BACKGROUND: Prostate epithelial cells accumulate a high level of aspartate that is utilized as a substrate for their unique function of production and secretion of enormously high levels of citrate. In most mammalian cells aspartate is synthesized; and, therefore is a non-essential amino acid. In contrast, in citrate-producing prostate cells, aspartate is an essential amino acid that must be derived from circulation. The prostate intracellular/extracellular conditions present a 40:1 concentration gradient. Therefore, these cells must possess a plasma membrane-associated aspartate uptake transport process to achieve their functional activity. In earlier kinetic studies we identified the existence of a unique Na+-dependent high-affinity L-aspartate transport process in rat prostate secretory epithelial cells. The present report is concerned with the identification of this putative L-aspartate transporter in rat and human prostate cells. RESULTS: The studies show for the first time that EAAC1 is expressed in normal rat prostate epithelial cells, in normal and hyperplastic human prostate glands, and in human malignant prostate cell lines. EAAC1 expression and high-affinity L-aspartate transport are correspondingly down-regulated by EAAC1 siRNA knock down. Exposure of prostate cells to physiological levels of prolactin or testosterone results in an up-regulation of EAAC1 expression and a corresponding increase in the high-affinity transport of L-aspartate into the cells. CONCLUSION: This study shows that EAAC1 functions as the high-affinity L-aspartate transporter that is responsible for the uptake and accumulation of aspartate in prostate cells. In other cells (predominantly excitable tissue cells), EAAC1 has been reported to function as a glutamate transporter rather than as an aspartate transporter. The regulation of EAAC1 expression and L-aspartate transport by testosterone and prolactin is consistent with their regulation of citrate production in prostate cells. The identification of EAAC1 as the high-affinity L-aspartate transporter now permits studies to elucidate the mechanism of hormonal regulation of EAAC1 gene expression, and to investigate the mechanism by which the cellular environment effects the functioning of EAAC1 as an aspartate transporter or as a glutamate transporter. [Abstract/Link to Full Text]

Cal S, Peinado JR, Llamazares M, Quesada V, Moncada-Pazos A, Garabaya C, López-Otín C
Identification and characterization of human polyserase-3, a novel protein with tandem serine-protease domains in the same polypeptide chain.
BMC Biochem. 2006;79.
BACKGROUND: We have previously described the identification and characterization of polyserase-1 and polyserase-2, two human serine proteases containing three different catalytic domains within the same polypeptide chain. Polyserase-1 shows a complex organization and it is synthesized as a membrane-bound protein which can generate three independent serine protease domains as a consequence of post-translational processing events. The two first domains are enzymatically active. By contrast, polyserase-2 is an extracellular glycosylated protein whose three protease domains remain embedded in the same chain, and only the first domain possesses catalytic activity. RESULTS: Following our interest in the study of the human degradome, we have cloned a human liver cDNA encoding polyserase-3, a new protease with tandem serine protease domains in the same polypeptide chain. Comparative analysis of polyserase-3 with the two human polyserases described to date, revealed that this novel polyprotein is more closely related to polyserase-2 than to polyserase-1. Thus, polyserase-3 is a secreted protein such as polyserase-2, but lacks additional domains like the type II transmembrane motif and the low-density lipoprotein receptor module present in the membrane-anchored polyserase-1. Moreover, analysis of post-translational mechanisms operating in polyserase-3 maturation showed that its two protease domains remain as integral parts of the same polypeptide chain. This situation is similar to that observed in polyserase-2, but distinct from polyserase-1 whose protease domains are proteolytically released from the original chain to generate independent units. Immunolocalization studies indicated that polyserase-3 is secreted as a non-glycosylated protein, thus being also distinct from polyserase-2, which is a heavily glycosylated protein. Enzymatic assays indicated that recombinant polyserase-3 degrades the alpha-chain of fibrinogen as well as pro-urokinase-type plasminogen activator (pro-uPA). Northern blot analysis showed that polyserase-3 exhibits a unique expression pattern among human polyserases, being predominantly detected in testis, liver, heart and ovary, as well as in several tumor cell lines. CONCLUSION: These findings contribute to define the growing group of human polyserine proteases composed at present by three different proteins. All of them share a complex structural design with several catalytic units in a single polypeptide but also show specific features in terms of enzymatic properties, expression patterns and post-translational maturation mechanisms. [Abstract/Link to Full Text]

Füllekrug J, Shevchenko A, Shevchenko A, Simons K
Identification of glycosylated marker proteins of epithelial polarity in MDCK cells by homology driven proteomics.
BMC Biochem. 2006;78.
BACKGROUND: MDCK cells derived from canine kidney are an important experimental model system for investigating epithelial polarity in mammalian cells. Monoclonal antibodies against apical gp114 and basolateral p58 have served as important tools in these studies. However, the molecular identity of these membrane glycoproteins has not been known. RESULTS: We have identified the sialoglycoprotein gp114 as a dog homologue of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family. Gp114 was enriched from tissue culture cells by subcellular fractionation and immunoaffinity chromatography. The identification was based on tandem mass spectrometry and homology based proteomics. In addition, the p58 basolateral marker glycoprotein was found to be the beta subunit of (Na+)(K+)-ATPase. CONCLUSION: Gp114 has been characterized previously regarding glycosylation dependent trafficking and lipid raft association. The identification as a member of the canine CEACAM family will enable synergy between the fields of epithelial cell biology and other research areas. Our approach exemplifies how membrane proteins can be identified from species with unsequenced genomes by homology based proteomics. This approach is applicable to any model system. [Abstract/Link to Full Text]

de Graaf K, Czajkowska H, Rottmann S, Packman LC, Lilischkis R, Lüscher B, Becker W
The protein kinase DYRK1A phosphorylates the splicing factor SF3b1/SAP155 at Thr434, a novel in vivo phosphorylation site.
BMC Biochem. 2006;77.
BACKGROUND: The U2 small nuclear ribonucleoprotein particle (snRNP) component SF3b1/SAP155 is the only spliceosomal protein known to be phosphorylated concomitant with splicing catalysis. DYRK1A is a nuclear protein kinase that has been localized to the splicing factor compartment. Here we describe the identification of DYRK1A as a protein kinase that phosphorylates SF3b1 in vitro and in cultivated cells. RESULTS: Overexpression of DYRK1A caused a markedly increased phosphorylation of SF3b1 in COS-7 cells as assessed by Western blotting with an antibody specific for phosphorylated Thr-Pro dipeptide motifs. Phosphopeptide mapping of metabolically labelled SF3b1 showed that the majority of the in vivo-phosphopeptides corresponded to sites also phosphorylated by DYRK1A in vitro. Phosphorylation with cyclin E/CDK2, a kinase previously reported to phosphorylate SF3b1, generated a completely different pattern of phosphopeptides. By mass spectrometry and mutational analysis of SF3b1, Thr434 was identified as the major phosphorylation site for DYRK1A. Overexpression of DYRK1A or the related kinase, DYRK1B, resulted in an enhanced phosphorylation of Thr434 in endogenous SF3b1 in COS-7 cells. Downregulation of DYRK1A in HEK293 cells or in HepG2 cells by RNA interference reduced the phosphorylation of Thr434 in SF3b1. CONCLUSION: The present data show that the splicing factor SF3b1 is a substrate of the protein kinase DYRK1A and suggest that DYRK1A may be involved in the regulation of pre mRNA-splicing. [Abstract/Link to Full Text]

Benetka W, Koranda M, Maurer-Stroh S, Pittner F, Eisenhaber F
Farnesylation or geranylgeranylation? Efficient assays for testing protein prenylation in vitro and in vivo.
BMC Biochem. 2006;76.
BACKGROUND: Available in vitro and in vivo methods for verifying protein substrates for posttranslational modifications via farnesylation or geranylgeranylation (for example, autoradiography with 3H-labeled anchor precursors) are time consuming (weeks/months), laborious and suffer from low sensitivity. RESULTS: We describe a new technique for detecting prenyl anchors in N-terminally glutathione S-transferase (GST)-labeled constructs of target proteins expressed in vitro in rabbit reticulocyte lysate and incubated with 3H-labeled anchor precursors. Alternatively, hemagglutinin (HA)-labeled constructs expressed in vivo (in cell culture) can be used. For registration of the radioactive marker, we propose to use a thin layer chromatography (TLC) analyzer. As a control, the protein yield is tested by Western blotting with anti-GST- (or anti-HA-) antibodies on the same membrane that has been previously used for TLC-scanning. These protocols have been tested with Rap2A, v-Ki-Ras2 and RhoA (variant RhoA63L) including the necessary controls. We show directly that RasD2 is a farnesylation target. CONCLUSION: Savings in time for experimentation and the higher sensitivity for detecting 3H-labeled lipid anchors recommend the TLC-scanning method with purified GST- (or HA-) tagged target proteins as the method of choice for analyzing their prenylation capabilities in vitro and in vivo and, possibly, also for studying the myristoyl and palmitoyl posttranslational modifications. [Abstract/Link to Full Text]

Kang J, Gocke CB, Yu H
Phosphorylation-facilitated sumoylation of MEF2C negatively regulates its transcriptional activity.
BMC Biochem. 2006;75.
BACKGROUND: Sumoylation has emerged as an important posttranslational regulatory mechanism for transcription factors and cofactors. Sumoylation of many transcription factors represses their transcriptional activities. The myocyte enhancer factor 2 (MEF2) family of transcription factors plays an important role in regulating gene expression during myogenesis and has been recently shown to be sumoylated. RESULTS: Consistent with earlier reports, we show that sumoylation of MEF2C at K391 inhibits its transcriptional activity. Sumoylation of MEF2C does not block its DNA-binding activity. A small C-terminal fragment of MEF2C containing K391, referred to as delta-N2-MEF2C, is efficiently sumoylated and, when targeted to DNA, represses transcription at neighbouring promoters. Because delta-N2-MEF2C lacks the binding site for class II histone deacetylases (HDACs), this result suggests that sumoylation of MEF2C may help to recruit transcriptional repressors other than these HDACs. Intriguingly, we show that phosphorylation of S396 in MEF2C, a residue in close proximity to the major sumoylation site (K391) and known to be phosphorylated in vivo, enhances sumoylation of delta- N2-MEF2C in vitro. The S396A mutation reduces sumoylation of MEF2C in vivo and enhances the transcription activity of MEF2C in reporter assays. CONCLUSION: We propose that phosphorylation of MEF2C at S396 facilitates its sumoylation at K391, which in turn recruits yet unidentified co-repressors to inhibit transcription. Our studies further suggest that sumoylation motifs containing a phosphorylated serine or an acidic residue at the +5 position might be more efficiently sumoylated. [Abstract/Link to Full Text]

---

Recent Articles in BMC Chemical Biology

---

No recent articles are currently available.

---

Recent Articles in BMC Molecular Biology

---

Mortusewicz O, Leonhardt H
XRCC1 and PCNA are loading platforms with distinct kinetic properties and different capacities to respond to multiple DNA lesions.
BMC Mol Biol. 2007;881.
BACKGROUND: Genome integrity is constantly challenged and requires the coordinated recruitment of multiple enzyme activities to ensure efficient repair of DNA lesions. We investigated the dynamics of XRCC1 and PCNA that act as molecular loading platforms and play a central role in this coordination. RESULTS: Local DNA damage was introduced by laser microirradation and the recruitment of fluorescent XRCC1 and PCNA fusion proteins was monitored by live cell microscopy. We found an immediate and fast recruitment of XRCC1 preceding the slow and continuous recruitment of PCNA. Fluorescence bleaching experiments (FRAP and FLIP) revealed a stable association of PCNA with DNA repair sites, contrasting the high turnover of XRCC1. When cells were repeatedly challenged with multiple DNA lesions we observed a gradual depletion of the nuclear pool of PCNA, while XRCC1 dynamically redistributed even to lesions inflicted last. CONCLUSION: These results show that PCNA and XRCC1 have distinct kinetic properties with functional consequences for their capacity to respond to successive DNA damage events. [Abstract/Link to Full Text]

Perehinec TM, Qazi SN, Gaddipati SR, Salisbury V, Rees CE, Hill PJ
Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria.
BMC Mol Biol. 2007;880.
BACKGROUND: The Gateway recombinatorial cloning system allows easy and rapid joining of DNA fragments. Here we report the construction and evaluation of three different Gram-positive vectors that can be used with the Multisite Gateway cloning system to rapidly produce new gene arrangements in plasmid constructs for use in a variety of Gram-positive bacteria. RESULTS: Comparison of patterns of reporter gene expression with conventionally constructed clones show that the presence of residual recombination (att) sites does not have an effect on patterns of gene expression, although overall levels of gene expression may vary. Rapid construction of these new vectors allowed vector/gene combinations to be optimized following evaluation of plasmid constructs in different bacterial cells and demonstrates the benefits of plasmid construction using Gateway cloning. CONCLUSION: The residual att sites present after Gateway cloning did not affect patterns of promoter induction in Gram-positive bacteria and there was no evidence of differences in mRNA stability of transcripts. However overall levels of gene expression may be reduced, possibly due to some post-transcriptional event. The new vectors described here allow faster, more efficient cloning in range of Gram-positive bacteria. [Abstract/Link to Full Text]

Koscianska E, Baev V, Skreka K, Oikonomaki K, Rusinov V, Tabler M, Kalantidis K
Prediction and preliminary validation of oncogene regulation by miRNAs.
BMC Mol Biol. 2007;879.
BACKGROUND: MicroRNAs (miRNAs) are one of the most abundant groups of regulatory genes in multicellular organisms, playing important roles in many fundamental cellular processes. More than four hundred miRNAs have been identified in humans and the deregulation of miRNA expression has been also shown in many cancers. Despite the postulated involvement of miRNAs in tumourigenesis, there are only a few examples where an oncogene or a tumour suppressor has been identified as a miRNA target. RESULTS: Here, we present an in silico analysis of potential miRNA- oncogene interactions. Moreover, we have tested the validity of two possible interactions of miRNAs with genes related to cancer. We present evidence for the down-regulation of c-MYC, one of the most potent and frequently deregulated oncogenes, by let-7 miRNA, via the predicted binding site in the 3'UTR, and verify the suppression of BCL-2 by miR16. CONCLUSION: In this work both bioinformatic and experimental approaches for the prediction and validation of possible targets for miRNAs have been used. A list of putative targets for different oncomirs, validation of which would be of special interest, is proposed and two such interactions have been experimentally validated. [Abstract/Link to Full Text]

Di Lisi R, Picard A, Ausoni S, Schiaffino S
GATA elements control repression of cardiac troponin I promoter activity in skeletal muscle cells.
BMC Mol Biol. 2007;878.
BACKGROUND: We reported previously that the cardiac troponin I (cTnI) promoter drives cardiac-specific expression of reporter genes in cardiac muscle cells and in transgenic mice, and that disruption of GATA elements inactivates the cTnI promoter in cultured cardiomyocytes. We have now examined the role of cTnI promoter GATA elements in skeletal muscle cells. RESULTS: Mutation or deletion of GATA elements induces a strong transcriptional activation of the cTnI promoter in regenerating skeletal muscle and in cultured skeletal muscle cells. Electrophoretic mobility shift assays show that proteins present in nuclear extracts of C2C12 muscle cells bind the GATA motifs present in the cTnI promoter. However, GATA protein complex formation is neither reduced nor supershifted by antibodies specific for GATA-2, -3 and -4, the only GATA transcripts present in muscle cells. CONCLUSION: These findings indicate that the cTnI gene promoter is repressed in skeletal muscle cells by GATA-like factors and open the way to further studies aimed at identifying these factors. [Abstract/Link to Full Text]

Angers-Loustau A, Rainy J, Wartiovaara K
PlasmaDNA: a free, cross-platform plasmid manipulation program for molecular biology laboratories.
BMC Mol Biol. 2007;877.
BACKGROUND: Most molecular biology experiments, and the techniques associated with this field of study, involve a great deal of engineering in the form of molecular cloning. Like all forms of engineering, perfect information about the starting material is crucial for successful completion of design and strategies. RESULTS: We have generated a program that allows complete in silico simulation of the cloning experiment. Starting with a primary DNA sequence, PlasmaDNA looks for restriction sites, open reading frames, primer annealing sequences, and various common domains. The databases are easily expandable by the user to fit his most common cloning needs. PlasmaDNA can manage and graphically represent multiple sequences at the same time, and keeps in memory the overhangs at the end of the sequences if any. This means that it is possible to virtually digest fragments, to add the digestion products to the project, and to ligate together fragments with compatible ends to generate the new sequences. Polymerase Chain Reaction (PCR) fragments can also be virtually generated using the primer database, automatically adding to the fragments any 5' extra sequences present in the primers. CONCLUSION: PlasmaDNA is a program available both on Windows and Apple operating systems, designed to facilitate molecular cloning experiments by building a visual map of the DNA. It then allows the complete planning and simulation of the cloning experiment. It also automatically updates the new sequences generated in the process, which is an important help in practice. The capacity to maintain multiple sequences in the same file can also be used to archive the various steps and strategies involved in the cloning of each construct. The program is freely available for download without charge or restriction. [Abstract/Link to Full Text]

Prigge JR, Schmidt EE
HAP1 can sequester a subset of TBP in cytoplasmic inclusions via specific interaction with the conserved TBP(CORE).
BMC Mol Biol. 2007;876.
BACKGROUND: Huntington's disease, spinal and bulbar muscular atrophy, and spinocerebellar ataxia 17 (SCA17) are caused by expansions in the polyglutamine (polyQ) repeats in Huntingtin protein (Htt), androgen receptor protein (AR), and TATA-binding protein (TBP), respectively. Htt-associated protein 1 (HAP1), a component of neuronal cytoplasmic stigmoid bodies (STBs), can sequester polyQ-expanded Htt and AR in STBs, thereby antagonizing formation of the nuclear aggregates associated with apoptotic neuron loss and disease progression. RESULTS: Clones of HAP1 were isolated from unbiased two-hybrid screens for proteins that interact with TBP. Domain mapping showed that regions between amino acids 157 and 261 and between amino acids 473 and 582 of mouse HAP1 both bind specifically to the conserved C-terminal TBP(CORE) domain, away from the TBP N-terminal polyQ region. When fluorescently tagged versions of HAP1 or TBP were expressed independently in COS-7, 293, or Neuro-2a cells, all TBP localized to the nucleus and all HAP1 assembled into cytoplasmic stigmoid-like bodies (STLBs). When co-expressed, a portion of the TBP was assembled into the HAP1 STLBs while the remainder was localized to the nucleus. Although the TBP N terminus, including the polyQ region, was unnecessary for TBP-HAP1 interaction, in mammalian cells, removal of the TBP Q(repeat) reduced the proportion of TBP that assembled into STLBs, whereas expansion of the Q(repeat) had no significant affect on TBP subcellular localization. CONCLUSION: HAP1 can sequester a subset of TBP protein away from the nucleus; extranuclear TBP sequestration is quantitatively influenced by the TBP polyQ repeat. These results suggest HAP1 could provide protection from SCA17 neuropathology. [Abstract/Link to Full Text]

Gundelach H, Braas D, Klempnauer KH
The promoter regions of the Myb-regulated Adora2B and Mcm4 genes co-localize with origins of DNA replication.
BMC Mol Biol. 2007;875.
BACKGROUND: The retroviral oncogene v-myb encodes a transcription factor (v-Myb) which is responsible for the transformation of myelomonocytic cells by avian myeloblastosis virus (AMV). v-Myb is thought to exert its biological effects by deregulating the expression of specific target genes. We have recently demonstrated that the chicken Gas41 gene, whose promoter co-localizes with an origin of DNA replication, is a bona fide Myb target gene. Because of this finding we have asked whether other Myb-regulated genes are also associated with DNA replication origins. RESULTS: We show that the promoter region of the chicken adenosine receptor 2B gene (Adora2B), a known Myb-target gene, acts as a DNA replication origin. Furthermore, we have examined known replication origins for the presence of Myb binding sites. We found that the intergenic region between the genes for the minichromosome maintenance 4 protein (Mcm4) and the catalytic subunit of DNA-dependent protein kinase (Prkdc), whose human counterpart has been identified as a replication origin, contains a number of Myb binding sites. Our data show that this region also acts as an origin of replication in chicken cells. Interestingly, we found that the chicken Mcm4 gene is also Myb-regulated. CONCLUSION: Our work identifies the chicken Mcm4 gene as a novel Myb target gene and presents evidence for the co-localization of two novel origins of DNA replication with Myb-regulated genes. Our work raises the possibility that a fraction of Myb target gene promoters is associated with DNA replication origins. [Abstract/Link to Full Text]

Chalmel F, Léveillard T, Jaillard C, Lardenois A, Berdugo N, Morel E, Koehl P, Lambrou G, Holmgren A, Sahel JA, Poch O
Rod-derived Cone Viability Factor-2 is a novel bifunctional-thioredoxin-like protein with therapeutic potential.
BMC Mol Biol. 2007;874.
BACKGROUND: Cone degeneration is the hallmark of the inherited retinal disease retinitis pigmentosa. We have previously identified a trophic factor "Rod-derived Cone Viability Factor (RdCVF) that is secreted by rods and promote cone viability in a mouse model of the disease. RESULTS: Here we report the bioinformatic identification and the experimental analysis of RdCVF2, a second trophic factor belonging to the Rod-derived Cone Viability Factor family. The mouse RdCVF gene is known to be bifunctional, encoding both a long thioredoxin-like isoform (RdCVF-L) and a short isoform with trophic cone photoreceptor viability activity (RdCVF-S). RdCVF2 shares many similarities with RdCVF in terms of gene structure, expression in a rod-dependent manner and protein 3D structure. Furthermore, like RdCVF, the RdCVF2 short isoform exhibits cone rescue activity that is independent of its putative thiol-oxydoreductase activity. CONCLUSION: Taken together, these findings define a new family of bifunctional genes which are: expressed in vertebrate retina, encode trophic cone viability factors, and have major therapeutic potential for human retinal neurodegenerative diseases such as retinitis pigmentosa. [Abstract/Link to Full Text]

Ferreira R, Eberharter A, Bonaldi T, Chioda M, Imhof A, Becker PB
Site-specific acetylation of ISWI by GCN5.
BMC Mol Biol. 2007;873.
BACKGROUND: The tight organisation of eukaryotic genomes as chromatin hinders the interaction of many DNA-binding regulators. The local accessibility of DNA is regulated by many chromatin modifying enzymes, among them the nucleosome remodelling factors. These enzymes couple the hydrolysis of ATP to disruption of histone-DNA interactions, which may lead to partial or complete disassembly of nucleosomes or their sliding on DNA. The diversity of nucleosome remodelling factors is reflected by a multitude of ATPase complexes with distinct subunit composition. RESULTS: We found further diversification of remodelling factors by posttranslational modification. The histone acetyltransferase GCN5 can acetylate the Drosophila remodelling ATPase ISWI at a single, conserved lysine, K753, in vivo and in vitro. The target sequence is strikingly similar to the N-terminus of histone H3, where the corresponding lysine, H3K14, can also be acetylated by GCN5. The acetylated form of ISWI represents a minor species presumably associated with the nucleosome remodelling factor NURF. CONCLUSION: Acetylation of histone H3 and ISWI by GCN5 is explained by the sequence similarity between the histone and ISWI around the acetylation site. The common motif RKT/SxGx(Kac)xPR/K differs from the previously suggested GCN5/PCAF recognition motif GKxxP. This raises the possibility of co-regulation of a nucleosome remodelling factor and its nucleosome substrate through acetylation of related epitopes and suggests a direct crosstalk between two distinct nucleosome modification principles. [Abstract/Link to Full Text]

Chen KL, Liu WH, Yang YY, Leu SJ, Shih NY
Characterization of novel transforming growth factor-beta type I receptors found in malignant pleural effusion tumor cells.
BMC Mol Biol. 2007;872.
BACKGROUND: Tumors expressing a transforming growth factor-beta type I receptor (T beta RI) mutant with sequence deletions in a nine-alanine (9A) stretch of the signal peptide are reported to be highly associated with disease progression. Expression of this mutant could interfere with endogenous TGFbeta signaling in the cell. However, little is known about the importance of the remaining part of the signal peptide on the cellular function of T beta RI. RESULTS: We cloned and identified four new in-frame deletion variants of T beta RI, designated DM1 to DM4, in pleural effusion-derived tumor cells. Intriguingly, DM1 and DM2, with a small region truncated in the putative signal peptide of T beta RI, had a serious defect in their protein expression compared with that of the wild-type receptor. Using serial deletion mutagenesis, we characterized a region encoded by nucleotides 16-51 as a key element controlling T beta RI protein expression. Consistently, both DM1 and DM2 have this peptide deleted. Experiments using cycloheximde and MG132 further confirmed its indispensable role for the protein stability of T beta RI. In contrast, truncation of the 9A-stretch itself or a region downstream to the stretch barely affected T beta RI expression. However, variants lacking a region C-terminal to the stretch completely lost their capability to conduct TGFbeta-induced transcriptional activation. Intriguingly, expression of DM3 in a cell sensitive to TGFbeta made it significantly refractory to TGFbeta-mediated growth inhibition. The effect of DM3 was to ablate the apoptotic event induced by TGFbeta. CONCLUSION: We identified four new transcript variants of T beta RI in malignant effusion tumor cells and characterized two key elements controlling its protein stability and transcriptional activation. Expression of one of variants bestowed cancer cells with a growth advantage in the presence of TGFbeta. These results highlight the potential roles of some naturally occurring T beta RI variants on the promotion of tumor malignancy. [Abstract/Link to Full Text]

Chabelskaya S, Gryzina V, Moskalenko S, Le Goff C, Zhouravleva G
Inactivation of NMD increases viability of sup45 nonsense mutants in Saccharomyces cerevisiae.
BMC Mol Biol. 2007;871.
BACKGROUND: The nonsense-mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature termination codons (PTCs). In yeast Saccharomyces cerevisiae, the activity of the NMD pathway depends on the recognition of the PTC by the translational machinery. Translation termination factors eRF1 (Sup45) and eRF3 (Sup35) participate not only in the last step of protein synthesis but also in mRNA degradation and translation initiation via interaction with such proteins as Pab1, Upf1, Upf2 and Upf3. RESULTS: In this work we have used previously isolated sup45 mutants of S. cerevisiae to characterize degradation of aberrant mRNA in conditions when translation termination is impaired. We have sequenced his7-1, lys9-A21 and trp1-289 alleles which are frequently used for analysis of nonsense suppression. We have established that sup45 nonsense and missense mutations lead to accumulation of his7-1 mRNA and CYH2 pre-mRNA. Remarkably, deletion of the UPF1 gene suppresses some sup45 phenotypes. In particular, sup45-n upf1Delta double mutants were less temperature sensitive, and more resistant to paromomycin than sup45 single mutants. In addition, deletion of either UPF2 or UPF3 restored viability of sup45-n double mutants. CONCLUSION: This is the first demonstration that sup45 mutations do not only change translation fidelity but also acts by causing a change in mRNA stability. [Abstract/Link to Full Text]

Lundell K, Thulin P, Hamsten A, Ehrenborg E
Alternative splicing of human peroxisome proliferator-activated receptor delta (PPAR delta): effects on translation efficiency and trans-activation ability.
BMC Mol Biol. 2007;870.
BACKGROUND: Peroxisome proliferator-activated receptor delta (PPAR delta) is a member of the nuclear receptor superfamily. Numerous studies have aimed at unravelling the physiological role of PPAR delta as a transcriptional regulator whereas the regulation of PPAR delta gene expression has been less studied. RESULTS: The principal transcription start site in the human PPAR delta gene identified here is positioned upstream of exon 1, although four alternative 5'-ends related to downstream exons were identified. The demonstration of multiple 5'-UTR splice variants of PPAR delta mRNA, with an impact on translation efficiency, suggests a translational regulation of human PPAR delta expression. Five untranslated exons identified in this study contribute to the variability among the 5'-UTRs of human PPAR delta mRNAs. Moreover, in vitro studies of a 3'-splice transcript encoding a truncated variant of PPAR delta (designated PPAR delta 2) show that this isoform constitutes a potential dominant negative form of the receptor. CONCLUSION: We propose that alternative splicing of human PPAR delta constitutes an intrinsic role for the regulation of PPAR delta expression and thus activity, and highlight the significance of alternative splicing of this nuclear receptor in physiology and disease. [Abstract/Link to Full Text]

Blasius M, Buob R, Shevelev IV, Hubscher U
Enzymes involved in DNA ligation and end-healing in the radioresistant bacterium Deinococcus radiodurans.
BMC Mol Biol. 2007;869.
BACKGROUND: Enzymes involved in DNA metabolic events of the highly radioresistant bacterium Deinococcus radiodurans are currently examined to understand the mechanisms that protect and repair the Deinococcus radiodurans genome after extremely high doses of gamma-irradiation. Although several Deinococcus radiodurans DNA repair enzymes have been characterised, no biochemical data is available for DNA ligation and DNA endhealing enzymes of Deinococcus radiodurans so far. DNA ligases are necessary to seal broken DNA backbones during replication, repair and recombination. In addition, ionizing radiation frequently leaves DNA strand-breaks that are not feasible for ligation and thus require end-healing by a 5'-polynucleotide kinase or a 3'-phosphatase. We expect that DNA ligases and end-processing enzymes play an important role in Deinococcus radiodurans DNA strand-break repair. RESULTS: In this report, we describe the cloning and expression of a Deinococcus radiodurans DNA ligase in Escherichia coli. This enzyme efficiently catalyses DNA ligation in the presence of Mn(II) and NAD+ as cofactors and lysine 128 was found to be essential for its activity. We have also analysed a predicted second DNA ligase from Deinococcus radiodurans that is part of a putative DNA repair operon and shows sequence similarity to known ATP-dependent DNA ligases. We show that this enzyme possesses an adenylyltransferase activity using ATP, but is not functional as a DNA ligase by itself. Furthermore, we identified a 5'-polynucleotide kinase similar to human polynucleotide kinase that probably prepares DNA termini for subsequent ligation. CONCLUSION: Deinococcus radiodurans contains a standard bacterial DNA ligase that uses NAD+ as a cofactor. Its enzymatic properties are similar to E. coli DNA ligase except for its preference for Mn(II) as a metal cofactor. The function of a putative second DNA ligase remains unclear, but its adenylyltransferase activity classifies it as a member of the nucleotidyltransferase family. Characterization of another protein from the same operon revealed a 5'-polynucleotide kinase with a possible role in DNA strand-break repair. [Abstract/Link to Full Text]

Buckley KM, Smith LC
Extraordinary diversity among members of the large gene family, 185/333, from the purple sea urchin, Strongylocentrotus purpuratus.
BMC Mol Biol. 2007;868.
BACKGROUND: Recent analysis of immune-related genes within the sea urchin genome revealed a number of large gene families with vertebrate homologues, such as the Toll-like and NOD/NALP-like receptor families and C-type lectins in addition to a rudimentary complement system. Therefore, the immune response of the purple sea urchin appears to be more complex than previously believed. Another component of the sea urchin immune response is an unusual family of mRNAs, known as 185/333, which is strongly upregulated in response to pathogen challenge. The work presented here indicates that this family of transcripts is derived from an unexpectedly diverse gene family. RESULTS: The 185/333 genes are small (< 2 kb) with only two exons. Their extraordinary diversity was exemplified by 121 unique sequences identified from 171 cloned genes. Sequences from the second exons were aligned optimally by introducing large gaps, which defined blocks of sequence known as elements. Genes were defined by the presence or absence of elements. Phylogenetic analysis defined five intron types which, when combined with the exon element patterns resulted in 31 gene patterns, 14 of which were not described previously. Sequence diversity was present in all elements, and was higher in the intron than the exons. Repeats within the sequence facilitated multiple alignments, of which two were analyzed in detail. Although the two alignments differed in length, number of elements, and number of patterns, both were about equally accurate at describing the 185/333 sequences. The genes were closely linked and flanked by short repeats. The repeats within and between the genes may promote their diversification through gene conversion, recombination, and meiotic mispairing. CONCLUSION: The diversity of the 185/333 gene family represents an intriguing addition to what is known about the S. purpuratus immune response, and provides further evidence that invertebrate immune systems are neither simple nor static. [Abstract/Link to Full Text]

Nygard AB, Jřrgensen CB, Cirera S, Fredholm M
Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR.
BMC Mol Biol. 2007;867.
BACKGROUND: Real-time quantitative PCR (qPCR) is a method for rapid and reliable quantification of mRNA transcription. Internal standards such as reference genes are used to normalise mRNA levels between different samples for an exact comparison of mRNA transcription level. Selection of high quality reference genes is of crucial importance for the interpretation of data generated by real-time qPCR. RESULTS: In this study nine commonly used reference genes were investigated in 17 different pig tissues using real-time qPCR with SYBR green. The genes included beta-actin (ACTB), beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L4 (RPL4), succinate dehydrogenase complex subunit A (SDHA), TATA box binding protein (TPB)and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ). The stability of these reference genes in different pig tissues was investigated using the geNorm application. The range of expression stability in the genes analysed was (from the most stable to the least stable): ACTB/RPL4, TBP, HPRT, HMBS, YWHAZ, SDHA, B2M and GAPDH. CONCLUSION: Expression stability varies greatly between genes. ACTB, RPL4, TPB and HPRT1 were found to have the highest stability across tissues. Based on both expression stability and expression level, our data suggest that ACTB and RPL4 are good reference genes for high abundant transcripts while TPB and HPRT1 are good reference genes for low abundant transcripts in expression studies across different pig tissues. [Abstract/Link to Full Text]

Ugrinova I, Monier K, Ivaldi C, Thiry M, Storck S, Mongelard F, Bouvet P
Inactivation of nucleolin leads to nucleolar disruption, cell cycle arrest and defects in centrosome duplication.
BMC Mol Biol. 2007;866.
BACKGROUND: Nucleolin is a major component of the nucleolus, but is also found in other cell compartments. This protein is involved in various aspects of ribosome biogenesis from transcription regulation to the assembly of pre-ribosomal particles; however, many reports suggest that it could also play an important role in non nucleolar functions. To explore nucleolin function in cell proliferation and cell cycle regulation we used siRNA to down regulate the expression of nucleolin. RESULTS: We found that, in addition to the expected effects on pre-ribosomal RNA accumulation and nucleolar structure, the absence of nucleolin results in a cell growth arrest, accumulation in G2, and an increase of apoptosis. Numerous nuclear alterations, including the presence of micronuclei, multiple nuclei or large nuclei are also observed. In addition, a large number of mitotic cells showed a defect in the control of centrosome duplication, as indicated by the presence of more than 2 centrosomes per cell associated with a multipolar spindle structure in the absence of nucleolin. This phenotype is very similar to that obtained with the inactivation of another nucleolar protein, B23. CONCLUSION: Our findings uncovered a new role for nucleolin in cell division, and highlight the importance of nucleolar proteins for centrosome duplication. [Abstract/Link to Full Text]

Boden SA, Shadiac N, Tucker EJ, Langridge P, Able JA
Expression and functional analysis of TaASY1 during meiosis of bread wheat (Triticum aestivum).
BMC Mol Biol. 2007;865.
BACKGROUND: Pairing and synapsis of homologous chromosomes is required for normal chromosome segregation and the exchange of genetic material via recombination during meiosis. Synapsis is complete at pachytene following the formation of a tri-partite proteinaceous structure known as the synaptonemal complex (SC). In yeast, HOP1 is essential for formation of the SC, and localises along chromosome axes during prophase I. Homologues in Arabidopsis (AtASY1), Brassica (BoASY1) and rice (OsPAIR2) have been isolated through analysis of mutants that display decreased fertility due to severely reduced synapsis of homologous chromosomes. Analysis of these genes has indicated that they play a similar role to HOP1 in pairing and formation of the SC through localisation to axial/lateral elements of the SC. RESULTS: The full length wheat cDNA and genomic clone, TaASY1, has been isolated, sequenced and characterised. TaASY1 is located on chromosome Group 5 and the open reading frame displays significant nucleotide sequence identity to OsPAIR2 (84%) and AtASY1 (63%). Transcript and protein analysis showed that expression is largely restricted to meiotic tissue, with elevated levels during the stages of prophase I when pairing and synapsis of homologous chromosomes occur. Immunolocalisation using transmission electron microscopy showed TaASY1 interacts with chromatin that is associated with both axial elements before SC formation as well as lateral elements of formed SCs. CONCLUSION: TaASY1 is a homologue of ScHOP1, AtASY1 and OsPAIR2 and is the first gene to be isolated from bread wheat that is involved in pairing and synapsis of homologous chromosomes. [Abstract/Link to Full Text]

Lafarge S, Hamzeh-Cognasse H, Chavarin P, Genin C, Garraud O, Cognasse F
A flow cytometry technique to study intracellular signals NF-kappaB and STAT3 in peripheral blood mononuclear cells.
BMC Mol Biol. 2007;864.
BACKGROUND: Cytokines have essential roles on intercellular communications and are effective in using a variety of intracellular pathways. Among this multitude of signalling pathways, the NF-kappaB (nuclear factor kappaB) and STAT (signal transducer and activator of transcription) families are among the most frequently investigated because of their importance. Indeed, they have important role in innate and adaptive immunity. Current techniques to study NF-kappaB and STAT rely on specific ELISAs, Western Blots and--most recently described--flow cytometry; so far, investigation of such signalling pathways are most commonly performed on homogeneous cells after purification. RESULTS: The present investigation aimed at developing a flow cytometry technique to study transcription factors in various cellular types such as mixtures of B-cells, T-lymphocytes and monocytes/macrophages stimulated in steady state conditions (in other words, as peripheral blood mononuclear cells). To achieve this goal, a two step procedure was carried out; the first one consisted of stimulating PBMCs with IL1beta, sCD40L and/or IL10 in such a manner that optimal stimulus was found for each cell subset (and subsequent signal transduction, therefore screened by specific ELISA); the second step consisted of assessing confirmation and fine delineation of technical conditions by specific Western-Blotting for either NF-kappaB or STAT products. We then went on to sensitize the detection technique for mixed cells using 4 color flow cytometry. CONCLUSION: In response to IL1beta, or IL10, the levels of phosphorylated NF-kappaB and STAT3--respectively--increased significantly for all the studied cell types. In contrast, B-cells and monocytes/macrophages--but, interestingly, not T-lymphocytes (in the context of PBMCs)--responded significantly to sCD40L by increasing phosphorylated NF-kappaB. [Abstract/Link to Full Text]

Klase Z, Kale P, Winograd R, Gupta MV, Heydarian M, Berro R, McCaffrey T, Kashanchi F
HIV-1 TAR element is processed by Dicer to yield a viral micro-RNA involved in chromatin remodeling of the viral LTR.
BMC Mol Biol. 2007;863.
BACKGROUND: RNA interference (RNAi) is a regulatory mechanism conserved in higher eukaryotes. The RNAi pathway generates small interfering RNA (siRNA) or micro RNA (miRNA) from either long double stranded stretches of RNA or RNA hairpins, respectively. The siRNA or miRNA then guides an effector complex to a homologous sequence of mRNA and regulates suppression of gene expression through one of several mechanisms. The suppression of gene expression through these mechanisms serves to regulate endogenous gene expression and protect the cell from foreign nucleic acids. There is growing evidence that many viruses have developed in the context of RNAi and express either a suppressor of RNAi or their own viral miRNA. RESULTS: In this study we investigated the possibility that the HIV-1 TAR element, a hairpin structure of ~50 nucleotides found at the 5' end of the HIV viral mRNA, is recognized by the RNAi machinery and processed to yield a viral miRNA. We show that the protein Dicer, the enzyme responsible for cleaving miRNA and siRNA from longer RNA sequences, is expressed in CD4+ T-cells. Interestingly, the level of expression of Dicer in monocytes is sub-optimal, suggesting a possible role for RNAi in maintaining latency in T-cells. Using a biotin labeled TAR element we demonstrate that Dicer binds to this structure. We show that recombinant Dicer is capable of cleaving the TAR element in vitro and that TAR derived miRNA is present in HIV-1 infected cell lines and primary T-cell blasts. Finally, we show that a TAR derived miRNA is capable of regulating viral gene expression and may be involved in repressing gene expression through transcriptional silencing. CONCLUSION: HIV-1 TAR element is processed by the Dicer enzyme to create a viral miRNA. This viral miRNA is detectable in infected cells and appears to contribute to viral latency. [Abstract/Link to Full Text]

Maccoux LJ, Clements DN, Salway F, Day PJ
Identification of new reference genes for the normalisation of canine osteoarthritic joint tissue transcripts from microarray data.
BMC Mol Biol. 2007;862.
BACKGROUND: Real-time reverse transcriptase quantitative polymerase chain reaction (real-time RT-qPCR) is the most accurate measure of gene expression in biological systems. The comparison of different samples requires the transformation of data through a process called normalisation. Reference or housekeeping genes are candidate genes which are selected on the basis of constitutive expression across samples, and allow the quantification of changes in gene expression. At present, no reference gene has been identified for any organism which is universally optimal for use across different tissue types or disease situations. We used microarray data to identify new reference genes generated from total RNA isolated from normal and osteoarthritic canine articular tissues (bone, ligament, cartilage, synovium and fat). RT-qPCR assays were designed and applied to each different articular tissue. Reference gene expression stability and ranking was compared using three different mathematical algorithms. RESULTS: Twelve new potential reference genes were identified from microarray data. One gene (mitochondrial ribosomal protein S7 [MRPS7]) was stably expressed in all five of the articular tissues evaluated. One gene HIRA interacting protein 5 isoform 2 [HIRP5]) was stably expressed in four of the tissues evaluated. A commonly used reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was not stably expressed in any of the tissues evaluated. Most consistent agreement between rank ordering of reference genes was observed between Bestkeeper(c) and geNorm, although each method tended to agree on the identity of the most stably expressed genes and the least stably expressed genes for each tissue. New reference genes identified using microarray data normalised in a conventional manner were more stable than those identified by microarray data normalised by using a real-time RT-qPCR methodology. CONCLUSION: Microarray data normalised by a conventional manner can be filtered using a simple stepwise procedure to identify new reference genes, some of which will demonstrate good measures of stability. Mitochondrial ribosomal protein S7 is a new reference gene worthy of investigation in other canine tissues and diseases. Different methods of reference gene stability assessment will generally agree on the most and least stably expressed genes, when co-regulation is not present. [Abstract/Link to Full Text]

van den Broek WJ, Wansink DG, Wieringa B
Somatic CTG*CAG repeat instability in a mouse model for myotonic dystrophy type 1 is associated with changes in cell nuclearity and DNA ploidy.
BMC Mol Biol. 2007;861.
BACKGROUND: Trinucleotide instability is a hallmark of degenerative neurological diseases like Huntington's disease, some forms of spinocerebellar ataxia and myotonic dystrophy type 1 (DM1). To investigate the effect of cell type and cell state on the behavior of the DM1 CTG*CAG repeat, we studied a knock-in mouse model for DM1 at different time points during ageing and followed how repeat fate in cells from liver and pancreas is associated with polyploidization and changes in nuclearity after the onset of terminal differentiation. RESULTS: After separation of liver hepatocytes and pancreatic acinar cells in pools with 2n, 4n or 8n DNA, we analyzed CTG*CAG repeat length variation by resolving PCR products on an automated PAGE system. We observed that somatic CTG*CAG repeat expansion in our DM1 mouse model occurred almost uniquely in the fraction of cells with high cell nuclearity and DNA ploidy and aggravated with aging. CONCLUSION: Our findings suggest that post-replicative and terminal-differentiation events, coupled to changes in cellular DNA content, form a preconditional state that influences the control of DNA repair or recombination events involved in trinucleotide expansion in liver hepatocytes and pancreatic acinar cells. [Abstract/Link to Full Text]

Cheung L, Andersen M, Gustavsson C, Odeberg J, Fernández-Pérez L, Norstedt G, Tollet-Egnell P
Hormonal and nutritional regulation of alternative CD36 transcripts in rat liver--a role for growth hormone in alternative exon usage.
BMC Mol Biol. 2007;860.
BACKGROUND: CD36 is a multiligand receptor involved in various metabolic pathways, including cellular uptake of long-chain fatty acids. Defect function or expression of CD36 can result in dyslipidemia or insulin resistance. We have previously shown that CD36 expression is female-predominant in rat liver. In the present study, hormonal and nutritional regulation of hepatic CD36 expression was examined in male and female rats. Since alternative transcription start sites have been described in murine and human Cd36, we investigated whether alternative CD36 transcripts are differentially regulated in rat liver during these conditions. RESULTS: Sequence information of the rat Cd36 5'-UTR was extended, showing that the gene structure of Cd36 in rat is similar to that previously described in mouse with at least two alternative first exons. The rat Cd36 exon 1a promoter was sequenced and found to be highly similar to murine and human Cd36. We show that alternative first exon usage is involved in the female-predominant expression of CD36 in rat liver and during certain hormonal states that induce CD36 mRNA abundance. Estrogen treatment or continuous infusion of growth hormone (GH) in male rats induced CD36 expression preferentially through the exon 1a promoter. Old age was associated with increased CD36 expression in male rats, albeit without any preferential first exon usage. Intermittent GH treatment in old male rats reversed this effect. Mild starvation (12 hours without food) reduced CD36 expression in female liver, whereas its expression was increased in skeletal muscle. CONCLUSION: The results obtained in this study confirm and extend our previous observation that GH is an important regulator of hepatic CD36, and depending on the mode of treatment (continuous or intermittent) the gene might be either induced or repressed. We suggest that the effects of continuous GH secretion in females (which is stimulatory) and intermittent GH secretion in males (which is inhibitory) explains the sex-different expression of this gene. Furthermore, a female-specific repression of hepatic CD36 in response to food deprivation was found, which was in contrast to a stimulatory effect in skeletal muscle. This demonstrates a tissue-specific regulation of Cd36. [Abstract/Link to Full Text]

Kim YG, Yoo JS, Kim JH, Kim CM, Oh JW
Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase.
BMC Mol Biol. 2007;859.
BACKGROUND: Japanese encephalitis virus (JEV) NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp) domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust in vitro RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated. RESULTS: To characterize the biochemical properties of JEV RdRp, we expressed in Escherichia coli and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A) RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U81, at the two nucleotides upstream of the 3'-end of the template. CONCLUSION: As a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the in vitro reconstitution of viral RNA replicase complex, we for the first time established an in vitro JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the development of anti-JEV agents. [Abstract/Link to Full Text]

Murphy MW, Zarkower D, Bardwell VJ
Vertebrate DM domain proteins bind similar DNA sequences and can heterodimerize on DNA.
BMC Mol Biol. 2007;858.
BACKGROUND: The DM domain is a zinc finger-like DNA binding motif first identified in the sexual regulatory proteins Doublesex (DSX) and MAB-3, and is widely conserved among metazoans. DM domain proteins regulate sexual differentiation in at least three phyla and also control other aspects of development, including vertebrate segmentation. Most DM domain proteins share little similarity outside the DM domain. DSX and MAB-3 bind partially overlapping DNA sequences, and DSX has been shown to interact with DNA via the minor groove without inducing DNA bending. DSX and MAB-3 exhibit unusually high DNA sequence specificity relative to other minor groove binding proteins. No detailed analysis of DNA binding by the seven vertebrate DM domain proteins, DMRT1-DMRT7 has been reported, and thus it is unknown whether they recognize similar or diverse DNA sequences. RESULTS: We used a random oligonucleotide in vitro selection method to determine DNA binding sites for six of the seven proteins. These proteins selected sites resembling that of DSX despite differences in the sequence of the DM domain recognition helix, but they varied in binding efficiency and in preferences for particular nucleotides, and some behaved anomalously in gel mobility shift assays. DMRT1 protein from mouse testis extracts binds the sequence we determined, and the DMRT proteins can bind their in vitro-defined sites in transfected cells. We also find that some DMRT proteins can bind DNA as heterodimers. CONCLUSION: Our results suggest that target gene specificity of the DMRT proteins does not derive exclusively from major differences in DNA binding specificity. Instead target specificity may come from more subtle differences in DNA binding preference between different homodimers, together with differences in binding specificity between homodimers versus heterodimers. [Abstract/Link to Full Text]

Adams AM, Harding PL, Iversen PL, Coleman C, Fletcher S, Wilton SD
Antisense oligonucleotide induced exon skipping and the dystrophin gene transcript: cocktails and chemistries.
BMC Mol Biol. 2007;857.
BACKGROUND: Antisense oligonucleotides (AOs) can interfere with exon recognition and intron removal during pre-mRNA processing, and induce excision of a targeted exon from the mature gene transcript. AOs have been used in vitro and in vivo to redirect dystrophin pre-mRNA processing in human and animal cells. Targeted exon skipping of selected exons in the dystrophin gene transcript can remove nonsense or frame-shifting mutations that would otherwise have lead to Duchenne Muscular Dystrophy, the most common childhood form of muscle wasting. RESULTS: Although many dystrophin exons can be excised using a single AO, several exons require two motifs to be masked for efficient or specific exon skipping. Some AOs were inactive when applied individually, yet pronounced exon excision was induced in transfected cells when the AOs were used in select combinations, clearly indicating synergistic rather than cumulative effects on splicing. The necessity for AO cocktails to induce efficient exon removal was observed with 2 different chemistries, 2'-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers. Similarly, other trends in exon skipping, as a consequence of 2'-O-methyl AO action, such as removal of additional flanking exons or variations in exon skipping efficiency with overlapping AOs, were also seen when the corresponding sequences were prepared as phosphorodiamidate morpholino oligomers. CONCLUSION: The combination of 2 AOs, directed at appropriate motifs in target exons was found to induce very efficient targeted exon skipping during processing of the dystrophin pre-mRNA. This combinatorial effect is clearly synergistic and is not influenced by the chemistry of the AOs used to induce exon excision. A hierarchy in exon skipping efficiency, observed with overlapping AOs composed of 2'-O-methyl modified bases, was also observed when these same sequences were evaluated as phosphorodiamidate morpholino oligomers, indicating design parameters established with one chemistry may be applied to the other. [Abstract/Link to Full Text]

Hojman P, Zibert JR, Gissel H, Eriksen J, Gehl J
Gene expression profiles in skeletal muscle after gene electrotransfer.
BMC Mol Biol. 2007;856.
BACKGROUND: Gene transfer by electroporation (DNA electrotransfer) to muscle results in high level long term transgenic expression, showing great promise for treatment of e.g. protein deficiency syndromes. However little is known about the effects of DNA electrotransfer on muscle fibres. We have therefore investigated transcriptional changes through gene expression profile analyses, morphological changes by histological analysis, and physiological changes by force generation measurements. DNA electrotransfer was obtained using a combination of a short high voltage pulse (HV, 1000 V/cm, 100 mus) followed by a long low voltage pulse (LV, 100 V/cm, 400 ms); a pulse combination optimised for efficient and safe gene transfer. Muscles were transfected with green fluorescent protein (GFP) and excised at 4 hours, 48 hours or 3 weeks after treatment. RESULTS: Differentially expressed genes were investigated by microarray analysis, and descriptive statistics were performed to evaluate the effects of 1) electroporation, 2) DNA injection, and 3) time after treatment. The biological significance of the results was assessed by gene annotation and supervised cluster analysis.Generally, electroporation caused down-regulation of structural proteins e.g. sarcospan and catalytic enzymes. Injection of DNA induced down-regulation of intracellular transport proteins e.g. sentrin. The effects on muscle fibres were transient as the expression profiles 3 weeks after treatment were closely related with the control muscles. Most interestingly, no changes in the expression of proteins involved in inflammatory responses or muscle regeneration was detected, indicating limited muscle damage and regeneration. Histological analysis revealed structural changes with loss of cell integrity and striation pattern in some fibres after DNA+HV+LV treatment, while HV+LV pulses alone showed preservation of cell integrity. No difference in the force generation capacity was observed in the muscles 2 weeks after DNA electrotransfer. CONCLUSION: The small and transient changes found in the gene expression profiles are of great importance, as this demonstrates that DNA electrotransfer is safe with minor effects on the muscle host cells. These findings are essential for introducing the DNA electrotransfer to muscle for clinical use. Indeed the HV+LV pulse combination used has been optimised to ensure highly efficient and safe DNA electrotransfer. [Abstract/Link to Full Text]

Said HM, Hagemann C, Stojic J, Schoemig B, Vince GH, Flentje M, Roosen K, Vordermark D
GAPDH is not regulated in human glioblastoma under hypoxic conditions.
BMC Mol Biol. 2007;855.
BACKGROUND: Gene expression studies related to cancer diagnosis and treatment are becoming more important. Housekeeping genes that are absolutely reliable are essential for these studies to normalize gene expression. An incorrect choice of housekeeping genes leads to interpretation errors of experimental results including evaluation and quantification of pathological gene expression. Here, we examined (a) the degree of regulation of GAPDH expression in human glioblastoma cells under hypoxic conditions in vitro in comparison to other housekeeping genes like beta-actin, serving as experimental loading controls, (b) the potential use of GAPDH as a target for tumor therapeutic approaches and (c) differences in GAPDH expression between low-grade astrocytomas and glioblastomas, for which modest and severe hypoxia, respectively, have been previously demonstrated. GAPDH and beta-actin expression was comparatively examined in vivo in human low-grade astrocytoma and glioblastoma on both protein and mRNA level, by Western blot and semiquantitative RT-PCR, respectively. Furthermore, the same proteins were determined in vitro in U373, U251 and GaMG human glioblastoma cells using the same methods. HIF-1alpha protein regulation under hypoxia was also determined on mRNA level in vitro in GaMG and on protein level in U251, U373 and GaMG cells. RESULTS: We observed no hypoxia-induced regulatory effect on GAPDH expression in the three glioblastoma cell lines studied in vitro. In addition, GAPDH expression was similar in patient tumor samples of low-grade astrocytoma and glioblastoma, suggesting a lack of hypoxic regulation in vivo. CONCLUSION: GAPDH represents an optimal choice of a housekeeping gene and/or loading control to determine the expression of hypoxia induced genes at least in glioblastoma. Because of the lack of GAPDH regulation under hypoxia, this gene is not an attractive target for tumor therapeutic approaches in human glioblastoma. [Abstract/Link to Full Text]

Glauser DA, Brun T, Gauthier BR, Schlegel W
Transcriptional response of pancreatic beta cells to metabolic stimulation: large scale identification of immediate-early and secondary response genes.
BMC Mol Biol. 2007;854.
BACKGROUND: Physiological long term adaptation of pancreatic beta cells is driven by stimuli such as glucose and incretin hormones acting via cAMP (e.g. GLP-1) and involves regulated gene expression. Several rapidly inducible immediate-early genes (IEGs) have been identified in beta cells. Many of these IEGs code for transcription factors and have the potential to control the transcription of downstream target genes likely involved in long term cellular adaptation. The identity of these target genes has not been determined, and the sequence of events occurring during beta cell adaptation is still unclear. RESULTS: We have developed a microarray-based strategy for the systematic search of targets. In Min6 insulin-secreting cells, we identified 592 targets and 1278 IEGs responding to a co-stimulation with glucose and cAMP. Both IEGs and targets were involved in a large panel of functions, including those important to beta cell physiology (metabolism, secretion). Nearly 200 IEGs were involved in signaling and transcriptional regulation. To find specific examples of the regulatory link between IEGs and targets, target promoter sequences were analyzed in silico. Statistically significant over-representation of AP-1 response elements notably suggested an important role for this transcription factor, which was experimentally verified. Indeed, cell stimulation altered expression of IEG-encoded components of the AP-1 complex, activating AP-1-dependent transcription. Loss and gain-of-function experiments furthermore allowed to validate a new AP-1 regulated gene (sulfiredoxin) among the targets. AP-1 and sulfiredoxin are sequentially induced also in primary cells from rat islets of Langerhans. CONCLUSION: By identifying IEGs and their downstream targets, this study brings a comprehensive description of the transcriptional response occurring after beta cell stimulation, as well as new mechanistic insights concerning the AP-1 transcription factor. [Abstract/Link to Full Text]

Ciudad L, Bellés X, Piulachs MD
Structural and RNAi characterization of the German cockroach lipophorin receptor, and the evolutionary relationships of lipoprotein receptors.
BMC Mol Biol. 2007;853.
BACKGROUND: Lipophorin receptors (LpRs) have been described in a number of insects, but functional studies have been reported only in locusts and mosquitoes. The aim of the present work was to characterize the LpR of the cockroach Blattella germanica, not only molecularly but also functionally using RNAi techniques, and to place LpRs in a phylogenetical context among lipoprotein receptors. RESULTS: We cloned a putative LpR from B. germanica (BgLpR) using RT-PCR methods. Two isoforms of BgLpR that differ from each other by an insertion/deletion of 24 amino acids were obtained from the fat body and the ovary. A phylogenetical analysis of lipoprotein receptors showed that BgLpR grouped with other sequences annotated as LpR in a cluster placed as a sister group of vertebrate low density lipoprotein receptors (LDLR) + lipoprotein receptor-related proteins 8 (LPR8) + vitellogenin receptors (VgR) + very low density lipoprotein receptors (VLDLR). The two BgLpR isoforms are expressed in different adult female tissues (fat body, ovary, brain, midgut, muscle) and in embryos. In ovaries and fat body, the two isoforms are similarly expressed during the first gonadotrophic cycle. mRNA levels in the fat body increase in parallel to vitellogenesis, whereas they decrease in the ovaries. BgLpR protein levels increase in parallel to vitellogenesis in both organs. Treatment with juvenile hormone increases BgLpR protein. RNAi experiments show that females with lower BgLpR expression have less lipophorin in the growing oocytes with respect to controls. CONCLUSION: The two isoforms of BgLpR are structurally similar to other LpRs. Phylogenetical analyses show that LpRs and the group formed by vertebrate LDLR + LPR8 + VgR + VLDLR, diverged from a common ancestor and diversified in parallel. The different expression patterns in the fat body and the ovary, comparing mRNA and protein, indicate that the corresponding mechanisms regulating BgLpR expression are different. Experiments with JH III suggest that such a hormone regulates the expression of BgLpR posttranscriptionally. RNAi experiments indicate that BgLpR is a functional lipophorin receptor. [Abstract/Link to Full Text]

Takahasi KR, Sakuraba Y, Gondo Y
Mutational pattern and frequency of induced nucleotide changes in mouse ENU mutagenesis.
BMC Mol Biol. 2007;852.
BACKGROUND: With the advent of sequence-based approaches in the mutagenesis studies, it is now possible to directly evaluate the genome-wide pattern of experimentally induced DNA sequence changes for a diverse array of organisms. To gain a more comprehensive understanding of the mutational bias inherent in mouse ENU mutagenesis, this study describes a detailed evaluation of the induced mutational pattern obtained from a sequence-based screen of ENU-mutagenized mice. RESULTS: Based on a large-scale screening data, we derive the sequence-based estimates of the nucleotide-specific pattern and frequency of ENU-induced base replacement mutation in the mouse germline, which are then combined with the pattern of codon usage in the mouse coding sequences to infer the spectrum of amino acid changes obtained by ENU mutagenesis. We detect a statistically significant difference between the mutational patterns in phenotype- versus sequence-based screens, which presumably reflects differential phenotypic effects caused by different amino acid replacements. We also demonstrate that the mutations exhibit strong strand asymmetry, and that this imbalance is generated by transcription, most likely as a by-product of transcription-coupled DNA repair in the germline. CONCLUSION: The results clearly illustrate the biased nature of ENU-induced mutations. We expect that a precise understanding of the mutational pattern and frequency of induced nucleotide changes would be of practical importance when designing sequence-based screening strategies to generate mutant mouse strains harboring amino acid variants at specific loci. More generally, by enhancing the collection of experimentally induced mutations in unambiguously defined genomic regions, sequence-based mutagenesis studies will further illuminate the molecular basis of mutagenic and repair mechanisms that preferentially produce a certain class of mutational changes over others. [Abstract/Link to Full Text]