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Recent Articles in Molecular Biology of the Cell

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Hodgson B, Calzada A, Labib K
Mrc1 and Tof1 regulate DNA replication forks in different ways during normal S phase.
Mol Biol Cell. 2007 Oct;18(10):3894-902.
The Mrc1 and Tof1 proteins are conserved throughout evolution, and in budding yeast they are known to associate with the MCM helicase and regulate the progression of DNA replication forks. Previous work has shown that Mrc1 is important for the activation of checkpoint kinases in responses to defects in S phase, but both Mrc1 and Tof1 also regulate the normal process of chromosome replication. Here, we show that these two important factors control the normal progression of DNA replication forks in distinct ways. The rate of progression of DNA replication forks is greatly reduced in the absence of Mrc1 but much less affected by loss of Tof1. In contrast, Tof1 is critical for DNA replication forks to pause at diverse chromosomal sites where nonnucleosomal proteins bind very tightly to DNA, and this role is not shared with Mrc1. [Abstract/Link to Full Text]

Muresan Z, Muresan V
The amyloid-beta precursor protein is phosphorylated via distinct pathways during differentiation, mitosis, stress, and degeneration.
Mol Biol Cell. 2007 Oct;18(10):3835-44.
Phosphorylation of amyloid-beta precursor protein (APP) at Thr(668) is a normal process linked to neurite extension and anterograde transport of vesicular cargo. By contrast, increased phosphorylation of APP is a pathological trait of Alzheimer's disease. APP is overexpressed in Down's syndrome, a condition that occasionally leads to increased APP phosphorylation, in cultured cells. Whether phosphorylation of APP in normal versus high APP conditions occurs by similar or distinct signaling pathways is not known. Here, we addressed this problem using brainstem-derived neurons (CAD cells). CAD cells that ectopically overexpress APP frequently show features of degenerating neurons. We found that, in degenerating cells, APP is hyperphosphorylated and colocalizes with early endosomes. By contrast, in normal CAD cells, phosphorylated APP (pAPP) is excluded from endosomes, and localizes to the Golgi apparatus and to transport vesicles within the neurites. Whereas the neuritic APP is phosphorylated by c-Jun NH(2)-terminal kinase through a pathway that is modulated by glycogen synthase kinase 3beta, the endosomal pAPP in degenerated CAD cells results from activation of cyclin-dependent kinase 5. Additional signaling pathways, leading to APP phosphorylation, become active during stress and mitosis. We conclude that distinct pathways of APP phosphorylation operate in proliferating, differentiating, stressed, and degenerating neurons. [Abstract/Link to Full Text]

Schaub S, Bohnet S, Laurent VM, Meister JJ, Verkhovsky AB
Comparative maps of motion and assembly of filamentous actin and myosin II in migrating cells.
Mol Biol Cell. 2007 Oct;18(10):3723-32.
To understand the mechanism of cell migration, one needs to know how the parts of the motile machinery of the cell are assembled and how they move with respect to each other. Actin and myosin II are thought to be the major structural and force-generating components of this machinery (Mitchison and Cramer, 1996; Parent, 2004). The movement of myosin II along actin filaments is thought to generate contractile force contributing to cell translocation, but the relative motion of the two proteins has not been investigated. We use fluorescence speckle and conventional fluorescence microscopy, image analysis, and computer tracking techniques to generate comparative velocity and assembly maps of actin and myosin II over the entire cell in a simple model system of persistently migrating fish epidermal keratocytes. The results demonstrate contrasting polarized assembly patterns of the two components, indicate force generation at the lamellipodium-cell body transition zone, and suggest a mechanism of anisotropic network contraction via sliding of myosin II assemblies along divergent actin filaments. [Abstract/Link to Full Text]

Sakato M, Sakakibara H, King SM
Chlamydomonas outer arm dynein alters conformation in response to Ca2+.
Mol Biol Cell. 2007 Sep;18(9):3620-34.
We have previously shown that Ca(2+) directly activates ATP-sensitive microtubule binding by a Chlamydomonas outer arm dynein subparticle containing the beta and gamma heavy chains (HCs). The gamma HC-associated LC4 light chain is a member of the calmodulin family and binds 1-2 Ca(2+) with K(Ca) = 3 x 10(-5) M in vitro, suggesting it may act as a Ca(2+) sensor for outer arm dynein. Here we investigate interactions between the LC4 light chain and gamma HC. Two IQ consensus motifs for binding calmodulin-like proteins are located within the stem domain of the gamma heavy chain. In vitro experiments indicate that LC4 undergoes a Ca(2+)-dependent interaction with the IQ motif domain while remaining tethered to the HC. LC4 also moves into close proximity of the intermediate chain IC1 in the presence of Ca(2+). The sedimentation profile of the gamma HC subunit changed subtly upon Ca(2+) addition, suggesting that the entire complex had become more compact, and electron microscopy of the isolated gamma subunit revealed a distinct alteration in conformation of the N-terminal stem in response to Ca(2+) addition. We propose that Ca(2+)-dependent conformational change of LC4 has a direct effect on the stem domain of the gamma HC, which eventually leads to alterations in mechanochemical interactions between microtubules and the motor domain(s) of the outer dynein arm. [Abstract/Link to Full Text]

Murphy SJ, Shapira KE, Henis YI, Leof EB
A unique element in the cytoplasmic tail of the type II transforming growth factor-beta receptor controls basolateral delivery.
Mol Biol Cell. 2007 Oct;18(10):3788-99.
Transforming growth factor (TGF)-beta receptors stimulate diverse signaling processes that control a wide range of biological responses. In polarized epithelia, the TGFbeta type II receptor (T2R) is localized at the basolateral membranes. Sequential cytoplasmic truncations resulted in receptor missorting to apical surfaces, and they indicated an essential targeting element(s) near the receptor's C terminus. Point mutations in the full-length receptor confirmed this prediction, and a unique basolateral-targeting region was elucidated between residues 529 and 538 (LTAxxVAxxR) that was distinct, but colocalized within a clinically significant signaling domain essential for TGFbeta-dependent activation of the Smad2/3 cascade. Transfer of a terminal 84 amino-acid fragment, containing the LTAxxVAxxR element, to the apically sorted influenza hemagglutinin (HA) protein was dominant and directed basolateral HA expression. Although delivery to the basolateral surfaces was direct and independent of any detectable transient apical localization, fluorescence recovery after photobleaching demonstrated similar mobility for the wild-type receptor and a missorted mutant lacking the targeting motif. This latter finding excludes the possibility that the domain acts as a cell membrane retention signal, and it supports the hypothesis that T2R sorting occurs from an intracellular compartment. [Abstract/Link to Full Text]

van der Weele CM, Tsai CW, Wolniak SM
Mago nashi is essential for spermatogenesis in Marsilea.
Mol Biol Cell. 2007 Oct;18(10):3711-22.
Spermatogenesis in Marsilea vestita is a rapid process that is activated by placing dry microspores into water. Nine division cycles produce seven somatic cells and 32 spermatids, where size and position define identity. Spermatids undergo de novo formation of basal bodies in a particle known as a blepharoplast. We are interested in mechanisms responsible for spermatogenous initial formation. Mago nashi (Mv-mago) is a highly conserved gene present as stored mRNA and stored protein in the microspore. Mv-mago protein increases in abundance during development and it localizes at discrete cytoplasmic foci (Mago-dots). RNA interference experiments show that new Mv-mago protein is required for development. With Mv-mago silenced, asymmetric divisions become symmetric, cell fate is disrupted, and development stops. The alpha-tubulin protein distribution, centrin translation, and Mv-PRP19 mRNA distribution are no longer restricted to the spermatogenous cells. Centrin aggregations, resembling blepharoplasts, occur in jacket cells. Mago-dots are undetectable after the silencing of Mv-mago, Mv-Y14, or Mv-eIF4AIII, three core components of the exon junction complex (EJC), suggesting that Mago-dots are either EJCs in the cytoplasm, or Mv-mago protein aggregations dependent on EJCs. Mv-mago protein and other EJC components apparently function in cell fate determination in developing male gametophytes of M. vestita. [Abstract/Link to Full Text]

Strub BR, Eswara MB, Pierce JB, Mangroo D
Utp8p is a nucleolar tRNA-binding protein that forms a complex with components of the nuclear tRNA export machinery in Saccharomyces cerevisiae.
Mol Biol Cell. 2007 Oct;18(10):3845-59.
Utp8p is an essential nucleolar component of the nuclear tRNA export machinery in Saccharomyces cerevisiae. It is thought to act at a step between tRNA maturation/aminoacylation and translocation of the tRNA across the nuclear pore complex. To understand the function of Utp8p in nuclear tRNA export, a comprehensive affinity purification analysis was conducted to identify proteins that interact with Utp8p in vivo. In addition to finding proteins that have been shown previously to copurify with Utp8p, a number of new interactions were identified. These interactions include aminoacyl-tRNA synthetases, the RanGTPase Gsp1p, and nuclear tRNA export receptors such as Los1p and Msn5p. Characterization of the interaction of Utp8p with a subset of the newly identified proteins suggests that Utp8p most likely transfer tRNAs to the nuclear tRNA export receptors by using a channeling mechanism. [Abstract/Link to Full Text]

Ogawa-Goto K, Tanaka K, Ueno T, Tanaka K, Kurata T, Sata T, Irie S
p180 is involved in the interaction between the endoplasmic reticulum and microtubules through a novel microtubule-binding and bundling domain.
Mol Biol Cell. 2007 Oct;18(10):3741-51.
p180 was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum membrane, although its precise role in animal cells has not yet been elucidated. Here, we characterized a new function of human p180 as a microtubule-binding and -modulating protein. Overexpression of p180 in mammalian cells induced an elongated morphology and enhanced acetylated microtubules. Consistently, electron microscopic analysis clearly revealed microtubule bundles in p180-overexpressing cells. Targeted depletion of endogenous p180 by small interfering RNAs led to aberrant patterns of microtubules and endoplasmic reticulum in mammalian cells, suggesting a specific interaction between p180 and microtubules. In vitro sedimentation assays using recombinant polypeptides revealed that p180 bound to microtubules directly and possessed a novel microtubule-binding domain (designated MTB-1). MTB-1 consists of a predicted coiled-coil region and repeat domain, and strongly promoted bundle formation both in vitro and in vivo when expressed alone. Overexpression of p180 induced acetylated microtubules in cultured cells in an MTB-1-dependent manner. Thus, our data suggest that p180 mediates interactions between the endoplasmic reticulum and microtubules mainly through the novel microtubule-binding and -bundling domain MTB-1. [Abstract/Link to Full Text]

Scott CM, Kruse KB, Schmidt BZ, Perlmutter DH, McCracken AA, Brodsky JL
ADD66, a gene involved in the endoplasmic reticulum-associated degradation of alpha-1-antitrypsin-Z in yeast, facilitates proteasome activity and assembly.
Mol Biol Cell. 2007 Oct;18(10):3776-87.
Antitrypsin deficiency is a primary cause of juvenile liver disease, and it arises from expression of the "Z" variant of the alpha-1 protease inhibitor (A1Pi). Whereas A1Pi is secreted from the liver, A1PiZ is retrotranslocated from the endoplasmic reticulum (ER) and degraded by the proteasome, an event that may offset liver damage. To better define the mechanism of A1PiZ degradation, a yeast expression system was developed previously, and a gene, ADD66, was identified that facilitates A1PiZ turnover. We report here that ADD66 encodes an approximately 30-kDa soluble, cytosolic protein and that the chymotrypsin-like activity of the proteasome is reduced in add66Delta mutants. This reduction in activity may arise from the accumulation of 20S proteasome assembly intermediates or from qualitative differences in assembled proteasomes. Add66p also seems to be a proteasome substrate. Consistent with its role in ER-associated degradation (ERAD), synthetic interactions are observed between the genes encoding Add66p and Ire1p, a transducer of the unfolded protein response, and yeast deleted for both ADD66 and/or IRE1 accumulate polyubiquitinated proteins. These data identify Add66p as a proteasome assembly chaperone (PAC), and they provide the first link between PAC activity and ERAD. [Abstract/Link to Full Text]

Kugler SJ, Nagel AC
putzig is required for cell proliferation and regulates notch activity in Drosophila.
Mol Biol Cell. 2007 Oct;18(10):3733-40.
We have identified the gene putzig (pzg) as a key regulator of cell proliferation and of Notch signaling in Drosophila. pzg encodes a Zn-finger protein that was found earlier within a macromolecular complex, including TATA-binding protein-related factor 2 (TRF2)/DNA replication-related element factor (DREF). This complex is involved in core promoter selection, where DREF functions as a transcriptional activator of replication-related genes. Here, we provide the first in vivo evidence that pzg is required for the expression of cell cycle and replication-related genes, and hence for normal developmental growth. Independent of its role in the TRF2/DREF complex, pzg acts as a positive regulator of Notch signaling that may occur by chromatin activation. Down-regulation of pzg activity inhibits Notch target gene activation, whereas Hedgehog (Hh) signal transduction and growth regulation are unaffected. Our findings uncover different modes of operation of pzg during imaginal development of Drosophila, and they provide a novel mechanism of Notch regulation. [Abstract/Link to Full Text]

Wanderling S, Simen BB, Ostrovsky O, Ahmed NT, Vogen SM, Gidalevitz T, Argon Y
GRP94 is essential for mesoderm induction and muscle development because it regulates insulin-like growth factor secretion.
Mol Biol Cell. 2007 Oct;18(10):3764-75.
Because only few of its client proteins are known, the physiological roles of the endoplasmic reticulum chaperone glucose-regulated protein 94 (GRP94) are poorly understood. Using targeted disruption of the murine GRP94 gene, we show that it has essential functions in embryonic development. grp94-/- embryos die on day 7 of gestation, fail to develop mesoderm, primitive streak, or proamniotic cavity. grp94-/- ES cells grow in culture and are capable of differentiation into cells representing all three germ layers. However, these cells do not differentiate into cardiac, smooth, or skeletal muscle. Differentiation cultures of mutant ES cells are deficient in secretion of insulin-like growth factor II and their defect can be complemented with exogenous insulin-like growth factors I or II. The data identify insulin-like growth factor II as one developmentally important protein whose production depends on the activity of GRP94. [Abstract/Link to Full Text]

Ando Y, Yasuda S, Oceguera-Yanez F, Narumiya S
Inactivation of Rho GTPases with Clostridium difficile toxin B impairs centrosomal activation of Aurora-A in G2/M transition of HeLa cells.
Mol Biol Cell. 2007 Oct;18(10):3752-63.
During G2 phase of cell cycle, centrosomes function as a scaffold for activation of mitotic kinases. Aurora-A is first activated at late G2 phase at the centrosome, facilitates centrosome maturation, and induces activation of cyclin B-Cdk1 at the centrosome for mitotic entry. Although several molecules including HEF1 and PAK are implicated in centrosomal activation of Aurora-A, signaling pathways leading to Aurora-A activation at the centrosome, and hence mitotic commitment in vertebrate cells remains largely unknown. Here, we have used Clostridium difficile toxin B and examined the role of Rho GTPases in G2/M transition of HeLa cells. Inactivation of Rho GTPases by the toxin B treatment delayed by 2 h histone H3 phosphorylation, Cdk1/cyclin B activation, and Aurora-A activation. Furthermore, PAK activation at the centrosome that was already present before the toxin addition was significantly attenuated for 2 h by the addition of toxin B, and HEF1 accumulation at the centrosome that occurred in late G2 phase was also delayed. These results suggest that Rho GTPases function in G2/M transition of mammalian cells by mediating multiple signaling pathways converging to centrosomal activation of Aurora-A. [Abstract/Link to Full Text]

Wong J, Nakajima Y, Westermann S, Shang C, Kang JS, Goodner C, Houshmand P, Fields S, Chan CS, Drubin D, Barnes G, Hazbun T
A protein interaction map of the mitotic spindle.
Mol Biol Cell. 2007 Oct;18(10):3800-9.
The mitotic spindle consists of a complex network of proteins that segregates chromosomes in eukaryotes. To strengthen our understanding of the molecular composition, organization, and regulation of the mitotic spindle, we performed a system-wide two-hybrid screen on 94 proteins implicated in spindle function in Saccharomyces cerevisiae. We report 604 predominantly novel interactions that were detected in multiple screens, involving 303 distinct prey proteins. We uncovered a pattern of extensive interactions between spindle proteins reflecting the intricate organization of the spindle. Furthermore, we observed novel connections between kinetochore complexes and chromatin-modifying proteins and used phosphorylation site mutants of NDC80/TID3 to gain insights into possible phospho-regulation mechanisms. We also present analyses of She1p, a novel spindle protein that interacts with the Dam1 kinetochore/spindle complex. The wealth of protein interactions presented here highlights the extent to which mitotic spindle protein functions and regulation are integrated with each other and with other cellular activities. [Abstract/Link to Full Text]

Sillibourne JE, Delaval B, Redick S, Sinha M, Doxsey SJ
Chromatin remodeling proteins interact with pericentrin to regulate centrosome integrity.
Mol Biol Cell. 2007 Sep;18(9):3667-80.
Pericentrin is an integral centrosomal component that anchors regulatory and structural molecules to centrosomes. In a yeast two-hybrid screen with pericentrin we identified chromodomain helicase DNA-binding protein 4 (CHD4/Mi2beta). CHD4 is part of the multiprotein nucleosome remodeling deacetylase (NuRD) complex. We show that many NuRD components interacted with pericentrin by coimmunoprecipitation and that they localized to centrosomes and midbodies. Overexpression of the pericentrin-binding domain of CHD4 or another family member (CHD3) dissociated pericentrin from centrosomes. Depletion of CHD3, but not CHD4, by RNA interference dissociated pericentrin and gamma-tubulin from centrosomes. Microtubule nucleation/organization, cell morphology, and nuclear centration were disrupted in CHD3-depleted cells. Spindles were disorganized, the majority showing a prometaphase-like configuration. Time-lapse imaging revealed mitotic failure before chromosome segregation and cytokinesis failure. We conclude that pericentrin forms complexes with CHD3 and CHD4, but a distinct CHD3-pericentrin complex is required for centrosomal anchoring of pericentrin/gamma-tubulin and for centrosome integrity. [Abstract/Link to Full Text]

Lerdrup M, Bruun S, Grandal MV, Roepstorff K, Kristensen MM, Hommelgaard AM, van Deurs B
Endocytic down-regulation of ErbB2 is stimulated by cleavage of its C-terminus.
Mol Biol Cell. 2007 Sep;18(9):3656-66.
High ErbB2 levels are associated with cancer, and impaired endocytosis of ErbB2 could contribute to its overexpression. Therefore, knowledge about the mechanisms underlying endocytic down-regulation of ErbB2 is warranted. The C-terminus of ErbB2 can be cleaved after various stimuli, and after inhibition of HSP90 with geldanamycin this cleavage is accompanied by proteasome-dependent endocytosis of ErbB2. However, it is unknown whether C-terminal cleavage is linked to endocytosis. To study ErbB2 cleavage and endocytic trafficking, we fused yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) to the N- and C-terminus of ErbB2, respectively (YFP-ErbB2-CFP). After geldanamycin stimulation YFP-ErbB2-CFP became cleaved in nonapoptotic cells in a proteasome-dependent manner, and a markedly larger relative amount of cleaved YFP-ErbB2-CFP was observed in early endosomes than in the plasma membrane. Furthermore, cleavage took place at the plasma membrane, and cleaved ErbB2 was internalized and degraded far more efficiently than full-length ErbB2. Concordantly, a C-terminally truncated ErbB2 was also readily endocytosed and degraded in lysosomes compared with full-length ErbB2. Altogether, we suggest that geldanamycin leads to C-terminal cleavage of ErbB2, which releases the receptor from a retention mechanism and causes endocytosis and lysosomal degradation of ErbB2. [Abstract/Link to Full Text]

Eisele YS, Baumann M, Klebl B, Nordhammer C, Jucker M, Kilger E
Gleevec increases levels of the amyloid precursor protein intracellular domain and of the amyloid-beta degrading enzyme neprilysin.
Mol Biol Cell. 2007 Sep;18(9):3591-600.
Amyloid-beta (Abeta) deposition is a major pathological hallmark of Alzheimer's disease. Gleevec, a known tyrosine kinase inhibitor, has been shown to lower Abeta secretion, and it is considered a potential basis for novel therapies for Alzheimer's disease. Here, we show that Gleevec decreases Abeta levels without the inhibition of Notch cleavage by a mechanism distinct from gamma-secretase inhibition. Gleevec does not influence gamma-secretase activity in vitro; however, treatment of cell lines leads to a dose-dependent increase in the amyloid precursor protein intracellular domain (AICD), whereas secreted Abeta is decreased. This effect is observed even in presence of a potent gamma-secretase inhibitor, suggesting that Gleevec does not activate AICD generation but instead may slow down AICD turnover. Concomitant with the increase in AICD, Gleevec leads to elevated mRNA and protein levels of the Abeta-degrading enzyme neprilysin, a potential target gene of AICD-regulated transcription. Thus, the Gleevec mediated-increase in neprilysin expression may involve enhanced AICD signaling. The finding that Gleevec elevates neprilysin levels suggests that its Abeta-lowering effect may be caused by increased Abeta-degradation. [Abstract/Link to Full Text]

Kesavapany S, Patel V, Zheng YL, Pareek TK, Bjelogrlic M, Albers W, Amin N, Jaffe H, Gutkind JS, Strong MJ, Grant P, Pant HC
Inhibition of Pin1 reduces glutamate-induced perikaryal accumulation of phosphorylated neurofilament-H in neurons.
Mol Biol Cell. 2007 Sep;18(9):3645-55.
Under normal conditions, the proline-directed serine/threonine residues of neurofilament tail-domain repeats are exclusively phosphorylated in axons. In pathological conditions such as amyotrophic lateral sclerosis (ALS), motor neurons contain abnormal perikaryal accumulations of phosphorylated neurofilament proteins. The precise mechanisms for this compartment-specific phosphorylation of neurofilaments are not completely understood. Although localization of kinases and phosphatases is certainly implicated, another possibility involves Pin1 modulation of phosphorylation of the proline-directed serine/threonine residues. Pin1, a prolyl isomerase, selectively binds to phosphorylated proline-directed serine/threonine residues in target proteins and isomerizes cis isomers to more stable trans configurations. In this study we show that Pin1 associates with phosphorylated neurofilament-H (p-NF-H) in neurons and is colocalized in ALS-affected spinal cord neuronal inclusions. To mimic the pathology of neurodegeneration, we studied glutamate-stressed neurons that displayed increased p-NF-H in perikaryal accumulations that colocalized with Pin1 and led to cell death. Both effects were reduced upon inhibition of Pin1 activity by the use of an inhibitor juglone and down-regulating Pin1 levels through the use of Pin1 small interfering RNA. Thus, isomerization of lys-ser-pro repeat residues that are abundant in NF-H tail domains by Pin1 can regulate NF-H phosphorylation, which suggests that Pin1 inhibition may be an attractive therapeutic target to reduce pathological accumulations of p-NF-H. [Abstract/Link to Full Text]

Sistla S, Pang JV, Wang CX, Balasundaram D
Multiple conserved domains of the nucleoporin Nup124p and its orthologs Nup1p and Nup153 are critical for nuclear import and activity of the fission yeast Tf1 retrotransposon.
Mol Biol Cell. 2007 Sep;18(9):3692-708.
The nucleoporin Nup124p is a host protein required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr in fission yeast. The human nucleoporin Nup153 and the Saccharomyces cerevisiae Nup1p were identified as orthologs of Nup124p. In this study, we show that all three nucleoporins share a large FG/FXFG-repeat domain and a C-terminal peptide sequence, GRKIxxxxxRRKx, that are absolutely essential for Tf1 retrotransposition. Though the FXFG domain was essential, the FXFG repeats themselves could be eliminated without loss of retrotransposon activity, suggesting the existence of a common element unrelated to FG/FXFG motifs. The Nup124p C-terminal peptide, GRKIAVPRSRRKR, was extremely sensitive to certain single amino acid changes within stretches of the basic residues. On the basis of our comparative study of Nup124p, Nup1p, and Nup153 domains, we have developed peptides that specifically knockdown retrotransposon activity by disengaging the Tf1-Gag from its host nuclear transport machinery without any harmful consequence to the host itself. Our results imply that those domains challenged a specific pathway affecting Tf1 transposition. Although full-length Nup1p or Nup153 does not complement Nup124p, the functionality of their conserved domains with reference to Tf1 activity suggests that these three proteins evolved from a common ancestor. [Abstract/Link to Full Text]

Pagant S, Kung L, Dorrington M, Lee MC, Miller EA
Inhibiting endoplasmic reticulum (ER)-associated degradation of misfolded Yor1p does not permit ER export despite the presence of a diacidic sorting signal.
Mol Biol Cell. 2007 Sep;18(9):3398-413.
Capture of newly synthesized proteins into endoplasmic reticulum (ER)-derived coat protomer type II (COPII) vesicles represents a critical juncture in the quality control of protein biogenesis within the secretory pathway. The yeast ATP-binding cassette transporter Yor1p is a pleiotropic drug pump that shows homology to the human cystic fibrosis transmembrane conductance regulator (CFTR). Deletion of a phenylalanine residue in Yor1p, equivalent to the major disease-causing mutation in CFTR, causes ER retention and degradation via ER-associated degradation. We have examined the relationship between protein folding, ERAD and forward transport during Yor1p biogenesis. Uptake of Yor1p into COPII vesicles is mediated by an N-terminal diacidic signal that likely interacts with the "B-site" cargo-recognition domain on the COPII subunit, Sec24p. Yor1p-DeltaF is subjected to complex ER quality control involving multiple cytoplasmic chaperones and degradative pathways. Stabilization of Yor1p-DeltaF by inhibiting its degradation does not permit access of Yor1p-DeltaF to COPII vesicles. We propose that the ER quality control checkpoint engages misfolded Yor1p even after it has been stabilized by inhibition of the degradative pathway. [Abstract/Link to Full Text]

Lee YJ, Hoe KL, Maeng PJ
Yeast cells lacking the CIT1-encoded mitochondrial citrate synthase are hypersusceptible to heat- or aging-induced apoptosis.
Mol Biol Cell. 2007 Sep;18(9):3556-67.
In Saccharomyces cerevisiae, the initial reaction of the tricarboxylic acid cycle is catalyzed by the mitochondrial citrate synthase Cit1. The function of Cit1 has previously been studied mainly in terms of acetate utilization and metabolon construction. Here, we report the relationship between the function of Cit1 and apoptosis. Yeast cells with cit1 deletion showed a temperature-sensitive growth phenotype, and they displayed a rapid loss in viability associated with typical apoptotic hallmarks, i.e., reactive oxygen species (ROS) accumulation and nuclear fragmentation, DNA breakage, and phosphatidylserine translocation, when exposed to heat stress. On long-term cultivation, cit1 null strains showed increased potentials for both aging-induced apoptosis and adaptive regrowth. Activation of the metacaspase Yca1 was detected during heat- or aging-induced apoptosis in cit1 null strains, and accordingly, deletion of YCA1 suppressed the apoptotic phenotype caused by cit1 null mutation. Cells with cit1 deletion showed higher tendency toward glutathione (GSH) depletion and subsequent ROS accumulation than the wild type, which was rescued by exogenous GSH, glutamate, or glutathione disulfide (GSSG). These results led us to conclude that GSH deficiency in cit1 null cells is caused by an insufficient supply of glutamate necessary for biosynthesis of GSH rather than the depletion of reducing power required for reduction of GSSG to GSH. [Abstract/Link to Full Text]

Duvezin-Caubet S, Koppen M, Wagener J, Zick M, Israel L, Bernacchia A, Jagasia R, Rugarli EI, Imhof A, Neupert W, Langer T, Reichert AS
OPA1 processing reconstituted in yeast depends on the subunit composition of the m-AAA protease in mitochondria.
Mol Biol Cell. 2007 Sep;18(9):3582-90.
The morphology of mitochondria in mammalian cells is regulated by proteolytic cleavage of OPA1, a dynamin-like GTPase of the mitochondrial inner membrane. The mitochondrial rhomboid protease PARL, and paraplegin, a subunit of the ATP-dependent m-AAA protease, were proposed to be involved in this process. Here, we characterized individual OPA1 isoforms by mass spectrometry, and we reconstituted their processing in yeast to identify proteases involved in OPA1 cleavage. The yeast homologue of OPA1, Mgm1, was processed both by PARL and its yeast homologue Pcp1. Neither of these rhomboid proteases cleaved OPA1. The formation of small OPA1 isoforms was impaired in yeast cells lacking the m-AAA protease subunits Yta10 and Yta12 and was restored upon expression of murine or human m-AAA proteases. OPA1 processing depended on the subunit composition of mammalian m-AAA proteases. Homo-oligomeric m-AAA protease complexes composed of murine Afg3l1, Afg3l2, or human AFG3L2 subunits cleaved OPA1 with higher efficiency than paraplegin-containing m-AAA proteases. OPA1 processing proceeded normally in murine cell lines lacking paraplegin or PARL. Our results provide evidence for different substrate specificities of m-AAA proteases composed of different subunits and reveal a striking evolutionary switch of proteases involved in the proteolytic processing of dynamin-like GTPases in mitochondria. [Abstract/Link to Full Text]

Nelson SA, Cooper JA
A novel pathway that coordinates mitotic exit with spindle position.
Mol Biol Cell. 2007 Sep;18(9):3440-50.
In budding yeast, the spindle position checkpoint (SPC) delays mitotic exit until the mitotic spindle moves into the neck between the mother and bud. This checkpoint works by inhibiting the mitotic exit network (MEN), a signaling cascade initiated and controlled by Tem1, a small GTPase. Tem1 is regulated by a putative guanine exchange factor, Lte1, but the function and regulation of Lte1 remains poorly understood. Here, we identify novel components of the checkpoint that operate upstream of Lte1. We present genetic evidence in agreement with existing biochemical evidence for the molecular mechanism of a pathway that links microtubule-cortex interactions with Lte1 and mitotic exit. Each component of this pathway is required for the spindle position checkpoint to delay mitotic exit until the spindle is positioned correctly. [Abstract/Link to Full Text]

Shirakihara T, Saitoh M, Miyazono K
Differential regulation of epithelial and mesenchymal markers by deltaEF1 proteins in epithelial mesenchymal transition induced by TGF-beta.
Mol Biol Cell. 2007 Sep;18(9):3533-44.
Epithelial-mesenchymal transition (EMT), a crucial event in cancer progression and embryonic development, is induced by transforming growth factor (TGF)-beta in mouse mammary NMuMG epithelial cells. Id proteins have previously been reported to inhibit major features of TGF-beta-induced EMT. In this study, we show that expression of the deltaEF1 family proteins, deltaEF1 (ZEB1) and SIP1, is gradually increased by TGF-beta with expression profiles reciprocal to that of E-cadherin. SIP1 and deltaEF1 each dramatically down-regulated the transcription of E-cadherin in NMuMG cells through direct binding to the E-cadherin promoter. Silencing of the expression of both SIP1 and deltaEF1, but not either alone, completely abolished TGF-beta-induced E-cadherin repression. However, expression of mesenchymal markers, including fibronectin, N-cadherin, and vimentin, was not affected by knockdown of SIP1 and deltaEF1. TGF-beta-induced the expression of Ets1, which in turn activated deltaEF1 promoter activity. Moreover, up-regulation of SIP1 and deltaEF1 expression by TGF-beta was suppressed by knockdown of Ets1 expression. In addition, Id2 suppressed the TGF-beta- and Ets1-induced up-regulation of deltaEF1. Taken together, these findings suggest that the deltaEF1 family proteins, SIP1 and deltaEF1, are necessary, but not sufficient, for TGF-beta-induced EMT and that Ets1 induced by TGF-beta may function as an upstream transcriptional regulator of SIP1 and deltaEF1. [Abstract/Link to Full Text]

Sato K, Noda Y, Yoda K
Pga1 is an essential component of Glycosylphosphatidylinositol-mannosyltransferase II of Saccharomyces cerevisiae.
Mol Biol Cell. 2007 Sep;18(9):3472-85.
The Saccharomyces cerevisiae essential gene YNL158w/PGA1 encodes an endoplasmic reticulum (ER)-localized membrane protein. We constructed temperature-sensitive alleles of PGA1 by error-prone polymerase chain reaction mutagenesis to explore its biological role. Pulse-chase experiments revealed that the pga1(ts) mutants accumulated the ER-form precursor of Gas1 protein at the restrictive temperature. Transport of invertase and carboxypeptidase Y were not affected. Triton X-114 phase separation and [(3)H]inositol labeling indicated that the glycosylphosphatidylinositol (GPI)-anchoring was defective in the pga1(ts) mutants, suggesting that Pga1 is involved in GPI synthesis or its transfer to target proteins. We found GPI18, which was recently reported to encode GPI-mannosyltransferase II (GPI-MT II), as a high-copy suppressor of the temperature sensitivity of pga1(ts). Both Gpi18 and Pga1 were detected in the ER by immunofluorescence, and they were coprecipitated from the Triton X-100-solubilized membrane. The gpi18(ts) and pga1(ts) mutants accumulated the same GPI synthetic intermediate at the restrictive temperature. From these results, we concluded that Pga1 is an additional essential component of the yeast GPI-MT II. [Abstract/Link to Full Text]

Wilhelmsen K, Litjens SH, Kuikman I, Margadant C, van Rheenen J, Sonnenberg A
Serine phosphorylation of the integrin beta4 subunit is necessary for epidermal growth factor receptor induced hemidesmosome disruption.
Mol Biol Cell. 2007 Sep;18(9):3512-22.
Hemidesmosomes (HDs) are multiprotein adhesion complexes that promote attachment of epithelial cells to the basement membrane. The binding of alpha6beta4 to plectin plays a central role in their assembly. We have defined three regions on beta4 that together harbor all the serine and threonine phosphorylation sites and show that three serines (S1356, S1360, and S1364), previously implicated in HD regulation, prevent the interaction of beta4 with the plectin actin-binding domain when phosphorylated. We have also established that epidermal growth factor receptor activation, which is known to function upstream of HD disassembly, results in the phosphorylation of only one or more of these three residues and the partial disassembly of HDs in keratinocytes. Additionally, we show that S1360 and S1364 of beta4 are the only residues phosphorylated by PKC and PKA in cells, respectively. Taken together, our studies indicate that multiple kinases act in concert to breakdown the structural integrity of HDs in keratinocytes, which is primarily achieved through the phosphorylation of S1356, S1360, and S1364 on the beta4 subunit. [Abstract/Link to Full Text]

Jorgensen P, Edgington NP, Schneider BL, Rupes I, Tyers M, Futcher B
The size of the nucleus increases as yeast cells grow.
Mol Biol Cell. 2007 Sep;18(9):3523-32.
It is not known how the volume of the cell nucleus is set, nor how the ratio of nuclear volume to cell volume (N/C) is determined. Here, we have measured the size of the nucleus in growing cells of the budding yeast Saccharomyces cerevisiae. Analysis of mutant yeast strains spanning a range of cell sizes revealed that the ratio of average nuclear volume to average cell volume was quite consistent, with nuclear volume being approximately 7% that of cell volume. At the single cell level, nuclear and cell size were strongly correlated in growing wild-type cells, as determined by three different microscopic approaches. Even in G1-phase, nuclear volume grew, although it did not grow quite as fast as overall cell volume. DNA content did not appear to have any immediate, direct influence on nuclear size, in that nuclear size did not increase sharply during S-phase. The maintenance of nuclear size did not require continuous growth or ribosome biogenesis, as starvation and rapamycin treatment had little immediate impact on nuclear size. Blocking the nuclear export of new ribosomal subunits, among other proteins and RNAs, with leptomycin B also had no obvious effect on nuclear size. Nuclear expansion must now be factored into conceptual and mathematical models of budding yeast growth and division. These results raise questions as to the unknown force(s) that expand the nucleus as yeast cells grow. [Abstract/Link to Full Text]

Oladipo A, Cowan A, Rodionov V
Microtubule motor Ncd induces sliding of microtubules in vivo.
Mol Biol Cell. 2007 Sep;18(9):3601-6.
The mitotic spindle is a microtubule (MT)-based molecular machine that serves for equal segregation of chromosomes during cell division. The formation of the mitotic spindle requires the activity of MT motors, including members of the kinesin-14 family. Although evidence suggests that kinesins-14 act by driving the sliding of MT bundles in different areas of the spindle, such sliding activity had never been demonstrated directly. To test the hypothesis that kinesins-14 can induce MT sliding in living cells, we developed an in vivo assay, which involves overexpression of the kinesin-14 family member Drosophila Ncd in interphase mammalian fibroblasts. We found that green fluorescent protein (GFP)-Ncd colocalized with cytoplasmic MTs, whose distribution was determined by microinjection of Cy3 tubulin into GFP-transfected cells. Ncd overexpression resulted in the formation of MT bundles that exhibited dynamic "looping" behavior never observed in control cells. Photobleaching studies and fluorescence speckle microscopy analysis demonstrated that neighboring MTs in bundles could slide against each other with velocities of 0.1 microm/s, corresponding to the velocities of movement of the recombinant Ncd in in vitro motility assays. Our data, for the first time, demonstrate generation of sliding forces between adjacent MTs by Ncd, and they confirm the proposed roles of kinesins-14 in the mitotic spindle morphogenesis. [Abstract/Link to Full Text]

Floyd S, Favre C, Lasorsa FM, Leahy M, Trigiante G, Stroebel P, Marx A, Loughran G, O'Callaghan K, Marobbio CM, Slotboom DJ, Kunji ER, Palmieri F, O'Connor R
The insulin-like growth factor-I-mTOR signaling pathway induces the mitochondrial pyrimidine nucleotide carrier to promote cell growth.
Mol Biol Cell. 2007 Sep;18(9):3545-55.
The insulin/insulin-like growth factor (IGF) signaling pathway to mTOR is essential for the survival and growth of normal cells and also contributes to the genesis and progression of cancer. This signaling pathway is linked with regulation of mitochondrial function, but how is incompletely understood. Here we show that IGF-I and insulin induce rapid transcription of the mitochondrial pyrimidine nucleotide carrier PNC1, which shares significant identity with the essential yeast mitochondrial carrier Rim2p. PNC1 expression is dependent on PI-3 kinase and mTOR activity and is higher in transformed fibroblasts, cancer cell lines, and primary prostate cancers than in normal tissues. Overexpression of PNC1 enhances cell size, whereas suppression of PNC1 expression causes reduced cell size and retarded cell cycle progression and proliferation. Cells with reduced PNC1 expression have reduced mitochondrial UTP levels, but while mitochondrial membrane potential and cellular ATP are not altered, cellular ROS levels are increased. Overall the data indicate that PNC1 is a target of the IGF-I/mTOR pathway that is essential for mitochondrial activity in regulating cell growth and proliferation. [Abstract/Link to Full Text]

Le Roux D, Lankar D, Yuseff MI, Vascotto F, Yokozeki T, Faure-André G, Mougneau E, Glaichenhaus N, Manoury B, Bonnerot C, Lennon-Duménil AM
Syk-dependent actin dynamics regulate endocytic trafficking and processing of antigens internalized through the B-cell receptor.
Mol Biol Cell. 2007 Sep;18(9):3451-62.
Antigen binding to the B-cell receptor (BCR) induces multiple signaling cascades that ultimately lead to B lymphocyte activation. In addition, the BCR regulates the key trafficking events that allow the antigen to reach endocytic compartments devoted to antigen processing, i.e., that are enriched for major histocompatibility factor class II (MHC II) and accessory molecules such as H2-DM. Here, we analyze the role in antigen processing and presentation of the tyrosine kinase Syk, which is activated upon BCR engagement. We show that convergence of MHC II- and H2-DM-containing compartments with the vesicles that transport BCR-uptaken antigens is impaired in cells lacking Syk activity. This defect in endocytic trafficking compromises the ability of Syk-deficient cells to form MHC II-peptide complexes from BCR-internalized antigens. Altered endocytic trafficking is associated to a failure of Syk-deficient cells to properly reorganize their actin cytoskeleton in response to BCR engagement. We propose that, by modulating the actin dynamics induced upon BCR stimulation, Syk regulates the positioning and transport of the vesicles that carry the molecules required for antigen processing and presentation. [Abstract/Link to Full Text]

Larina O, Bhat P, Pickett JA, Launikonis BS, Shah A, Kruger WA, Edwardson JM, Thorn P
Dynamic regulation of the large exocytotic fusion pore in pancreatic acinar cells.
Mol Biol Cell. 2007 Sep;18(9):3502-11.
Loss of granule content during exocytosis requires the opening of a fusion pore between the secretory granule and plasma membrane. In a variety of secretory cells, this fusion pore has now been shown to subsequently close. However, it is still unclear how pore closure is physiologically regulated and contentious as to how closure relates to granule content loss. Here, we examine the behavior of the fusion pore during zymogen granule exocytosis in pancreatic acinar cells. By using entry of high-molecular-weight dyes from the extracellular solution into the granule lumen, we show that the fusion pore has a diameter of 29-55 nm. We further show that by 5 min after granule fusion, many granules have a closed fusion pore with evidence indicating that pore closure is a prelude to endocytosis and that in granules with a closed fusion pore the chymotrypsinogen content is low. Finally, we show that latrunculin B treatment promotes pore closure, suggesting F-actin affects pore dynamics. Together, our data do not support the classical view in acinar cells that exocytosis ends with granule collapse. Instead, for many granules the fusion pore closes, probably as a transition to endocytosis, and likely involving an F-actin-dependent mechanism. [Abstract/Link to Full Text]

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Recent Articles in Molecular and Cellular Biology

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Bandyopadhyay S, Ashraf MZ, Daher P, Howe PH, DiCorleto PE
HOXA9 participates in the transcriptional activation of E-selectin in endothelial cells.
Mol Cell Biol. 2007 Jun;27(12):4207-16.
The homeobox gene HOXA9 has recently been shown to be an important regulator of endothelial cell (EC) differentiation and activation in addition to its role in embryonic development and hematopoiesis. In this report, we have determined that the EC-leukocyte adhesion molecule E-selectin is a key target for HOXA9. The depletion of HOXA9 protein in ECs resulted in a significant and specific decrease in tumor necrosis factor alpha (TNF-alpha)-induced E-selectin gene expression. In addition, HOXA9 specifically activated the E-selectin gene promoter in ECs. Progressive deletional analyses together with site-specific mutagenesis of the E-selectin promoter indicated that the Abd-B-like HOX DNA-binding motif, CAATTTTATTAA, located in the proximal region spanning bp -210 to -221 upstream of the transcription start site was crucial for the promoter induction by HOXA9. Both HOXA9 in EC nuclear extract and recombinant HOXA9 protein bound to this sequence in vitro. Moreover, we showed that HOXA9 binds temporally, in a TNF-alpha-dependent manner, to the region containing this Abd-B-like element in vivo. We have thus identified a novel and functionally critical cis-regulatory element for TNF-alpha-mediated transient expression of the E-selectin gene. Further, we provide evidence that HOXA9 acts as an obligate proinflammatory factor by mediating cytokine induction of E-selectin. [Abstract/Link to Full Text]

Dong X, Sweet J, Challis JR, Brown T, Lye SJ
Transcriptional activity of androgen receptor is modulated by two RNA splicing factors, PSF and p54nrb.
Mol Cell Biol. 2007 Jul;27(13):4863-75.
Nuclear receptors regulate gene activation or repression through dynamic interactions with coregulators. The interactions between nuclear receptors and RNA splicing factors link gene transcription initiation with pre-mRNA splicing, providing a coordinated control of the products of gene transcription. Here we report that two RNA splicing factors, PTB-associated splicing factor (PSF) and p54nrb, synergistically form protein complexes with the androgen receptor (AR) in a ligand-independent manner and inhibit its transcriptional activity. PSF does not affect AR protein stability, as in the case of the progesterone receptor, but impedes the interaction of AR with the androgen response element. Both splicing factors interact directly with mSin3A and attract mSin3A to the AR complex in a synergistic manner. The suppression of AR transcriptional activity by PSF and p54nrb is reversed by the inhibition of histone deacetylase activity. These data demonstrated that PSF and p54nrb complex with AR and play a key role in modulating AR-mediated gene transcription. [Abstract/Link to Full Text]

Susaki E, Nakayama K, Nakayama KI
Cyclin D2 translocates p27 out of the nucleus and promotes its degradation at the G0-G1 transition.
Mol Cell Biol. 2007 Jul;27(13):4626-40.
The nuclear export and cytoplasmic degradation of the cyclin-dependent kinase inhibitor p27 are required for effective progression of the cell cycle through the G(0)-G(1) transition. The mechanism responsible for this translocation of p27 has remained unclear, however. We now show that cyclin D2 directly links growth signaling with the nuclear export of p27 at the G(0)-G(1) transition in some cell types. The up-regulation of cyclin D2 in response to mitogenic stimulation was found to occur earlier than that of other D-type cyclins and in parallel with down-regulation of p27 at the G(0)-G(1) transition. RNA interference-mediated depletion of cyclin D2 inhibited the nuclear export of p27 and delayed its degradation at the G(0)-G(1) transition. In contrast, overexpression of cyclin D2 in G(0) phase shifted the localization of p27 from the nucleus to the cytoplasm and reduced the stability of p27. Overexpression of the cyclin D2(T280A) mutant, whose export from the nucleus is impaired, prevented the translocation and degradation of p27. These results indicate that cyclin D2 translocates p27 from the nucleus into the cytoplasm for its KPC-dependent degradation at the G(0)-G(1) transition. [Abstract/Link to Full Text]

Niendorf S, Oksche A, Kisser A, Löhler J, Prinz M, Schorle H, Feller S, Lewitzky M, Horak I, Knobeloch KP
Essential role of ubiquitin-specific protease 8 for receptor tyrosine kinase stability and endocytic trafficking in vivo.
Mol Cell Biol. 2007 Jul;27(13):5029-39.
Posttranslational modification by ubiquitin controls multiple cellular functions and is counteracted by the activities of deubiquitinating enzymes. UBPy (USP8) is a growth-regulated ubiquitin isopeptidase that interacts with the HRS-STAM complex. Using Cre-loxP-mediated gene targeting in mice, we show that lack of UBPy results in embryonic lethality, whereas its conditional inactivation in adults causes fatal liver failure. The defect is accompanied by a strong reduction or absence of several growth factor receptor tyrosine kinases (RTKs), like epidermal growth factor receptor, hepatocyte growth factor receptor (c-met), and ERBB3. UBPy-deficient cells exhibit aberrantly enlarged early endosomes colocalizing with enhanced ubiquitination and have reduced levels of HRS and STAM2. Congruently immortalized cells gradually stop proliferation upon induced deletion of UBPy. These results unveil a central and nonredundant role of UBPy in growth regulation, endosomal sorting, and the control of RTKs in vivo. [Abstract/Link to Full Text]

Wiederschain D, Chen L, Johnson B, Bettano K, Jackson D, Taraszka J, Wang YK, Jones MD, Morrissey M, Deeds J, Mosher R, Fordjour P, Lengauer C, Benson JD
Contribution of polycomb homologues Bmi-1 and Mel-18 to medulloblastoma pathogenesis.
Mol Cell Biol. 2007 Jul;27(13):4968-79.
Bmi-1 and Mel-18 are structural homologues that belong to the Polycomb group of transcriptional regulators and are believed to stably maintain repression of gene expression by altering the state of chromatin at specific promoters. While a number of clinical and experimental observations have implicated Bmi-1 in human tumorigenesis, the role of Mel-18 in cancer cell growth has not been investigated. We report here that short hairpin RNA-mediated knockdown of either Bmi-1 or Mel-18 in human medulloblastoma DAOY cells results in the inhibition of proliferation, loss of clonogenic survival, anchorage-independent growth, and suppression of tumor formation in nude mice. Furthermore, overexpression of both Bmi-1 and Mel-18 significantly increases the clonogenic survival of Rat1 fibroblasts. In contrast, stable downregulation of Bmi-1 or Mel-18 alone does not affect the growth of normal human WI38 fibroblasts. Proteomics-based characterization of Bmi-1 and Mel-18 protein complexes isolated from cancer cells revealed substantial similarities in their respective compositions. Finally, gene expression analysis identified a number of cancer-relevant pathways that may be controlled by Bmi-1 and Mel-18 and also showed that these Polycomb proteins regulate a set of common gene targets. Taken together, these results suggest that Bmi-1 and Mel-18 may have overlapping functions in cancer cell growth. [Abstract/Link to Full Text]

Chen X, Ruggiero C, Li S
Yeast Rpb9 plays an important role in ubiquitylation and degradation of Rpb1 in response to UV-induced DNA damage.
Mol Cell Biol. 2007 Jul;27(13):4617-25.
Rpb9, a nonessential subunit of RNA polymerase II (Pol II), has multiple transcription-related functions in Saccharomyces cerevisiae, including transcription elongation and transcription-coupled repair (TCR). Here we show that, in response to UV radiation, Rpb9 also functions in promoting ubiquitylation and degradation of Rpb1, the largest subunit of Pol II. This function of Rpb9 is not affected by any pathways of nucleotide excision repair, including TCR mediated by Rpb9 itself and by Rad26. Rpb9 is composed of three distinct domains: the N-terminal Zn1, the C-terminal Zn2, and the central linker. The Zn2 domain, which is dispensable for transcription elongation and TCR functions, is essential for Rpb9 to promote Rpb1 degradation, whereas the Zn1 and linker domains, which are essential for transcription elongation and TCR functions, play a subsidiary role in Rpb1 degradation. Coimmunoprecipitation analysis suggests that almost the full length of Rpb9 is required for a strong interaction with the core Pol II: deletion of the Zn2 domain causes dramatically weakened interaction, whereas deletion of Zn1 and the linker resulted in undetectable interaction. Furthermore, we show that Rpb1, rather than the whole Pol II complex, is degraded in response to UV radiation and that the degradation is primarily mediated by the 26S proteasome. [Abstract/Link to Full Text]

McClellan KA, Ruzhynsky VA, Douda DN, Vanderluit JL, Ferguson KL, Chen D, Bremner R, Park DS, Leone G, Slack RS
Unique requirement for Rb/E2F3 in neuronal migration: evidence for cell cycle-independent functions.
Mol Cell Biol. 2007 Jul;27(13):4825-43.
The cell cycle regulatory retinoblastoma (Rb) protein is a key regulator of neural precursor proliferation; however, its role has been expanded to include a novel cell-autonomous role in mediating neuronal migration. We sought to determine the Rb-interacting factors that mediate both the cell cycle and migration defects. E2F1 and E2F3 are likely Rb-interacting candidates that we have shown to be deregulated in the absence of Rb. Using mice with compound null mutations of Rb and E2F1 or E2F3, we asked to what extent either E2F1 or E2F3 interacts with Rb in neurogenesis. Here, we report that E2F1 and E2F3 are both functionally relevant targets in neural precursor proliferation, cell cycle exit, and laminar patterning. Each also partially mediates the Rb requirement for neuronal survival. Neuronal migration, however, is specifically mediated through E2F3, beyond its role in cell cycle regulation. This study not only outlines overlapping and distinct functions for E2Fs in neurogenesis but also is the first to establish a physiologically relevant role for the Rb/E2F pathway beyond cell cycle regulation in vivo. [Abstract/Link to Full Text]

Nawathean P, Stoleru D, Rosbash M
A small conserved domain of Drosophila PERIOD is important for circadian phosphorylation, nuclear localization, and transcriptional repressor activity.
Mol Cell Biol. 2007 Jul;27(13):5002-13.
We identify in this study a 27-amino-acid motif which is conserved between the Drosophila melanogaster period protein (PER) and the three mammalian PERs. Characterization of PER lacking this motif (PER Delta) shows that it is important for phosphorylation of Drosophila PER by casein kinase I epsilon (CKI epsilon; doubletime protein or DBT) and CKII. S2 cell assays indicate that the domain also contributes significantly to PER nuclear localization as well as to PER transcriptional repressor activity. These two phenomena appear linked, since PER Delta transcriptional repressor activity in S2 cells was restored when nuclear localization was facilitated. Two less direct assays of PER Delta activity in flies can be interpreted similarly. The separate assay of nuclear import and export suggests that the domain functions in part to facilitate PER phosphorylation within the cytoplasm, which in turn promotes nuclear entry. As there is evidence that the kinases also function within the nucleus to promote transcriptional repression, we suggest that there is a subsequent collaboration between phosphorylated PER and the kinases to repress CLK-CYC activity, probably through the phosphorylation of CLK. This is then followed by additional PER phosphorylation, which occurs within the nucleus and leads to PER degradation. [Abstract/Link to Full Text]

Brown SE, Szyf M
Epigenetic programming of the rRNA promoter by MBD3.
Mol Cell Biol. 2007 Jul;27(13):4938-52.
Within the human genome there are hundreds of copies of the rRNA gene, but only a fraction of these genes are active. Silencing through epigenetics has been extensively studied; however, it is essential to understand how active rRNA genes are maintained. Here, we propose a role for the methyl-CpG binding domain protein MBD3 in epigenetically maintaining active rRNA promoters. We show that MBD3 is localized to the nucleolus, colocalizes with upstream binding factor, and binds to unmethylated rRNA promoters. Knockdown of MBD3 by small interfering RNA results in increased methylation of the rRNA promoter coupled with a decrease in RNA polymerase I binding and pre-rRNA transcription. Conversely, overexpression of MBD3 results in decreased methylation of the rRNA promoter. Additionally, overexpression of MBD3 induces demethylation of nonreplicating plasmids containing the rRNA promoter. We demonstrate that this demethylation occurs following the overexpression of MBD3 and its increased interaction with the methylated rRNA promoter. This is the first demonstration that MBD3 is involved in inducing and maintaining the demethylated state of a specific promoter. [Abstract/Link to Full Text]

Delpuech O, Griffiths B, East P, Essafi A, Lam EW, Burgering B, Downward J, Schulze A
Induction of Mxi1-SR alpha by FOXO3a contributes to repression of Myc-dependent gene expression.
Mol Cell Biol. 2007 Jul;27(13):4917-30.
Forkhead transcription factors of the O class (FOXOs) are important targets of the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. FOXOs have been implicated in the regulation of cell cycle progression, oxidative stress resistance, and apoptosis. Using DNA microarrays, we analyzed the transcriptional response to FOXO3a activation by gene expression analysis in DLD-1 colon cancer cells stably expressing a FOXO3a.A3-ER fusion protein. We found that activation of FOXO3a resulted in repression of a number of previously identified Myc target genes. Furthermore, FOXO3a activation induced expression of several members of the Mad/Mxd family of transcriptional repressors, most notably Mxi1. The induction of Mxi1 by FOXO3a was specific to the Mxi1-SR alpha isoform and was mediated by three highly conserved FOXO binding sites within the first intron of the gene. Activation of FOXO3a in response to inhibition of Akt also resulted in activation of Mxi1-SR alpha expression. Silencing of Mxi1 by small interfering RNA (siRNA) reduced FOXO3a-mediated repression of a number of Myc target genes. We also observed that FOXO3a activation induced a switch in promoter occupancy from Myc to Mxi1 on the E-box containing promoter regions of two Myc target genes, APEX and FOXM1. siRNA-mediated transient silencing of Mxi1 or all Mad/Mxd proteins reduced exit from S phase in response to FOXO3a activation, and stable silencing of Mxi1 or Mad1 reduced the growth inhibitory effect of FOXO3a. We conclude that induction of Mad/Mxd proteins contributes to the inhibition of proliferation in response to FOXO3a activation. Our results provide evidence of direct regulation of Mxi1 by FOXO3a and imply an additional mechanism through which the PI3-kinase/Akt/FOXO pathway can modulate Myc function. [Abstract/Link to Full Text]

Morita M, Suzuki T, Nakamura T, Yokoyama K, Miyasaka T, Yamamoto T
Depletion of mammalian CCR4b deadenylase triggers elevation of the p27Kip1 mRNA level and impairs cell growth.
Mol Cell Biol. 2007 Jul;27(13):4980-90.
The stability of mRNA influences the abundance of cellular transcripts and proteins. Deadenylases play critical roles in mRNA turnover and thus are important for the regulation of various biological events. Here, we report the identification and characterization of CCR4b/CNOT6L, which is homologous to yeast CCR4 mRNA deadenylase. CCR4b is localized mainly in the cytoplasm and displays deadenylase activity both in vitro and in vivo. CCR4b forms a multisubunit complex similar to the yeast CCR4-NOT complex. Suppression of CCR4b by RNA interference results in growth retardation of NIH 3T3 cells accompanied by elevation of both p27(Kip1) mRNA and p27(Kip1) protein. Reintroduction of wild-type CCR4b, but not mutant CCR4b lacking deadenylase activity, restores the growth of CCR4b-depleted NIH 3T3 cells. The data suggest that CCR4b regulates cell growth in a manner dependent on its deadenylase activity. We also show that p27(Kip1) mRNA is stabilized and its poly(A) tail is preserved in CCR4b-depleted cells. Our findings provide evidence that CCR4b deadenylase is a constituent of the mammalian CCR4-NOT complex and regulates the turnover rate of specific target mRNAs. Thus, CCR4b may be involved in various cellular events that include cell proliferation. [Abstract/Link to Full Text]

Kim EY, Ko HW, Yu W, Hardin PE, Edery I
A DOUBLETIME kinase binding domain on the Drosophila PERIOD protein is essential for its hyperphosphorylation, transcriptional repression, and circadian clock function.
Mol Cell Biol. 2007 Jul;27(13):5014-28.
A common feature of animal circadian clocks is the progressive phosphorylation of PERIOD (PER) proteins from hypo- to hyperphosphorylated species, events that are highly dependent on casein kinase 1 epsilon (termed DOUBLETIME [DBT] in Drosophila melanogaster) and necessary for normal clock progression. Drosophila PER (dPER) functions in the negative limb of the clockworks by presumably binding to the transcription factor CLOCK (CLK) and inhibiting its transactivation activity. Here, we identify a small region on dPER that is conserved with mammalian PERs and contains the major in vivo DBT binding domain, termed dPDBD (for dPER DBT binding domain). This domain is required for the manifestation of molecular and behavioral rhythms in vivo. In the absence of the dPDBD, the dPER protein is present at constant high levels throughout a daily cycle, undergoes little phosphorylation, and is severely impaired in its ability to function as a transcriptional repressor. Our findings indicate that the binding of dPER to CLK is not sufficient for transcriptional inhibition, implicating a more indirect mode of action whereby dPER acts as a molecular bridge to "deliver" DBT and/or other factors that directly repress CLK-dependent gene expression. [Abstract/Link to Full Text]

Hsu M, Mabaera R, Lowrey CH, Martin DI, Fiering S
CpG hypomethylation in a large domain encompassing the embryonic beta-like globin genes in primitive erythrocytes.
Mol Cell Biol. 2007 Jul;27(13):5047-54.
There is little evidence addressing the role of CpG methylation in transcriptional control of genes that do not contain CpG islands. This is reflected in the ongoing debate about whether CpG methylation merely suppresses retroelements or if it also plays a role in developmental and tissue-specific gene regulation. The genes of the beta-globin locus are an important model of mammalian developmental gene regulation and do not contain CpG islands. We have analyzed the methylation status of regions in the murine beta-like globin locus in uncultured primitive and definitive erythroblasts and other cultured primary and transformed cell types. A large ( approximately 20-kb) domain is hypomethylated only in primitive erythroid cells; it extends from the region just past the locus control region to before beta-major and encompasses the embryonic genes Ey, beta h1, and beta h0. Even retrotransposons in this region are hypomethylated in primitive erythroid cells. The existence of this large developmentally regulated domain of hypomethylation supports a mechanistic role for DNA methylation in developmental regulation of globin genes. [Abstract/Link to Full Text]

Swaminathan S, Kile AC, MacDonald EM, Koepp DM
Yra1 is required for S phase entry and affects Dia2 binding to replication origins.
Mol Cell Biol. 2007 Jul;27(13):4674-84.
The Saccharomyces cerevisiae F-box protein Dia2 is important for DNA replication and genomic stability. Using an affinity approach, we identified Yra1, a transcription-coupled mRNA export protein, as a Dia2 interaction partner. We find that yra1 mutants are sensitive to DIA2 expression levels. Like Dia2, Yra1 associates with chromatin and binds replication origins, suggesting that they may function together in DNA replication. Consistent with this idea, Yra1 and Dia2 coimmunoprecipitate with Hys2, a subunit of DNA polymerase delta. The C terminus of Yra1 is required to interact with Dia2. A yra1 mutant that lacks this domain is temperature sensitive yet has no apparent defect in RNA export. Remarkably, this mutant also fails to enter S phase at the nonpermissive temperature. Significantly, other mutants in transcription-coupled export do not exhibit S phase entry defects or sensitivity to DIA2 expression levels. Together, these results indicate that Yra1 has a role in DNA replication distinct from its role in mRNA export. Furthermore, Dia2 binding to replication origins is significantly reduced when association with Yra1 is compromised, suggesting that one aspect of the role of Yra1 in DNA replication may involve recruiting Dia2 to chromatin. [Abstract/Link to Full Text]

Rudra D, Mallick J, Zhao Y, Warner JR
Potential interface between ribosomal protein production and pre-rRNA processing.
Mol Cell Biol. 2007 Jul;27(13):4815-24.
It has become clear that in Saccharomyces cerevisiae the transcription of ribosomal protein genes, which makes up a major proportion of the total transcription by RNA polymerase II, is controlled by the interaction of three transcription factors, Rap1, Fhl1, and Ifh1. Of these, only Rap1 binds directly to DNA and only Ifh1 is absent when transcription is repressed. We have examined further the nature of this interaction and find that Ifh1 is actually associated with at least two complexes. In addition to its association with Rap1 and Fhl1, Ifh1 forms a complex (CURI) with casein kinase 2 (CK2), Utp22, and Rrp7. Fhl1 is loosely associated with the CURI complex; its absence partially destabilizes the complex. The CK2 within the complex phosphorylates Ifh1 in vitro but no other members of the complex. Two major components of this complex, Utp22 and Rrp7, are essential participants in the processing of pre-rRNA. Depletion of either protein, but not of other proteins in the early processing steps, brings about a substantial increase in ribosomal protein mRNA. We propose a model in which the CURI complex is a key mediator between the two parallel pathways necessary for ribosome synthesis: the transcription and processing of pre-rRNA and the transcription of ribosomal protein genes. [Abstract/Link to Full Text]

Sagar GD, Gereben B, Callebaut I, Mornon JP, Zeöld A, da Silva WS, Luongo C, Dentice M, Tente SM, Freitas BC, Harney JW, Zavacki AM, Bianco AC
Ubiquitination-induced conformational change within the deiodinase dimer is a switch regulating enzyme activity.
Mol Cell Biol. 2007 Jul;27(13):4774-83.
Ubiquitination is a critical posttranslational regulator of protein stability and/or subcellular localization. Here we show that ubiquitination can also regulate proteins by transiently inactivating enzymatic function through conformational change in a dimeric enzyme, which can be reversed upon deubiquitination. Our model system is the thyroid hormone-activating type 2 deiodinase (D2), an endoplasmic reticulum-resident type 1 integral membrane enzyme. D2 exists as a homodimer maintained by interacting surfaces at its transmembrane and globular cytosolic domains. The D2 dimer associates with the Hedgehog-inducible ubiquitin ligase WSB-1, the ubiquitin conjugase UBC-7, and VDU-1, a D2-specific deubiquitinase. Upon binding of T4, its natural substrate, D2 is ubiquitinated, which inactivates the enzyme by interfering with D2's globular interacting surfaces that are critical for dimerization and catalytic activity. This state of transient inactivity and change in dimer conformation persists until deubiquitination. The continuous association of D2 with this regulatory protein complex supports rapid cycles of deiodination, conjugation to ubiquitin, and enzyme reactivation by deubiquitination, allowing tight control of thyroid hormone action. [Abstract/Link to Full Text]

Jones N, Hardy WR, Friese MB, Jorgensen C, Smith MJ, Woody NM, Burden SJ, Pawson T
Analysis of a Shc family adaptor protein, ShcD/Shc4, that associates with muscle-specific kinase.
Mol Cell Biol. 2007 Jul;27(13):4759-73.
Shc family proteins serve as phosphotyrosine adaptor molecules in various receptor-mediated signaling pathways. In mammals, three distinct Shc genes have been described that encode proteins characterized by two phosphotyrosine-interaction modules, an amino-terminal phosphotyrosine binding (PTB) domain and a carboxy-terminal Src homology 2 domain. Here, we report the analysis of an uncharacterized fourth Shc family protein, ShcD/Shc4, that is expressed in adult brain and skeletal muscle. Consistent with this expression pattern, we find that ShcD can associate via its PTB domain with the phosphorylated muscle-specific kinase (MuSK) receptor tyrosine kinase and undergo tyrosine phosphorylation downstream of activated MuSK. Interestingly, additional sites of tyrosine phosphorylation, including a novel Grb2 binding site, are present on ShcD that are not found in other Shc family proteins. Activation of MuSK upon agrin binding at the neuromuscular junction (NMJ) induces clustering and tyrosine phosphorylation of acetylcholine receptors (AChRs) required for synaptic transmission. ShcD is coexpressed with MuSK in the postsynaptic region of the NMJ, and in cultured myotubes stimulated with agrin, expression of ShcD appears to be important for early tyrosine phosphorylation of the AChR. Thus, we have characterized a new member of the Shc family of docking proteins, which may mediate a specific aspect of signaling downstream of the MuSK receptor. [Abstract/Link to Full Text]

Qiang L, Wang H, Farmer SR
Adiponectin secretion is regulated by SIRT1 and the endoplasmic reticulum oxidoreductase Ero1-L alpha.
Mol Cell Biol. 2007 Jul;27(13):4698-707.
Adiponectin is secreted from adipose tissue in response to metabolic effectors in order to sensitize the liver and muscle to insulin. Reduced circulating levels of adiponectin that usually accompany obesity contribute to the associated insulin resistance. The molecular mechanisms controlling the production of adiponectin are essentially unknown. In this report, we demonstrate that the endoplasmic reticulum (ER) oxidoreductase Ero1-L alpha and effectors modulating peroxisome proliferator-activated receptor gamma (PPAR gamma) and SIRT1 activities regulate secretion of adiponectin from 3T3-L1 adipocytes. Specifically, adiponectin secretion and Ero1-L alpha expression are induced during the early phase of adipogenesis but are then down-regulated during the terminal phase, coincident with an increased expression of SIRT1. Suppression of SIRT1 or activation of PPAR gamma enhances Ero1-L alpha expression and stimulates secretion of high-molecular-weight complexes of adiponectin in mature adipocytes. Suppression of Ero1-L alpha through expression of a corresponding small interfering RNA reduces adiponectin secretion during the differentiation of 3T3-L1 preadipocytes. Moreover, ectopic expression of Ero1-L alpha in Ero1-L alpha-deficient 3T3 fibroblasts stimulates the secretion of adiponectin following their conversion into adipocytes and prevents the suppression of adiponectin secretion in response to activation of SIRT1 by exposure to resveratrol. These findings provide a framework to understand the mechanisms by which adipocytes regulate secretion of adiponectin in response to various metabolic states. [Abstract/Link to Full Text]

Dershowitz A, Snyder M, Sbia M, Skurnick JH, Ong LY, Newlon CS
Linear derivatives of Saccharomyces cerevisiae chromosome III can be maintained in the absence of autonomously replicating sequence elements.
Mol Cell Biol. 2007 Jul;27(13):4652-63.
Replication origins in Saccharomyces cerevisiae are spaced at intervals of approximately 40 kb. However, both measurements of replication fork rate and studies of hypomorphic alleles of genes encoding replication initiation proteins suggest the question of whether replication origins are more closely spaced than should be required. We approached this question by systematically deleting replicators from chromosome III. The first significant increase in loss rate detected for the 315-kb full-length chromosome occurred only after all five efficient chromosomal replicators in the left two-thirds of the chromosome (ARS305, ARS306, ARS307, ARS309, and ARS310) had been deleted. The removal of the inefficient replicator ARS308 from this originless region caused little or no additional increase in loss rate. Chromosome fragmentations that removed the normally inactive replicators on the left end of the chromosome or the replicators distal to ARS310 on the right arm showed that both groups of replicators contribute significantly to the maintenance of the originless chromosome. Surprisingly, a 142-kb derivative of chromosome III, lacking all sequences that function as autonomously replicating sequence elements in plasmids, replicated and segregated properly 97% of the time. Both the replication initiation protein ORC and telomeres or a linear topology were required for the maintenance of chromosome fragments lacking replicators. [Abstract/Link to Full Text]

Fiumera HL, Broadley SA, Fox TD
Translocation of mitochondrially synthesized Cox2 domains from the matrix to the intermembrane space.
Mol Cell Biol. 2007 Jul;27(13):4664-73.
The N-terminal and C-terminal domains of mitochondrially synthesized cytochrome c oxidase subunit II, Cox2, are translocated through the inner membrane to the intermembrane space (IMS). We investigated the distinct mechanisms of N-tail and C-tail export by analysis of epitope-tagged Cox2 variants encoded in Saccharomyces cerevisiae mitochondrial DNA. Both the N and C termini of a truncated protein lacking the Cox2 C-terminal domain were translocated to the IMS via a pathway dependent upon the conserved translocase Oxa1. The topology of this Cox2 variant, accumulated at steady state, was largely but not completely unaffected in mutants lacking proteins required for export of the C-tail domain, Cox18 and Mss2. C-tail export was blocked by truncation of the last 40 residues from the C-tail domain, indicating that sequence and/or structural features of this domain are required for its translocation. Mss2, a peripheral protein bound to the inner surface of the inner membrane, coimmunoprecipitated with full-length newly synthesized Cox2, whose leader peptide had already been cleaved in the IMS. Our data suggest that the C-tail domain is recognized posttranslationally by a specialized translocation apparatus after the N-tail has been translocated by Oxa1. [Abstract/Link to Full Text]

Pick E, Lau OS, Tsuge T, Menon S, Tong Y, Dohmae N, Plafker SM, Deng XW, Wei N
Mammalian DET1 regulates Cul4A activity and forms stable complexes with E2 ubiquitin-conjugating enzymes.
Mol Cell Biol. 2007 Jul;27(13):4708-19.
DET1 (de-etiolated 1) is an essential negative regulator of plant light responses, and it is a component of the Arabidopsis thaliana CDD complex containing DDB1 and COP10 ubiquitin E2 variant. Human DET1 has recently been isolated as one of the DDB1- and Cul4A-associated factors, along with an array of WD40-containing substrate receptors of the Cul4A-DDB1 ubiquitin ligase. However, DET1 differs from conventional substrate receptors of cullin E3 ligases in both biochemical behavior and activity. Here we report that mammalian DET1 forms stable DDD-E2 complexes, consisting of DDB1, DDA1 (DET1, DDB1 associated 1), and a member of the UBE2E group of canonical ubiquitin-conjugating enzymes. DDD-E2 complexes interact with multiple ubiquitin E3 ligases. We show that the E2 component cannot maintain the ubiquitin thioester linkage once bound to the DDD core, rendering mammalian DDD-E2 equivalent to the Arabidopsis CDD complex. While free UBE2E-3 is active and able to enhance UbcH5/Cul4A activity, the DDD core specifically inhibits Cul4A-dependent polyubiquitin chain assembly in vitro. Overexpression of DET1 inhibits UV-induced CDT1 degradation in cultured cells. These findings demonstrate that the conserved DET1 complex modulates Cul4A functions by a novel mechanism. [Abstract/Link to Full Text]

Zhang S, Fei T, Zhang L, Zhang R, Chen F, Ning Y, Han Y, Feng XH, Meng A, Chen YG
Smad7 antagonizes transforming growth factor beta signaling in the nucleus by interfering with functional Smad-DNA complex formation.
Mol Cell Biol. 2007 Jun;27(12):4488-99.
Smad7 plays an essential role in the negative-feedback regulation of transforming growth factor beta (TGF-beta) signaling by inhibiting TGF-beta signaling at the receptor level. It can interfere with binding to type I receptors and thus activation of receptor-regulated Smads or recruit the E3 ubiquitin ligase Smurf to receptors and thus target them for degradation. Here, we report that Smad7 is predominantly localized in the nucleus of Hep3B cells. The targeted expression of Smad7 in the nucleus conferred superior inhibitory activity on TGF-beta signaling, as determined by reporter assay in mammalian cells and by its effect on zebrafish embryogenesis. Furthermore, Smad7 repressed Smad3/4-, Smad2/4-, and Smad1/4-enhanced reporter gene expression, indicating that Smad7 can function independently of type I receptors. An oligonucleotide precipitation assay revealed that Smad7 can specifically bind to the Smad-responsive element via its MH2 domain, and DNA-binding activity was further confirmed in vivo with the promoter of PAI-1, a TGF-beta target gene, by chromatin immunoprecipitation. Finally, we provide evidence that Smad7 disrupts the formation of the TGF-beta-induced functional Smad-DNA complex. Our findings suggest that Smad7 inhibits TGF-beta signaling in the nucleus by a novel mechanism. [Abstract/Link to Full Text]

Boerries M, Most P, Gledhill JR, Walker JE, Katus HA, Koch WJ, Aebi U, Schoenenberger CA
Ca2+ -dependent interaction of S100A1 with F1-ATPase leads to an increased ATP content in cardiomyocytes.
Mol Cell Biol. 2007 Jun;27(12):4365-73.
S100A1, a Ca(2+)-sensing protein of the EF-hand family that is expressed predominantly in cardiac muscle, plays a pivotal role in cardiac contractility in vitro and in vivo. It has recently been demonstrated that by restoring Ca(2+) homeostasis, S100A1 was able to rescue contractile dysfunction in failing rat hearts. Myocardial contractility is regulated not only by Ca(2+) homeostasis but also by energy metabolism, in particular the production of ATP. Here, we report a novel interaction of S100A1 with mitochondrial F(1)-ATPase, which affects F(1)-ATPase activity and cellular ATP production. In particular, cardiomyocytes that overexpress S100A1 exhibited a higher ATP content than control cells, whereas knockdown of S100A1 expression decreased ATP levels. In pull-down experiments, we identified the alpha- and beta-chain of F(1)-ATPase to interact with S100A1 in a Ca(2+)-dependent manner. The interaction was confirmed by colocalization studies of S100A1 and F(1)-ATPase and the analysis of the S100A1-F(1)-ATPase complex by gel filtration chromatography. The functional impact of this association is highlighted by an S100A1-mediated increase of F(1)-ATPase activity. Consistently, ATP synthase activity is reduced in cardiomyocytes from S100A1 knockout mice. Our data indicate that S100A1 might play a key role in cardiac energy metabolism. [Abstract/Link to Full Text]

Tanji T, Hu X, Weber AN, Ip YT
Toll and IMD pathways synergistically activate an innate immune response in Drosophila melanogaster.
Mol Cell Biol. 2007 Jun;27(12):4578-88.
The inducible expression of antimicrobial peptide genes in Drosophila melanogaster is regulated by the conserved Toll and peptidoglycan recognition protein LC/immune deficiency (PGRP-LC/IMD) signaling pathways. It has been proposed that the two pathways have independent functions and mediate the specificity of innate immune responses towards different microorganisms. Scattered evidence also suggests that some antimicrobial target genes can be activated by both Toll and IMD, albeit to different extents. This dual activation can be mediated by independent stimulation or by cross-regulation of the two pathways. We show in this report that the Toll and IMD pathways can interact synergistically, demonstrating that cross-regulation occurs. The presence of Spätzle (the Toll ligand) and gram-negative peptidoglycan (the PGRP-LC ligand) together caused synergistic activation of representative target genes of the two pathways, including Drosomycin, Diptericin, and AttacinA. Constitutive activation of Toll and PGRP-LC/IMD could mimic the synergistic stimulation. RNA interference assays and promoter analyses demonstrate that cooperation of different NF-kappaB-related transcription factors mediates the synergy. These results illustrate how specific ligand binding by separate upstream pattern recognition receptors can be translated into a broad-spectrum host response, a hallmark of innate immunity. [Abstract/Link to Full Text]

Gao J, Maison SF, Wu X, Hirose K, Jones SM, Bayazitov I, Tian Y, Mittleman G, Matthews DB, Zakharenko SS, Liberman MC, Zuo J
Orphan glutamate receptor delta1 subunit required for high-frequency hearing.
Mol Cell Biol. 2007 Jun;27(12):4500-12.
The function of the orphan glutamate receptor delta subunits (GluRdelta1 and GluRdelta2) remains unclear. GluRdelta2 is expressed exclusively in the Purkinje cells of the cerebellum, and GluRdelta1 is prominently expressed in inner ear hair cells and neurons of the hippocampus. We found that mice lacking the GluRdelta1 protein displayed significant cochlear threshold shifts for frequencies of >16 kHz. These deficits correlated with a substantial loss of type IV spiral ligament fibrocytes and a significant reduction of endolymphatic potential in high-frequency cochlear regions. Vulnerability to acoustic injury was significantly enhanced; however, the efferent innervation of hair cells and the classic efferent inhibition of outer hair cells were unaffected. Hippocampal and vestibular morphology and function were normal. Our findings show that the orphan GluRdelta1 plays an essential role in high-frequency hearing and ionic homeostasis in the basal cochlea, and the locus encoding GluRdelta1 represents a candidate gene for congenital or acquired high-frequency hearing loss in humans. [Abstract/Link to Full Text]

Zhang S, Cagatay T, Amanai M, Zhang M, Kline J, Castrillon DH, Ashfaq R, Oz OK, Wharton KA
Viable mice with compound mutations in the Wnt/Dvl pathway antagonists nkd1 and nkd2.
Mol Cell Biol. 2007 Jun;27(12):4454-64.
Gradients of Wnt/beta-catenin signaling coordinate development and physiological homeostasis in metazoan animals. Proper embryonic development of the fruit fly Drosophila melanogaster requires the Naked cuticle (Nkd) protein to attenuate a gradient of Wnt/beta-catenin signaling across each segmental anlage. Nkd inhibits Wnt signaling by binding the intracellular protein Dishevelled (Dsh). Mice and humans have two nkd homologs, nkd1 and nkd2, whose encoded proteins can bind Dsh homologs (the Dvl proteins) and inhibit Wnt signaling. To determine whether nkd genes are necessary for murine development, we replaced nkd exons that encode Dvl-binding sequences with IRES-lacZ/neomycin cassettes. Mutants homozygous for each nkd(lacZ) allele are viable with slightly reduced mean litter sizes. Surprisingly, double-knockout mice are viable, with subtle alterations in cranial bone morphology that are reminiscent of mutation in another Wnt/beta-catenin antagonist, axin2. Our data show that nkd function in the mouse is dispensable for embryonic development. [Abstract/Link to Full Text]

Iida J, Ishizaki H, Okamoto-Tanaka M, Kawata A, Sumita K, Ohgake S, Sato Y, Yorifuji H, Nukina N, Ohashi K, Mizuno K, Tsutsumi T, Mizoguchi A, Miyoshi J, Takai Y, Hata Y
Synaptic scaffolding molecule alpha is a scaffold to mediate N-methyl-D-aspartate receptor-dependent RhoA activation in dendrites.
Mol Cell Biol. 2007 Jun;27(12):4388-405.
Synaptic scaffolding molecule (S-SCAM) interacts with a wide variety of molecules at excitatory and inhibitory synapses. It comprises three alternative splicing variants, S-SCAMalpha, -beta, and -gamma. We generated mutant mice lacking specifically S-SCAMalpha. S-SCAMalpha-deficient mice breathe and feed normally but die within 24 h after birth. Primary cultured hippocampal neurons from mutant mice have abnormally elongated dendritic spines. Exogenously expressed S-SCAMalpha corrects this abnormal morphology, while S-SCAMbeta and -gamma have no effect. Active RhoA decreases in cortical neurons from mutant mice. Constitutively active RhoA and ROCKII shift the length of dendritic spines toward the normal level, whereas ROCK inhibitor (Y27632) blocks the effect by S-SCAMalpha. S-SCAMalpha fails to correct the abnormal spine morphology under the treatment of N-methyl-d-aspartate (NMDA) receptor inhibitor (AP-5), Ca(2+)/calmodulin kinase inhibitor (KN-62), or tyrosine kinase inhibitor (PP2). NMDA treatment increases active RhoA in dendrites in wild-type hippocampal neurons, but not in mutant neurons. The ectopic expression of S-SCAMalpha, but not -beta, recovers the NMDA-responsive accumulation of active RhoA in dendrites. Phosphorylation of extracellular signal-regulated kinase 1/2 and Akt and calcium influx in response to NMDA are not impaired in mutant neurons. These data indicate that S-SCAMalpha is a scaffold required to activate RhoA protein in response to NMDA receptor signaling in dendrites. [Abstract/Link to Full Text]

Kinyamu HK, Archer TK
Proteasome activity modulates chromatin modifications and RNA polymerase II phosphorylation to enhance glucocorticoid receptor-mediated transcription.
Mol Cell Biol. 2007 Jul;27(13):4891-904.
The 26S proteasome modulates steroid hormone receptor-dependent gene transcription at least in part by regulating turnover and recycling of receptor/transcriptional DNA complexes, thereby ensuring continued hormone response. For the glucocorticoid receptor (GR), inhibition of proteasome-mediated proteolysis or RNA interference-mediated depletion of specific proteasome subunits results in an increase in gene expression. To facilitate transcription, proteasome inhibition alters at least two features associated with modification of chromatin architecture and gene transcription. First, proteasome inhibition increases trimethyl histone H3K4 levels with a corresponding accumulation of this modification on GR-regulated promoters in vivo. Secondly, global levels of phosphorylated RNA polymerase II (Pol II) increase, together with hormone-dependent association of the phosphorylated Pol II, with the promoter and the body of the activated gene. We propose that apart from modulating receptor turnover, the proteasome directly influences both the transcription machinery and chromatin structure, factors integral to nuclear receptor-regulated gene transcription. [Abstract/Link to Full Text]

Hoar K, Chakravarty A, Rabino C, Wysong D, Bowman D, Roy N, Ecsedy JA
MLN8054, a small-molecule inhibitor of Aurora A, causes spindle pole and chromosome congression defects leading to aneuploidy.
Mol Cell Biol. 2007 Jun;27(12):4513-25.
Aurora A kinase plays an essential role in the proper assembly and function of the mitotic spindle, as its perturbation causes defects in centrosome separation, spindle pole organization, and chromosome congression. Moreover, Aurora A disruption leads to cell death via a mechanism that involves aneuploidy generation. However, the link between the immediate functional consequences of Aurora A inhibition and the development of aneuploidy is not clearly defined. In this study, we delineate the sequence of events that lead to aneuploidy following Aurora A inhibition using MLN8054, a selective Aurora A small-molecule inhibitor. Human tumor cells treated with MLN8054 show a high incidence of abnormal mitotic spindles, often with unseparated centrosomes. Although these spindle defects result in mitotic delays, cells ultimately divide at a frequency near that of untreated cells. We show that many of the spindles in the dividing cells are bipolar, although they lack centrosomes at one or more spindle poles. MLN8054-treated cells frequently show alignment defects during metaphase, lagging chromosomes in anaphase, and chromatin bridges during telophase. Consistent with the chromosome segregation defects, cells treated with MLN8054 develop aneuploidy over time. Taken together, these results suggest that Aurora A inhibition kills tumor cells through the development of deleterious aneuploidy. [Abstract/Link to Full Text]

Taniguchi K, Kohno R, Ayada T, Kato R, Ichiyama K, Morisada T, Oike Y, Yonemitsu Y, Maehara Y, Yoshimura A
Spreds are essential for embryonic lymphangiogenesis by regulating vascular endothelial growth factor receptor 3 signaling.
Mol Cell Biol. 2007 Jun;27(12):4541-50.
Spred/Sprouty family proteins negatively regulate growth factor-induced ERK activation. Although the individual physiological roles of Spred-1 and Spred-2 have been investigated using gene-disrupted mice, the overlapping functions of Spred-1 and Spred-2 have not been clarified. Here, we demonstrate that the deletion of both Spred-1 and Spred-2 resulted in embryonic lethality at embryonic days 12.5 to 15.5 with marked subcutaneous hemorrhage, edema, and dilated lymphatic vessels filled with erythrocytes. This phenotype resembled that of Syk(-/-) and SLP-76(-/-) mice with defects in the separation of lymphatic vessels from blood vessels. The number of LYVE-1-positive lymphatic vessels and lymphatic endothelial cells increased markedly in Spred-1/2-deficient embryos compared with WT embryos, while the number of blood vessels was not different. Ex vivo colony assay revealed that Spred-1/2 suppressed lymphatic endothelial cell proliferation and/or differentiation. In cultured cells, the overexpression of Spred-1 or Spred-2 strongly suppressed vascular endothelial growth factor-C (VEGF-C)/VEGF receptor (VEGFR)-3-mediated ERK activation, while Spred-1/2-deficient cells were extremely sensitive to VEGFR-3 signaling. These data suggest that Spreds play an important role in lymphatic vessel development by negatively regulating VEGF-C/VEGFR-3 signaling. [Abstract/Link to Full Text]

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Recent Articles in Journal of Cell Science

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Tran AD, Marmo TP, Salam AA, Che S, Finkelstein E, Kabarriti R, Xenias HS, Mazitschek R, Hubbert C, Kawaguchi Y, Sheetz MP, Yao TP, Bulinski JC
HDAC6 deacetylation of tubulin modulates dynamics of cellular adhesions.
J Cell Sci. 2007 Apr 15;120(Pt 8):1469-79.
Genetic or pharmacological alteration of the activity of the histone deacetylase 6 (HDAC6) induces a parallel alteration in cell migration. Using tubacin to block deacetylation of alpha-tubulin, and not other HDAC6 substrates, yielded a motility reduction equivalent to agents that block all NAD-independent HDACs. Accordingly, we investigated how the failure to deacetylate tubulin contributes to decreased motility in HDAC6-inhibited cells. Testing the hypothesis that motility is reduced because cellular adhesion is altered, we found that inhibiting HDAC6 activity towards tubulin rapidly increased total adhesion area. Next, we investigated the mechanism of the adhesion area increase. Formation of adhesions proceeded normally and cell spreading was more rapid in the absence of active HDAC6; however, photobleaching assays and adhesion breakdown showed that adhesion turnover was slower. To test the role of hyperacetylated tubulin in altering adhesion turnover, we measured microtubule dynamics in HDAC6-inhibited cells because dynamic microtubules are required to target adhesions for turnover. HDAC6 inhibition yielded a decrease in microtubule dynamics that was sufficient to decrease focal adhesion turnover. Thus, our results suggest a scenario in which the decreased dynamics of hyperacetylated microtubules in HDAC6-inhibited cells compromises their capacity to mediate the focal adhesion dynamics required for rapid cell migration. [Abstract/Link to Full Text]

Amessou M, Fradagrada A, Falguičres T, Lord JM, Smith DC, Roberts LM, Lamaze C, Johannes L
Syntaxin 16 and syntaxin 5 are required for efficient retrograde transport of several exogenous and endogenous cargo proteins.
J Cell Sci. 2007 Apr 15;120(Pt 8):1457-68.
Retrograde transport allows proteins and lipids to leave the endocytic pathway to reach other intracellular compartments, such as trans-Golgi network (TGN)/Golgi membranes, the endoplasmic reticulum and, in some instances, the cytosol. Here, we have used RNA interference against the SNARE proteins syntaxin 5 and syntaxin 16, combined with recently developed quantitative trafficking assays, morphological approaches and cell intoxication analysis to show that these SNARE proteins are not only required for efficient retrograde transport of Shiga toxin, but also for that of an endogenous cargo protein - the mannose 6-phosphate receptor - and for the productive trafficking into cells of cholera toxin and ricin. We have found that the function of syntaxin 16 was specifically required for, and restricted to, the retrograde pathway. Strikingly, syntaxin 5 RNA interference protected cells particularly strongly against Shiga toxin. Since our trafficking analysis showed that apart from inhibiting retrograde endosome-to-TGN transport, the silencing of syntaxin 5 had no additional effect on Shiga toxin endocytosis or trafficking from TGN/Golgi membranes to the endoplasmic reticulum, we hypothesize that syntaxin 5 also has trafficking-independent functions. In summary, our data demonstrate that several cellular and exogenous cargo proteins use elements of the same SNARE machinery for efficient retrograde transport between early/recycling endosomes and TGN/Golgi membranes. [Abstract/Link to Full Text]

Cottle DL, McGrath MJ, Cowling BS, Coghill ID, Brown S, Mitchell CA
FHL3 binds MyoD and negatively regulates myotube formation.
J Cell Sci. 2007 Apr 15;120(Pt 8):1423-35.
MyoD initiates muscle differentiation and promotes skeletal myogenesis by regulating temporal gene expression. MyoD-interacting proteins induce regulatory effects, and the identification of new MyoD-binding partners may provide mechanistic insights into the regulation of gene expression during myogenesis. FHL3 is one of three members of the FHL protein family that are expressed in skeletal muscle, but its function in myogenesis is unknown. Overexpression of human FHL3 in mouse C2C12 cells retarded myotube formation and decreased the expression of muscle-specific regulatory genes such as myogenin but not MyoD. By contrast, short interfering RNA (siRNA)-mediated FHL3 protein knockdown enhanced myoblast differentiation associated with increased myogenin, but not MyoD protein expression, early during differentiation. We demonstrate that FHL3 is a MyoD-associated protein by direct binding assays, colocalisation in the nucleus of myoblasts and GST pull-down studies. Moreover, we determined that FHL3 interacts with MyoD, functioning as its potent negative co-transcriptional regulator. Ectopic expression of FHL3 in myoblasts impaired MyoD-mediated transcriptional activity and muscle gene expression. By contrast, siRNA-mediated FHL3 knockdown enhanced MyoD transcriptional activity in a dose-dependent manner. These findings reveal that FHL3 association with MyoD may contribute to the regulation of MyoD-dependent transcription of muscle genes and thereby myogenesis. [Abstract/Link to Full Text]

Diaz FJ, Wigglesworth K, Eppig JJ
Oocytes determine cumulus cell lineage in mouse ovarian follicles.
J Cell Sci. 2007 Apr 15;120(Pt 8):1330-40.
The two principal functions of ovarian follicles are developmental and endocrine. The cumulus cells surrounding the oocyte are specialized to serve the development of the oocyte and steroidogenesis is a principal role of mural granulosa cells that line the follicle wall. The findings in this report demonstrate that oocytectomy or treatment with an inhibitor of SMAD2/3 activation results in decreased cumulus marker mRNA transcript levels and allows FSH to induce mural marker transcripts in cumulus cells. In addition, SMAD2/3 signaling is involved in enabling cumulus expansion and EGF-induced increases in Ptx3, Ptgs2 and Has2 mRNA levels. By contrast, follicle-stimulating hormone (FSH) stimulated expression of mural transcripts, but suppressed levels of cumulus transcripts. Thus, FSH and oocyte-stimulated SMAD2/3 signaling establish opposing gradients of influence in the follicle. These specify the mural and cumulus granulosa cell phenotypes that are pivotal for appropriate endocrine function and oocyte development. [Abstract/Link to Full Text]

Ohkawa N, Fujitani K, Tokunaga E, Furuya S, Inokuchi K
The microtubule destabilizer stathmin mediates the development of dendritic arbors in neuronal cells.
J Cell Sci. 2007 Apr 15;120(Pt 8):1447-56.
The regulation of microtubule dynamics is important for the appropriate arborization of neuronal dendrites during development, which in turn is critical for the formation of functional neural networks. Here we show that stathmin, a microtubule destabilizing factor, is downregulated at both the expression and activity levels during cerebellar development, and this down-regulation contributes to dendritic arborization. Stathmin overexpression drastically limited the dendritic growth of cultured Purkinje cells. The stathmin activity was suppressed by neural activity and CaMKII-dependent phosphorylation at Ser16, which led to dendritic arborization. Stathmin phosphorylation at Ser16 was mediated by the activation of voltage-gated calcium channels and metabotropic glutamate receptor 1. Although overexpression of SCG10, a member of the stathmin family, also limited the dendritic arborization, SCG10 did not mediate the CaMKII regulation of dendritic development. These results suggest that calcium elevation activates CaMKII, which in turn phosphorylates stathmin at Ser16 to stabilize dendritic microtubules. siRNA knockdown of endogenous stathmin significantly reduced dendritic growth in Purkinje cells. Thus, these data suggest that proper regulation of stathmin activity is a key factor for controlling the dendritic microtubule dynamics that are important for neuronal development. [Abstract/Link to Full Text]

Joo JY, Kim BW, Lee JS, Park JY, Kim S, Yun YJ, Lee SH, Lee SH, Rhim H, Son H
Activation of NMDA receptors increases proliferation and differentiation of hippocampal neural progenitor cells.
J Cell Sci. 2007 Apr 15;120(Pt 8):1358-70.
The prolonged effects of N-methyl-D-aspartate (NMDA) receptor activation on the proliferation and differentiation of hippocampal neural progenitor cells (NPCs) were studied. Under conditions of mitogen-mediated proliferation, a single NMDA pulse (5 microM) increased the fraction of 5-bromo-2-deoxyuridine (BrdU)-positive (BrdU(+)) cells after a delay of 72 hours. Similarly, a single systemic injection of NMDA (100 mg/kg) increased the number of BrdU(+) cells in the dentate gyrus (DG) after 28 days, but not after 3 days. NMDA receptor activation induced an immediate influx of Ca(2+) into the NPCs and the NPCs expressed and released vascular endothelial growth factor (VEGF) in an NMDA receptor-dependent manner within 72 hours. With repetitive stimulation at the same dose, NMDA stimulated the acquisition of a neuronal phenotype accompanied by an increase in the expression of proneural basic helix-loop-helix (bHLH) factors. Together these findings suggest that neurogenesis in the developing brain is likely to be both directly and indirectly regulated by complex interactions between Ca(2+) influx and excitation-releasable cytokines, even at mild levels of excitation. In addition, our results are the first to show that stimulation of NPCs may lead to either proliferation or neuronal differentiation, depending on the level of NMDA receptor activation. [Abstract/Link to Full Text]

Basto R, Gergely F, Draviam VM, Ohkura H, Liley K, Raff JW
Hsp90 is required to localise cyclin B and Msps/ch-TOG to the mitotic spindle in Drosophila and humans.
J Cell Sci. 2007 Apr 1;120(Pt 7):1278-87.
During mitosis, cyclin B is extremely dynamic and although it is concentrated at the centrosomes and spindle microtubules (MTs) in organisms ranging from yeast to humans, the mechanisms that determine its localisation are poorly understood. To understand how cyclin B is targeted to different locations in the cell we have isolated proteins that interact with cyclin B in Drosophila embryo extracts. Here we show that cyclin B interacts with the molecular chaperone Hsp90 and with the MT-associated protein (MAP) Mini spindles (Msps; the Drosophila orthologue of XMAP215/ch-TOG). Both Hsp90 and Msps are concentrated at centrosomes and spindles, and we show that Hsp90, but not Msps, is required for the efficient localisation of cyclin B to these structures. We find that, unlike what happens with other cell cycle proteins, Hsp90 is not required to stabilise cyclin B or Msps during mitosis. Thus, we propose that Hsp90 plays a novel role in regulating the localisation of cyclin B and Msps during mitosis. [Abstract/Link to Full Text]

Castańares C, Redondo-Horcajo M, Magán-Marchal N, ten Dijke P, Lamas S, Rodríguez-Pascual F
Signaling by ALK5 mediates TGF-beta-induced ET-1 expression in endothelial cells: a role for migration and proliferation.
J Cell Sci. 2007 Apr 1;120(Pt 7):1256-66.
Endothelin-1 (ET-1) is a potent endothelial-derived 21-amino-acid vasoconstrictor peptide and its expression is potently regulated by the cytokine transforming growth factor-beta (TGF-beta). Most cell types contain a TGF-beta type I receptor form known as activin receptor-like kinase 5 (ALK5). However, endothelial cells coexpress an additional type I receptor named ALK1. These forms do not constitute redundant receptors with the same function, but they activate different Smad-mediated expression programmes leading to specific endothelial phenotypes. The aim of our study was to characterize the TGF-beta-induced pathway leading to ET-1 expression in endothelial cells and the contribution of the TGF-beta-mediated enhancement of ET-1 to the regulation of the endothelial cell migration and proliferation capacity. Our experiments indicate that TGF-beta induces ET-1 expression preferentially through the ALK5/Smad3 pathway. Specific ALK5 inhibition totally blocked the anti-angiogenic effect of TGF-beta. Antagonism of ET receptors partially reverted the effect of TGF-beta, indicating that a significant portion of the anti-migratory and anti-proliferative actions of this cytokine is mediated by ET-1 acting in an autocrine manner on endothelial cells. [Abstract/Link to Full Text]

Bertling E, Quintero-Monzon O, Mattila PK, Goode BL, Lappalainen P
Mechanism and biological role of profilin-Srv2/CAP interaction.
J Cell Sci. 2007 Apr 1;120(Pt 7):1225-34.
Profilin and cyclase-associated protein (CAP, known in yeast as Srv2) are ubiquitous and abundant actin monomer-binding proteins. Profilin catalyses the nucleotide exchange on actin monomers and promotes their addition to filament barbed ends. Srv2/CAP recycles newly depolymerized actin monomers from ADF/cofilin for subsequent rounds of polymerization. Srv2/CAP also harbors two proline-rich motifs and has been suggested to interact with profilin. However, the mechanism and biological role of the possible profilin-Srv2/CAP interaction has not been investigated. Here, we show that Saccharomyces cerevisiae Srv2 and profilin interact directly (K(D) approximately 1.3 microM) and demonstrate that a specific proline-rich motif in Srv2 mediates this interaction in vitro and in vivo. ADP-actin monomers and profilin do not interfere with each other's binding to Srv2, suggesting that these three proteins can form a ternary complex. Genetic and cell biological analyses on an Srv2 allele (srv2-201) defective in binding profilin reveals that a direct interaction with profilin is not essential for Srv2 cellular function. However, srv2-201 causes a moderate increase in cell size and partially suppresses the cell growth and actin organization defects of an actin binding mutant profilin (pfy1-4). Together these data suggest that Srv2 is an important physiological interaction partner of profilin. [Abstract/Link to Full Text]

Tessadori F, Chupeau MC, Chupeau Y, Knip M, Germann S, van Driel R, Fransz P, Gaudin V
Large-scale dissociation and sequential reassembly of pericentric heterochromatin in dedifferentiated Arabidopsis cells.
J Cell Sci. 2007 Apr 1;120(Pt 7):1200-8.
Chromocenters in Arabidopsis thaliana are discrete nuclear domains of mainly pericentric heterochromatin. They are characterized by the presence of repetitive sequences, methylated DNA and dimethylated histone H3K9. Here we show that dedifferentiation of specialized mesophyll cells into undifferentiated protoplasts is accompanied by the disruption of chromocenter structures. The dramatic reduction of heterochromatin involves the decondensation of all major repeat regions, also including the centromeric 180 bp tandem repeats. Only the 45S rDNA repeat remained in a partly compact state in most cells. Remarkably, the epigenetic indicators for heterochromatin, DNA methylation and H3K9 dimethylation, did not change upon decondensation. Furthermore, the decondensation of pericentric heterochromatin did not result in transcriptional reactivation of silent genomic elements. The decondensation process was reversible upon prolonged culturing. Strikingly, recondensation of heterochromatin into chromocenters is a stepwise process. Compaction of the tandemly arranged 45S rDNA regions occurs first, followed by the centromeric 180 bp and the 5S rDNA repeats and finally the dispersed repeats, including transposons. The sequence of reassembly seems to be correlated to the size of the repeat domains. Our results indicate that different types of pericentromeric repeats form different types of heterochromatin, which subsequently merge to form a chromocenter. [Abstract/Link to Full Text]

Murphy C
Endo-fin-ally a SARA for BMP receptors.
J Cell Sci. 2007 Apr 1;120(Pt 7):1153-5. [Abstract/Link to Full Text]

Shi W, Chang C, Nie S, Xie S, Wan M, Cao X
Endofin acts as a Smad anchor for receptor activation in BMP signaling.
J Cell Sci. 2007 Apr 1;120(Pt 7):1216-24.
Signaling through receptors of the transforming growth factor beta (TGFbeta) superfamily is mediated by cytoplasmic Smad proteins. It has been demonstrated that Smad anchor for receptor activation (SARA) facilitates TGFbeta and activin/nodal signaling by recruiting and presenting Smad2/3 to the receptor complex. SARA does not bind Smad1 and hence does not enhance bone morphogenetic protein (BMP) signaling. Here we report for the first time that the endosome-associated FYVE-domain protein endofin acts as a Smad anchor for receptor activation in BMP signaling. We demonstrate that endofin binds Smad1 preferentially and enhances Smad1 phosphorylation and nuclear localization upon BMP stimulation. Silencing of endofin by RNAi resulted in a reduction in BMP-dependent Smad1 phosphorylation. Moreover, disruption of the membrane-anchoring FYVE motif by point mutation led to a reduction of BMP-responsive gene expression in cell culture and Xenopus ectodermal explants. Furthermore, we demonstrate that endofin contains a protein-phosphatase-binding motif, which functions to negatively modulate BMP signals through receptor dephosphorylation. Taken together, our results suggest that endofin plays an important role in both positive and negative feedback regulation of the BMP signaling pathway. [Abstract/Link to Full Text]

Buchsbaum RJ
Rho activation at a glance.
J Cell Sci. 2007 Apr 1;120(Pt 7):1149-52. [Abstract/Link to Full Text]

Paramio JM, Santos M, Jorcano JL
The ends of a conundrum?
J Cell Sci. 2007 Apr 1;120(Pt 7):1145-7; author reply 1147-8. [Abstract/Link to Full Text]

Chen J, Cheng X, Merched-Sauvage M, Caulin C, Roop DR, Koch PJ
An unexpected role for keratin 10 end domains in susceptibility to skin cancer.
J Cell Sci. 2006 Dec 15;119(Pt 24):5067-76.
Keratin 10 (K10) is a type I keratin that is expressed in post-mitotic suprabasal keratinocytes of the skin. Based on cell culture experiments and transgenic mouse studies, it has been proposed that K10 suppresses cell proliferation and tumor formation in the skin. Furthermore, the ability of K10 to suppress cell proliferation was mapped to its unique N- and C-terminal protein domains. In the present study, we modified the endogenous keratin 14 (K14) gene of mice using a knock-in approach to encode a chimeric keratin that consists of the K14 rod domain fused to the K10 head and tail domains (K1014chim). This transgene was expressed in the basal layer of the epidermis and the outer root sheath of hair follicles. Unexpectedly, we found that the K10 end domains had no effect on basal keratinocyte proliferation in vivo. Moreover, when subjected to a chemical skin carcinogenesis protocol, papilloma formation in mutant mice was accelerated instead of being inhibited. Our data suggest that the increased tumor susceptibility of K1014chim mice is in part due to a suppression of apoptosis in mutant keratinocytes. Our results support the notion that intermediate filaments, in addition to their function as cytoskeletal components, affect tumor susceptibility of epithelial cells. [Abstract/Link to Full Text]

Nazarenko I, Schäfer R, Sers C
Mechanisms of the HRSL3 tumor suppressor function in ovarian carcinoma cells.
J Cell Sci. 2007 Apr 15;120(Pt 8):1393-404.
HRSL3 (also known as H-REV107-1) belongs to a class II tumor suppressor gene family and is downregulated in several human tumors including ovarian carcinomas. To unravel the mechanism of HRSL3 tumor suppressor action, we performed a yeast two-hybrid screen and identified the alpha-isoform of the regulatory subunit A of protein phosphatase 2A (PR65alpha) as a new interaction partner of HRSL3. Interaction between HRSL3 and PR65alpha was confirmed in vitro and by co-immunoprecipitation in mammalian cells. We demonstrate that HRSL3 binds to the endogenous PR65alpha, thereby partially sequestering the catalytic subunit PR36 from the PR65 protein complex, and inhibiting PP2A catalytic activity. Furthermore, binding of HRSL3 to PR65 induces apoptosis in ovarian carcinoma cells in a caspase-dependent manner. Using several mutant HRSL3 constructs, we identified the N-terminal proline-rich region within the HRSL3 protein as the domain that is relevant for both binding of PR65alpha and induction of programmed cell death. This suggests that the negative impact of HRSL3 onto PP2A activity is important for the HRSL3 pro-apoptotic function and indicates a role of PP2A in survival of human ovarian carcinomas. The analysis of distinct PP2A target molecules revealed PKCzeta as being involved in HRSL3 action. These data implicate HRSL3 as a signaling regulatory molecule, which is functionally involved in the oncogenic network mediating growth and survival of ovarian cancer cells. [Abstract/Link to Full Text]

Hayashi M, Nimura K, Kashiwagi K, Harada T, Takaoka K, Kato H, Tamai K, Kaneda Y
Comparative roles of Twist-1 and Id1 in transcriptional regulation by BMP signaling.
J Cell Sci. 2007 Apr 15;120(Pt 8):1350-7.
Basic helix-loop-helix (bHLH) transcription factors are known as key regulators for mesenchymal differentiation. The present study showed that overexpression of Twist-1, a bHLH transcription factor, suppresses bone morphogenetic protein (BMP)-induced osteoblast differentiation, and downregulation of endogenous Twist-1 enhances BMP signaling. Maximal inhibition of BMP signaling was observed when Twist-1 was bound to E47, which markedly enhanced the stability of Twist-1. Co-immunoprecipitation assays revealed that Twist-1 formed a complex with Smad4 and histone deacetylase (HDAC) 1 in MC3T3-E1 cells stably expressing Twist-1. With trichostatin, an HDAC inhibitor, osteogenic factors such as alkaline phosphatase, Runx2 and osteopontin increased. Those results suggested that Twist-1 inhibited BMP signaling by recruiting HDAC1 to Smad4. Furthermore, the inhibitory effects of Twist-1 on BMP signaling were overcome by Id1 through induction of Twist-1 degradation. These findings suggest that Twist-1 can act as an inhibitor of BMP signaling, and Id1 can regulate BMP signaling through a positive feedback loop repressing Twist-1 function. These two molecules may therefore regulate differentiation of mesenchymal cells into progeny such as osteoblasts by controlling BMP signaling. [Abstract/Link to Full Text]

Voltmer-Irsch S, Kneissel S, Adenot PG, Schmidt-Zachmann MS
Regulatory mechanisms governing the oocyte-specific synthesis of the karyoskeletal protein NO145.
J Cell Sci. 2007 Apr 15;120(Pt 8):1412-22.
Given the prominence and the biological importance of the nucleus it is remarkable how little is still known about structure-forming proteins in the nuclear interior. The karyoskeletal protein NO145 has been identified as a major constituent of a filamentous network surrounding the amplified nucleoli of Xenopus laevis oocytes. We now show that an orthologous protein also occurs in female germ cells of a wide range of other vertebrates, where it forms dot-like structures. Using the Xenopus oocyte system we further report a specific regulatory mechanism responsible for (1) the rapid degradation of the NO145 protein during meiotic maturation, and (2) the cell-type-dependent translation of NO145 mRNA. Microinjection experiments have revealed that NO145 is a target of proteasomes and the use of the rapid amplification of cDNA ends-polyadenylation test (RACE-PAT) has disclosed the existence of NO145 mRNAs differing in their 3' UTRs. Reporter systems as well as polyribosome profiling experiments have revealed the regulatory importance of the 3' UTRs, which affect the translational efficiency as well as the stability of the encoded protein. The highly conserved cell-type specificity and the extremely tight temporal regulation of NO145 synthesis suggest an important role of this protein in female meiotic prophase. [Abstract/Link to Full Text]

Rodríguez M, Torrent G, Bosch M, Rayne F, Dubremetz JF, Ribó M, Benito A, Vilanova M, Beaumelle B
Intracellular pathway of Onconase that enables its delivery to the cytosol.
J Cell Sci. 2007 Apr 15;120(Pt 8):1405-11.
Onconase is an RNase with a very specific property because it is selectively toxic to transformed cells. This toxin is thought to recognize cell surface receptors, and the protection conferred by metabolic poisons against Onconase toxicity indicated that this RNase relies on endocytic uptake to kill cells. Nevertheless, its internalization pathway has yet to be unraveled. We show here that Onconase enters cells using AP-2/clathrin-mediated endocytosis. It is then routed, together with transferrin, to the receptor recycling compartment. Increasing the Onconase concentration in this structure using tetanus toxin light chain expression enhanced Onconase toxicity, indicating that recycling endosomes are a key compartment for Onconase cytosolic delivery. This intracellular destination is specific to Onconase because other (and much less toxic) RNases follow the default pathway to late endosomes/lysosomes. Drugs neutralizing endosomal pH increased Onconase translocation efficiency from purified endosomes during cell-free translocation assays by preventing Onconase dissociation from its receptor at endosomal pH. Consistently, endosome neutralization enhanced Onconase toxicity up to 100-fold. Onconase translocation also required cytosolic ATP hydrolysis. This toxin therefore shows an unusual entry process that relies on clathrin-dependent endocytic uptake and then neutralization of low endosomal pH for efficient translocation from the endosomal lumen to the cytosol. [Abstract/Link to Full Text]

Lardi-Studler B, Smolinsky B, Petitjean CM, Koenig F, Sidler C, Meier JC, Fritschy JM, Schwarz G
Vertebrate-specific sequences in the gephyrin E-domain regulate cytosolic aggregation and postsynaptic clustering.
J Cell Sci. 2007 Apr 15;120(Pt 8):1371-82.
Gephyrin is a multifunctional protein contributing to molybdenum cofactor (Moco) synthesis and postsynaptic clustering of glycine and GABA(A) receptors. It contains three major functional domains (G-C-E) and forms cytosolic aggregates and postsynaptic clusters by unknown mechanisms. Here, structural determinants of gephyrin aggregation and clustering were investigated by neuronal transfection of EGFP-tagged deletion and mutant gephyrin constructs. EGFP-gephyrin formed postsynaptic clusters containing endogenous gephyrin and GABA(A)-receptors. Isolated GC- or E-domains failed to aggregate and exerted dominant-negative effects on endogenous gephyrin clustering. A construct interfering with intermolecular E-domain dimerization readily auto-aggregated but showed impaired postsynaptic clustering. Finally, two mutant constructs with substitution of vertebrate-specific E-domain sequences with homologue bacterial MoeA sequences uncovered a region crucial for gephyrin clustering. One construct failed to aggregate, but retained Moco biosynthesis capacity, demonstrating the independence of gephyrin enzymatic activity and aggregation. Reinserting two vertebrate-specific residues restored gephyrin aggregation and increased formation of postsynaptic clusters containing GABA(A) receptors at the expense of PSD-95 clusters - a marker of glutamatergic synapses. These results underscore the key role of specific E-domain regions distinct from the known dimerization interface for controlling gephyrin aggregation and postsynaptic clustering and suggest that formation of gephyrin clusters influences the homeostatic balance between inhibitory and excitatory synapses. [Abstract/Link to Full Text]

Bax DV, Mahalingam Y, Cain S, Mellody K, Freeman L, Younger K, Shuttleworth CA, Humphries MJ, Couchman JR, Kielty CM
Cell adhesion to fibrillin-1: identification of an Arg-Gly-Asp-dependent synergy region and a heparin-binding site that regulates focal adhesion formation.
J Cell Sci. 2007 Apr 15;120(Pt 8):1383-92.
We have defined the molecular basis of cell adhesion to fibrillin-1, the major structural component of extracellular microfibrils that are associated with elastic fibres. Using human dermal fibroblasts, and recombinant domain swap fragments containing the Arg-Gly-Asp motif, we have demonstrated a requirement for upstream domains for integrin-alpha(5)beta(1)-mediated cell adhesion and migration. An adjacent heparin-binding site, which supports focal adhesion formation, was mapped to the fibrillin-1 TB5 motif. Site-directed mutagenesis revealed two arginine residues that are crucial for heparin binding, and confirmed their role in focal adhesion formation. These integrin and syndecan adhesion motifs juxtaposed on fibrillin-1 are evolutionarily conserved and reminiscent of similar functional elements on fibronectin, highlighting their crucial functional importance. [Abstract/Link to Full Text]

Williams SA, Xia L, Cummings RD, McEver RP, Stanley P
Fertilization in mouse does not require terminal galactose or N-acetylglucosamine on the zona pellucida glycans.
J Cell Sci. 2007 Apr 15;120(Pt 8):1341-9.
Fertilization in mammals requires sperm to bind to the zona pellucida (ZP) that surrounds the egg. Galactose (Gal) or N-acetylglucosamine (GlcNAc) residues on the glycans of ZP protein 3 (ZP3) have been implicated as mouse sperm receptors. However, Mgat1(-/-) eggs with modified N-glycans lacking terminal Gal and GlcNAc residues are fertilized. To determine if Gal and GlcNAc on O-glycans of the ZP are required for fertilization, a conditional allele of the T-synthase gene (T-syn(F)) was generated. T-syn encodes core 1 beta1,3-galactosyltransferase 1 (T-synthase), which initiates the synthesis of core-1-derived O-glycans, the only O-glycans on mouse ZP3. T-syn(F/F):ZP3Cre females in which T-syn(F) was deleted at the beginning of oogenesis generated eggs lacking core-1-derived O-glycans. Nevertheless, T-syn(F/F):ZP3Cre females were fertile and their eggs bound sperm similarly to controls. In addition, T-syn(-/-) embryos generated from T-syn null eggs developed until approximately E12.5. Thus, core-1-derived O-glycans are not required for blastogenesis, implantation, or development prior to midgestation. Moreover, T-syn(-/-)Mgat1(-/-) eggs lacking complex and hybrid N-glycans as well as core-1-derived O-glycans were fertilized. The combined data show that mouse ZP3 does not require terminal Gal or GlcNAc on either N- or O-glycans for fertilization. [Abstract/Link to Full Text]

Heubes S, Stemmann O
The AAA-ATPase p97-Ufd1-Npl4 is required for ERAD but not for spindle disassembly in Xenopus egg extracts.
J Cell Sci. 2007 Apr 15;120(Pt 8):1325-9.
The highly abundant AAA-ATPase p97 is required for diverse cellular processes, of which ER-associated protein degradation (ERAD) is understood best. Previously, a new role of p97 in spindle disassembly at the end of mitosis has been reported. However, we show that neither addition of dominant-negative p97 mutants nor depletion of crucial p97 adaptors impairs transition of meiotic spindles into interphase arrays of microtubules. The dominant-negative approach is validated by inhibition of ERAD, which we reconstitute for the first time in the powerful biochemical system of Xenopus egg extracts. The role of p97 in spindle disassembly during meiotic exit should therefore be reconsidered. [Abstract/Link to Full Text]

Beznoussenko GV, Dolgikh VV, Seliverstova EV, Semenov PB, Tokarev YS, Trucco A, Micaroni M, Di Giandomenico D, Auinger P, Senderskiy IV, Skarlato SO, Snigirevskaya ES, Komissarchik YY, Pavelka M, De Matteis MA, Luini A, Sokolova YY, Mironov AA
Analogs of the Golgi complex in microsporidia: structure and avesicular mechanisms of function.
J Cell Sci. 2007 Apr 1;120(Pt 7):1288-98.
Microsporidia are obligatory intracellular parasites, most species of which live in the host cell cytosol. They synthesize and then transport secretory proteins from the endoplasmic reticulum to the plasma membrane for formation of the spore wall and the polar tube for cell invasion. However, microsporidia do not have a typical Golgi complex. Here, using quick-freezing cryosubstitution and chemical fixation, we demonstrate that the Golgi analogs of the microsporidia Paranosema (Antonospora) grylli and Paranosema locustae appear as 300-nm networks of thin (25- to 40-nm diameter), branching or varicose tubules that display histochemical features of a Golgi, but that do not have vesicles. Vesicles are not formed even if membrane fusion is inhibited. These tubular networks are connected to the endoplasmic reticulum, the plasma membrane and the forming polar tube, and are positive for Sec13, gammaCOP and analogs of giantin and GM130. The spore-wall and polar-tube proteins are transported from the endoplasmic reticulum to the target membranes through these tubular networks, within which they undergo concentration and glycosylation. We suggest that the intracellular transport of secreted proteins in microsporidia occurs by a progression mechanism that does not involve the participation of vesicles generated by coat proteins I and II. [Abstract/Link to Full Text]

Bidla G, Dushay MS, Theopold U
Crystal cell rupture after injury in Drosophila requires the JNK pathway, small GTPases and the TNF homolog Eiger.
J Cell Sci. 2007 Apr 1;120(Pt 7):1209-15.
The prophenoloxidase-activating cascade is a key component of arthropod immunity. Drosophila prophenoloxidase is stored in crystal cells, a specialized class of blood cells from which it is released through cell rupture. Within minutes after bleeding, prophenoloxidase is activated leading to visible melanization of the clot matrix. Using crystal cell rupture and melanization as readouts to screen mutants in signal transduction pathways, we show that prophenoloxidase release requires Jun N-terminal kinase, small Rho GTPases and Eiger, the Drosophila homolog of tumor necrosis factor. We also provide evidence that in addition to microbial products, endogenous signals from dying hemocytes contribute to triggering and/or assembly of the prophenoloxidase-activating cascade, and that this process can be inhibited in vitro and in vivo using the viral apoptotic inhibitor p35. Our results provide a more comprehensive view of immune signal transduction pathways, with implications for immune reactions where cell death is used as a terminal mode of cell activation. [Abstract/Link to Full Text]

Jing R, Wilhelmsson U, Goodwill W, Li L, Pan Y, Pekny M, Skalli O
Synemin is expressed in reactive astrocytes in neurotrauma and interacts differentially with vimentin and GFAP intermediate filament networks.
J Cell Sci. 2007 Apr 1;120(Pt 7):1267-77.
Immature astrocytes and astrocytoma cells contain synemin and three other intermediate filament (IF) proteins: glial fibrillary acidic protein (GFAP), vimentin and nestin. Here, we show that, after neurotrauma, reactive astrocytes produce synemin and thus propose synemin as a new marker of reactive astrocytes. Comparison of synemin mRNA and protein levels in brain tissues and astrocyte cultures from wild-type, Vim(-)(/)(-) and Gfap(-)(/)(-)Vim(-)(/)(-) mice showed that in the absence of vimentin, synemin protein was undetectable although synemin mRNA was present at wild-type levels. By contrast, in Gfap(-)(/)(-) astrocytes, synemin protein and mRNA levels, as well as synemin incorporation into vimentin IFs, were unaltered. Biochemical assays with purified proteins suggested that synemin interacts with GFAP IFs like an IF-associated protein rather than like a polymerization partner, whereas the opposite was true for synemin interaction with vimentin. In transfection experiments, synemin did not incorporate into normal, filamentous GFAP networks, but integrated into vimentin and GFAP heteropolymeric networks. Thus, alongside GFAP, vimentin and nestin, reactive astrocytes contain synemin, whose accumulation is suppressed post-transcriptionally in the absence of a polymerization partner. In astrocytes, this partner is vimentin and not GFAP, which implies a functional difference between these two type III IF proteins. [Abstract/Link to Full Text]

Legrand-Poels S, Kustermans G, Bex F, Kremmer E, Kufer TA, Piette J
Modulation of Nod2-dependent NF-kappaB signaling by the actin cytoskeleton.
J Cell Sci. 2007 Apr 1;120(Pt 7):1299-310.
Actin disruption by CytochalasinD (CytD) and LatrunculinB (LatB) induced NF-kappaB activation in myelomonocytic and intestinal epithelial cells. In an attempt to elucidate the mechanism by which actin disruption induced IKK activation, we studied the human Nod2 protein, which was able to induce NF-kappaB activation and whose expression was restricted to myelomonocytic and intestinal epithelial cells. Nod2 is thought to play key roles in pathogen defence through sensing bacteria and generating an inflammatory immune response. We showed that actin disruption by CytD significantly and specifically increased Nod2-mediated NF-kappaB signaling. Nod2 was fully partitioned in the Triton-X-100-insoluble fraction but translocated into the soluble fraction after CytD treatment, demonstrating that the presence of Nod2 in the detergent-insoluble pellet was specific to actin cytoskeleton. Confocal analysis also revealed a Nod2 colocalization with membrane-associated F-actin. Colocalization and co-immunoprecipitation assays with endogenous Rac1 have shown that Nod2 associated with activated Rac1 in membrane ruffles through both its N-terminal caspase recruitment domains (CARD) and C-terminal leucine-rich repeats (LRRs). Membrane ruffle disruption by a Rac1 dominant negative form primed Nod2-dependent NF-kappaB signaling. The recruitment of Nod2 in Rac-induced dynamic cytoskeletal structures could be a strategy to both repress the Nod2-dependent NF-kappaB signaling in unstimulated cells and rapidly mobilize Nod2 during bacterial infection. [Abstract/Link to Full Text]

Lipp JJ, Hirota T, Poser I, Peters JM
Aurora B controls the association of condensin I but not condensin II with mitotic chromosomes.
J Cell Sci. 2007 Apr 1;120(Pt 7):1245-55.
The assembly of mitotic chromosomes is controlled by condensin complexes. In vertebrates, condensin I binds to chromatin in prometaphase, confers rigidity to chromosomes and enables the release of cohesin complexes from chromosome arms, whereas condensin II associates with chromosomes in prophase and promotes their condensation. Both complexes are essential for chromosome segregation in anaphase. Although the association of condensins with chromatin is important for the assembly and segregation of mitotic chromosomes, it is poorly understood how this process is controlled. Here we show that the mitotic kinase Aurora B regulates the association of condensin I, but not the interaction of condensin II with chromatin. Quantitative time-lapse imaging of cells expressing GFP-tagged condensin subunits revealed that Aurora B is required for efficient loading of condensin I onto chromosomes in prometaphase and for maintenance of the complex on chromosomes in later stages of mitosis. The three non-SMC subunits of condensin I are Aurora B substrates in vitro and their mitosis-specific phosphorylation depends on Aurora B in vivo. Our data indicate that Aurora B contributes to chromosome rigidity and segregation by promoting the binding of condensin I to chromatin. We have also addressed how Aurora B might mediate the dissociation of cohesin from chromosome arms. [Abstract/Link to Full Text]

Schober JM, Komarova YA, Chaga OY, Akhmanova A, Borisy GG
Microtubule-targeting-dependent reorganization of filopodia.
J Cell Sci. 2007 Apr 1;120(Pt 7):1235-44.
Interaction between the microtubule system and actin cytoskeleton has emerged as a fundamental process required for spatial regulation of cell protrusion and retraction activities. In our current studies, analysis of digital fluorescence images revealed targeting of microtubules to filopodia in B16F1 melanoma cells and fibroblasts. We investigated the functional consequence of targeting on filopodia reorganization and examined mechanisms by which microtubules may be guided to, or interact with, filopodia. Live cell imaging studies show that targeting events in lamellipodia wings temporally correlated with filopodia turning toward the lamellipodium midline and with filopodia merging. Rapid uncoupling of targeting with nocodazole decreased filopodia merging events and increased filopodia density. Total internal reflection fluorescence microscopy identified microtubules near the ventral surface and upward movement of targeted filopodia. The role of adhesion sites and microtubule plus-end proteins in targeting was investigated. Correlation of adhesion sites with microtubule targeting to filopodia was not observed and depletion of microtubule plus-end proteins did not significantly alter targeting frequency. We propose that microtubules target filopodia, independent of focal adhesions and plus-end proteins, causing filopodia movement and microtubules regulate filopodia density in lamellipodia wings through filopodia merging events. [Abstract/Link to Full Text]


The more the merrier. By Caveman.
J Cell Sci. 2007 Jan 15;120(Pt 2):201-3. [Abstract/Link to Full Text]

Keady BT, Kuo P, Martínez SE, Yuan L, Hake LE
MAPK interacts with XGef and is required for CPEB activation during meiosis in Xenopus oocytes.
J Cell Sci. 2007 Mar 15;120(Pt 6):1093-103.
Meiotic progression in Xenopus oocytes, and all other oocytes investigated, is dependent on polyadenylation-induced translation of stockpiled maternal mRNAs. Early during meiotic resumption, phosphorylation of CPE-binding protein (CPEB) is required for polyadenylation-induced translation of mRNAs encoding cell cycle regulators. Xenopus Gef (XGef), a Rho-family guanine-exchange factor, influences the activating phosphorylation of CPEB. An exchange-deficient version of XGef does not, therefore implicating Rho-family GTPase function in early meiosis. We show here that Clostridium difficile Toxin B, a Rho-family GTPase inhibitor, does not impair early CPEB phosphorylation or progression to germinal vesicle breakdown, indicating that XGef does not influence these events through activation of a Toxin-B-sensitive GTPase. Using the inhibitors U0126 for mitogen-activated protein kinase (MAPK), and ZM447439 for Aurora kinase A and Aurora kinase B, we found that MAPK is required for phosphorylation of CPEB, whereas Aurora kinases are not. Furthermore, we do not detect active Aurora kinase A in early meiosis. By contrast, we observe an early, transient activation of MAPK, independent of Mos protein expression. MAPK directly phosphorylates CPEB on four residues (T22, T164, S184, S248), but not on S174, a key residue for activating CPEB function. Notably, XGef immunoprecipitates contain MAPK, and this complex can phosphorylate CPEB. MAPK may prime CPEB for phosphorylation on S174 by an as-yet-unidentified kinase or may activate this kinase. [Abstract/Link to Full Text]

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Recent Articles in Journal of Cellular and Molecular Medicine

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Andryushkova AA, Kuznetsova IA, Bineva VN, Toporkova LB, Sakhno LV, Tikhonova MA, Chernykh ER, Orlovskaya IA, Nevinsky GA
Formation of different abzymes in autoimmune-prone MRL-lpr/lpr mice is associated with changes in colony formation of haematopoietic progenitors.
J Cell Mol Med. 2007 May-Jun;11(3):531-51.
It was shown that IgGs from the sera of 2-7-month-old control non-autoimmune (CBA x C57BL)F1 and BALB/c mice and 2-3-month-old autoimmune prone MRL-lpr/lpr mice (conditionally healthy mice) are catalytically inactive. During spontaneous development of deep systemic lupus erythematosus (SLE)-like pathology a specific reorganization of immune system of these mice leads to conditions associated with a production of IgGs hydrolyzing DNA, ATP and polysaccharides with low catalytic activities (conditionally pre-diseased mice).A significant increase in DNase, ATPase and amylase IgG relative activities associated with a transition from pre-diseased to deep diseased mice is correlated with additional changes in differentiation and proliferation of mice bone marrow haematopoietic stem cells (HSCs) and lymphocyte proliferation in different organs.The highest increase in all abzyme activities was found in mice immunized with DNA, which in comparison with pre-diseased and diseased mice are characterized by a different profile of HSC differentiation and by a suppression of cell apoptosis. Abzyme activities in the serum of pregnant females were comparable with those for pre-diseased mice, but the profile of HSC differentiation and cell apoptosis levels in pregnant and pre-diseased mice were quite different. Right after the beginning of lactation (4 days after delivery) and in a late time of lactation (14 days after delivery) there was an observed increase in cell apoptosis and two different stages of significant change in the HSC differentiation profiles; the first stage was accompanied with a significant increase and the second with a remarkable decrease in abzyme activities. Overall, all mouse groups investigated are characterized by a specific relationship between abzyme activities, HSC differentiation profiles, levels of lymphocyte proliferation, and cell apoptosis in different organs. From our point of view, the appearance of ATPase, DNase activities may be considered the earliest statistically significant marker of mouse spontaneous SLE and a further significant increase in their activities correlates with the appearance of SLE visible markers and with an increase in concentrations of anti-DNA Abs and urine protein. However, development of autoimmune (AI)-reactions and the increase in the sera anti-DNA antibodies (Abs) and in the abzyme activities in pregnant and lactating mice do not associate with SLE visible markers and proteinuria. The possible differences in immune system reorganizations during pre-disease, disease, pregnancy and lactation leading to production of different auto-antibodies and abzymes are discussed. [Abstract/Link to Full Text]

Wiehe JM, Ponsaerts P, Rojewski MT, Homann JM, Greiner J, Kronawitter D, Schrezenmeier H, Hombach V, Wiesneth M, Zimmermann O, Torzewski J
mRNA-mediated gene delivery into human progenitor cells promotes highly efficient protein expression.
J Cell Mol Med. 2007 May-Jun;11(3):521-30.
Gene transfer into human CD34+ haematopoietic progenitor cells (HPC) and multi-potent mesenchymal stromal cells (MSC) is an essential tool for numerous in vitro and in vivo applications including therapeutic strategies, such as tissue engineering and gene therapy. Virus based methods may be efficient, but bear risks like tumorigenesis and activation of immune responses. A safer alternative is non-viral gene transfer, which is considered to be less efficient and accomplished with high cell toxicity. The truncated low affinity nerve growth factor receptor (ALNGFR) is a marker gene approved for human in vivo application. Human CD34+ HPC and human MSC were transfected with in vitro-transcribed mRNA for DeltaLNGFR using the method of nucleofection. Transfection efficiency and cell viability were compared to plasmid-based nucleofection. Protein expression was assessed using flow cytometry over a time period of 10 days. Nucleofection of CD34+ HPC and MSC with mRNA resulted in significantly higher transfection efficiencies compared to plasmid transfection. Cell differentiation assays were performed after selecting DeltaLNGFR positive cells using a fluorescent activating cell sorter. Neither cell differentiation of MSC into chondrocytes, adipocytes and osteoblasts, nor differentiation of HPC into burst forming unit erythroid (BFU-E) colony forming unit-granulocyte, erythrocyte, macrophage and megakaryocyte (CFU-GEMM), and CFU-granulocyte-macrophage (GM) was reduced. mRNA based nucleofection is a powerful, highly efficient and non-toxic approach for transient labelling of human progenitor cells or, via transfection of selective proteins, for transient manipulation of stem cell function. It may be useful to transiently manipulate stem cell characteristics and thus combine principles of gene therapy and tissue engineering. [Abstract/Link to Full Text]

Reeve JL, Szegezdi E, Logue SE, Chonghaile TN, O'Brien T, Ritter T, Samali A
Distinct mechanisms of cardiomyocyte apoptosis induced by doxorubicin and hypoxia converge on mitochondria and are inhibited by Bcl-xL.
J Cell Mol Med. 2007 May-Jun;11(3):509-20.
Hypoxia and doxorubicin can cause cardiotoxicity and loss of myocardial function. These effects are due, in part, to an induction of apoptosis. Herein we identify the apoptotic pathways activated in H9c2 cells in response to hypoxia (O(2)/N(2)/CO(2), 0.5:94.5:5) and doxorubicin (0.5 muM). Although the apoptosis induced was accompanied by induction of Fas and Fas ligand, the death receptor pathway was not critical for caspase activation by either stimulus. Hypoxia induced the expression of endoplasmic reticulum (ER) stress mediators and processed ER-resident pro-caspase-12 whereas doxorubicin did not induce an ER stress response. Most importantly, both stimuli converged on mitochondria to promote apoptosis. Accumulation of cytochrome c in the cytosol coincided with the processing of pro-caspase-9 and -3. Increasing the expression of the anti-apoptotic protein Bcl-x(L), either by dexamethasone or adenovirus-mediated transduction, protected H9c2 cells from doxorubicin- and hypoxia-induced apoptosis. Bcl-x(L) attenuated mitochondrial cytochrome crelease and reduced downstream pro-caspase processing and apoptosis. These data demonstrate that two distinct cardiomyocyte-damaging stimuli converge on mitochondria thus presenting this organelle as a potentially important therapeutic target for anti-apoptotic strategies for cardiovascular diseases. [Abstract/Link to Full Text]

P?unescu V, Deak E, Herman D, Siska IR, T?nasie G, Bunu C, Anghel S, Tatu CA, Oprea TI, Henschler R, Rüster B, Bistrian R, Seifried E
In vitro differentiation of human mesenchymal stem cells to epithelial lineage.
J Cell Mol Med. 2007 May-Jun;11(3):502-8.
Our study examined whether human bone marrow-derived MSCs are able to differentiate, in vitro, into functional epithelial-like cells. MSCs were isolated from the sternum of 8 patients with different hematological disorders. The surface phenotype of these cells was characterized.To induce epithelial differentiation, MSCs were cultured using Epidermal Growth Factor, Keratinocyte Growth Factor, Hepatocyte Growth Factor and Insulin-like growth Factor-II. Differentiated cells were further characterized both morphologically and functionally by their capacity to express markers with specificity for epithelial lineage. The expression of cytokeratin 19 was assessed by immunocytochemistry, and cytokeratin 18 was evaluated by quantitative RT-PCR (Taq-man). The data demonstrate that human MSCs isolated from human bone marrow can differentiate into epithelial-like cells and may thus serve as a cell source for tissue engineering and cell therapy of epithelial tissue. [Abstract/Link to Full Text]

Faussone-Pellegrini MS, Vannucchi MG, Alaggio R, Strojna A, Midrio P
Morphology of the interstitial cells of Cajal of the human ileum from foetal to neonatal life.
J Cell Mol Med. 2007 May-Jun;11(3):482-94.
The so-called interstitial cells of Cajal myenteric plexus (ICC-MP), interstitial cells of Cajal intramuscular (ICC-IM) and interstitial cells of Cajal deep muscular plexus (ICC-DMP) are the three types of ICC endowed within the intestinal muscle coat where they play different roles in gut motility. Studies on ICC ontogenesis showed ICC-MP in the human ileum by 7-9 weeks while information on ICC-IM and ICC-DMP in foetuses and newborns are not exhaustive. Functional recordings in the fasting state of prematurely born babies aged 28-37 weeks showed immature ileal motility. To gain more information on the time of appearance of the three ICC types in the human ileum and on the steps of the acquisition of mature features, we studied by c-kit immuno-histochemistry foetuses aged 17-27 weeks and newborns aged 36-41 weeks. In parallel, the maturative steps of enteric plexuses and muscle layers were immunohistochemically examined by using anti-neuron specific enolase (NSE), anti-S-100 and anti-alpha smooth muscle actin (alphaSMA) antibodies. The appearance and differentiation of all the ICC types were seen to occur in concomitance with those of the related nerve plexuses and muscle layers. ICC-MP appeared first, ICC-IM and ICC-DMP later and their differentiation was incomplete at birth. In conclusion, the ICC-MP, the intestinal pacemaker cells, in spite of absence of food intake, are already present during the foetal life and the ICC-IM appear by pre-term life, thus ensuring neurotransmission. The ICC-DMP and their related nerve plexus and smooth muscle cells, i.e. the intestinal stretch receptor, begin to differentiate at birth. These findings might help in predicting neonatal ileal motor behaviour and in interpreting the role of ICC abnormalities in the pathophysiology of intestinal motile disorders of neonates and young children. [Abstract/Link to Full Text]

Schauvliege R, Janssens S, Beyaert R
Pellino proteins: novel players in TLR and IL-1R signalling.
J Cell Mol Med. 2007 May-Jun;11(3):453-61.
Members of the Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) family play important roles in immunity and inflammation. They initiate common intracellular signalling cascades leading to the activation of nuclear factor-?B (NF-?B) and other transcription factors that stimulate the expression of a variety of genes that shape an appropriate immune response. TLR/IL-1R signalling involves multiple proteinprotein interactions, but the mechanisms that regulate these interactions are still largely unclear. In this context, Pellino proteins have been suggested to function as evolutionary conserved scaffold proteins in TLR/IL-1R signalling. However, recently Pellino proteins were also proposed to function as novel ubiquitin ligases for IL-1R associated kinase 1 (IRAK-1). Here we review our current knowledge on the expression, biological role and mechanism of action of Pellino proteins in TLR/IL-1R-induced signalling. [Abstract/Link to Full Text]

Vreys V, David G
Mammalian heparanase: what is the message?
J Cell Mol Med. 2007 May-Jun;11(3):427-52.
Heparan sulphate proteoglycans are ubiquitous macromolecules of cell surfaces and extracellular matrices. Numerous extracellular matrix proteins, growth factors, morphogens, cytokines, chemokines and coagulation factors are bound and regulated by heparan sulphate. Degradation of heparan sulphate thus potentially profoundly affects cell and tissue function. Although there is evidence that several heparan sulphate-degrading endoglucuronidases (heparanases) might exist, so far only one transcript encoding a functional heparanase has been identified: heparanase-1. In the first part of this review, we discuss the current knowledge about heparan sulphate proteoglycans and the functional importance of their versatile interactions. In the second part, we summarize recent findings that have contributed to the characterization of heparanase-1, focusing on the molecular properties, working mechanism, substrate specificity, expression pattern, cellular activation and localization of this enzyme. Additionally, we review data implicating heparanase-1 in several normal and pathological processes, focusing on tumour metastasis and angiogenesis, and on evidence for a potentially direct signalling function of the molecule. In that context, we also briefly discuss heparanase-2, an intriguing close homologue of heparanase-1, for which, so far, no heparan sulphate-degrading activity could be demonstrated. [Abstract/Link to Full Text]

Schröder R, Vrabie A, Goebel HH
Primary desminopathies.
J Cell Mol Med. 2007 May-Jun;11(3):416-26.
Mutations of the human desmin gene on chromosome 2q35 cause a familial or sporadic form of skeletal myopathy frequently associated with cardiac abnormalities. Skeletal and cardiac muscle from patients with primary desminopathies characteristically display cytoplasmic accumulation of desmin-immunoreactive material and myofibrillar changes. However, desmin-positive protein aggregates in conjunction with myofibrillar abnormalities are also the morphological hallmark of the large group of secondary desminopathies (synonyms: myofibrillar myopathies, desmin-related myopathies), which comprise sporadic and familial neuromuscular conditions of considerable clinical and genetic heterogeneity. Here, we will give an overview on the functional role of desmin in striated muscle as well as the main clinical, myopathological, genetic and patho-physiological aspects of primary desminopathies. Furthermore, we will discuss recent genetic and biochemical advances in distinguishing primary from secondary desminopathies. [Abstract/Link to Full Text]

Hermann M, Margreiter R, Hengster P
Molecular and cellular key players in human islet transplantation.
J Cell Mol Med. 2007 May-Jun;11(3):398-415.
Human islet transplantation could represent an attractive alternative to insulin injections for the treatment of diabetes type 1. However, such an approach requires a better understanding of the molecular and cellular switches controlling ?-cell function in general as well as after transplantation into the liver. Although much research has been done into the suitability of stem or progenitor cells to generate a limitless supply of human ?-cells, a reproducible and efficient protocol for the differentiation of such cells into stably insulin-secreting ?-cells suitable for transplantation has yet to be reported. Fueled by recent findings showing that mature ?-cells are able to regenerate, many efforts have been undertaken to expand this cell pool. Unfortunately, also these approaches had problems to yield sufficiently differentiated human islet cells. The aim of this review is to summarize recent findings describing some of the molecular and cellular key players of islet biology. A more complete understanding of their orchestration and the use of new methods such as real time confocal imaging for the assessment of islet quality may yield the necessary advancements for more successful human islet transplantation. [Abstract/Link to Full Text]

Paknikar KM
Landmark discoveries in intracellular transport and secretion.
J Cell Mol Med. 2007 May-Jun;11(3):393-7.
Cellular protein transport and secretion is fundamental to the very existence of an organism, regulating important physiological functions such as reproduction, digestion, energy production, growth, neurotransmission, hormone release, water and ion transport, etc., all required for the survival and maintenance of homeostasis within an organism. Molecular understanding of transport and secretion of intracellular product has therefore been of paramount importance and aggressively investigated for over six decades. Only in the last 20 years, the general molecular mechanism of the process has come to light, following discovery of key proteins involved in ER-Golgi transport, and discovery of the porosome the universal secretion machinery in cells. [Abstract/Link to Full Text]

Reid PC, Urano Y, Kodama T, Hamakubo T
Alzheimer's disease: cholesterol, membrane rafts, isoprenoids and statins.
J Cell Mol Med. 2007 May-Jun;11(3):383-92.
Alzheimer's disease (AD) is a heterogeneous neurodegenerative disorder and the most prevalent form of dementia worldwide. AD is characterized pathologically by amyloid-? plaques, neurofibrillary tangles and neuronal loss, and clinically by a progressive loss of cognitive abilities. At present, the fundamental molecular mechanisms underlying the disease are unclear and no treatment for AD is known. Epidemiological evidence continues to mount linking vascular diseases, such as hypertension and diabetes, and hypercholesterolaemia with an increased risk for developing AD. A growing amount of evidence suggests a mechanistic link between cholesterol metabolism in the brain and the formation of amyloid plaques in AD development. Cholesterol and statins clearly modulate ?-amyloid precursor protein (?APP) processing in cell culture and animal models. Statins not only reduce endogenous cholesterol synthesis but also exert other various pleiotrophic effects, such as the reduction in protein isoprenylation. Through these effects statins modulate a variety of cellular functions involving both cholesterol (and membrane rafts) and isoprenylation. Although clearly other factors, such as vascular inflammation, oxidative stress and genetic factors, are intimately linked with the progression of AD, this review focuses on the present research findings describing the effect of cholesterol, membrane rafts and isoprenylation in regulating ?APP processing and in particular ?-secretase complex assembly and function and AD progression, along with consideration for the potential role statins may play in modulating these events. [Abstract/Link to Full Text]

Medina MA, Muńoz-Chápuli R, Quesada AR
Challenges of antiangiogenic cancer therapy: trials and errors, and renewed hope.
J Cell Mol Med. 2007 May-Jun;11(3):374-82.
Angiogenesis inhibition has been proposed as a general strategy to fight cancer. However, in spite of the promising preclinical results, a first generation of antiangiogenic compounds yielded poor results in clinical trials. Conceptual errors and mistakes in the design of trials and in the definition of clinical end-points could account for these negative results. In this context of discouraging results, a second generation of antiangiogenic therapies is showing positive results in phases II and III trials at the beginning of the twenty-first century. In fact, several combined treatments with conventional chemotherapy and antiangiogenic compounds have been recently approved. The discovery and pharmacological development of future generations of angiogenesis inhibitors will benefit from further advances in the understanding of the mechanisms involved in human angiogenesis. New styles of trials are necessary, to avoid missing potential therapeutic effects. Different clinical end-points, new surrogate biomarkers and methods of imaging will be helpful in this process. Real efficacy in clinical trials may come with the combined use of antiangiogenic agents with conventional chemotherapy or radiotherapy, and combinations of several antiangiogenic compounds with different mechanisms of action. Finally, the existing antiangiogenic strategies should include other approaches such as vascular targeting or angioprevention. [Abstract/Link to Full Text]

Mihai S, Sitaru C
Immunopathology and molecular diagnosis of autoimmune bullous diseases.
J Cell Mol Med. 2007 May-Jun;11(3):462-81.
Autoimmune bullous diseases are associated with autoimmunity against structural components maintaining cell-cell and cell matrix adhesion in the skin and mucous membranes. Pemphigus diseases are characterized by autoantibodies against the intercellular junctions and intraepithelial blisters. In pemphigoid diseases and epidermolysis bullosa acquisita, sub-epidermal blistering is associated with autoantibodies targeting proteins of the hemidesmosomal anchoring complex. The autoantigens in autoimmune blistering diseases have been extensively characterized over the past three decades. In general, the pathogenicity of autoantibodies, already suggested by clinical observations, has been conclusively demonstrated experimentally. Detection of tissue-bound and circulating serum autoantibodies and characterization of their molecular specificity is mandatory for the diagnosis of autoimmune blistering diseases. For this purpose, various immunofluorescence methods as well as immunoassays, including immunoblotting, enzyme-linked immunosorbent assay and immunoprecipitation have been developed. This review article describes the immunopathological features of autoimmune bullous diseases and the immunological and molecular tests used for their diagnosis and monitoring. [Abstract/Link to Full Text]

Kanda S, Kanetake H, Miyata Y
Downregulation of Fes inhibits VEGF-A-induced chemotaxis and capillary-like morphogenesis by cultured endothelial cells.
J Cell Mol Med. 2007 May-Jun;11(3):495-501.
The aim of this study was to determine whether the downregulation of endogenous Fes by siRNA in cultured endothelial cells affects vascular endothelial growth factor-A (VEGF-A)-induced chemotaxis and capillary-like morphogenesis, which are considered as angiogenic cellular responses in vitro. VEGF-A-treatment induced autophosphorylation of Fes in cultured endothelial cells. LY294002, a phosphoinositide 3-kinase inhibitor, significantly inhibited VEGF-A-induced chemotaxis and capillary-like morphogenesis. Downregulation of Fes attenuated these VEGF-A-induced cellular responses but LY294002 did not produce further inhibition of these responses. Downregulation of Fes neither affected VEGF-A-induced autophosphorylation of VEGF receptor 2 nor mitogen-activated protein kinase activation, but markedly decreased Akt activation. Taken together, our novel results indicate the involvement of Fes in VEGF-A-induced cellular responses by cultured endothelial cells. [Abstract/Link to Full Text]

Paslaru L, Davidson S, Popescu I, Morange M
The effect of Cyclosporine A on cardiomyocytes differentiation.
J Cell Mol Med. 2007 Mar-Apr;11(2):369-71.
Cyclosporine A (CsA) is a powerful immunosuppressive drug which significantly improved the success of organ transplantation; however, the major limiting factors for the drug's clinical use are its long and short term adverse effects. The present study was conducted to examine, in a dose-dependent manner, in a model of cardiogenesis, the effect of CsA on cardiomyocytes differentiation. [Abstract/Link to Full Text]

Pistol G, Matache C, Calugaru A, Stavaru C, Tanaseanu S, Ionescu R, Dumitrache S, Stefanescu M
Roles of CD147 on T lymphocytes activation and MMP-9 secretion in systemic lupus erythematosus.
J Cell Mol Med. 2007 Mar-Apr;11(2):339-48.
The cellular and molecular mechanisms involved in many abnormalities described in Systemic Lupus Erythematosus (SLE) are still unclear. Some of these abnormalities referred to the hyperactivation of T lymphocytes and the enhanced secretion of MMP-9 by peripheral blood mononuclear cells (PBMCs). Therefore, in this paper we investigated the potential role of CD147 molecule in these abnormalities. Our results demonstrated that CD147 molecule is overexpressed on CD3+T lymphocytes from SLE patients when compared with CD3+T lymphocytes from healthy donors. Monoclonal anti-CD147 antibodies, MEM-M6/1 clone, were able to inhibit protein tyrosine phosphorylation only in CD3 x CD28 costimulated T lymphocytes from SLE patients. However, this monoclonal antibody was unable to inhibit the enhanced activity of MMP-9 secreted by SLE PBMCs. [Abstract/Link to Full Text]

Griffoni C, Spisni E, Strillacci A, Toni M, Bachschmid MM, Tomasi V
Selective inhibition of prostacyclin synthase activity by rofecoxib.
J Cell Mol Med. 2007 Mar-Apr;11(2):327-38.
The development of cyclooxygenase-2 (COX-2) selective inhibitors prompted studies aimed at treating chronic inflammatory diseases and cancer by using this new generation of drugs.Yet, several recent reports pointed out that long-term treatment of patients with COX-2 selective inhibitors (especially rofecoxib) caused severe cardiovascular complicances. The aim of this study was to ascertain whether, in addition to inhibiting COX-2, rofecoxib may also affect prostacyclin (PGI2) level by inhibiting PGI2 forming enzyme (prostacyclin synthase, PGIS). In order to evaluate if selective (celecoxib, rofecoxib) and non-selective (aspirin, naproxen) anti-inflammatory compounds could decrease PGI2 production in endothelial cells by inhibiting PGIS, we analyzed the effect of anti-inflammatory compounds on the enzyme activity by ELISA assay after addition of exogenous substrate, on PGIS protein levels by Western blotting and on its subcellular distribution by confocal microscopy. We also analyzed the effect of rofecoxib on PGIS activity in bovine aortic microsomal fractions enriched in PGIS. This study demonstrates an inhibitory effect of rofecoxib on PGIS activity in human umbilical vein endothelial (HUVE) cells and in PGIS-enriched bovine aortic microsomal fractions, which is not observed by using other anti-inflammatory compounds. The inhibitory effect of rofecoxib is associated neither to a decrease of PGIS protein levels nor to an impairment of the enzyme intracellular localization. The results of this study may explain the absence of a clear relationship between COX-2 selectivity and cardiovascular side effects. Moreover, in the light of these results we propose that novel selective COX-2 inhibitors should be tested on PGI2 synthase activity inhibition. [Abstract/Link to Full Text]

Tamizhselvi R, Moore PK, Bhatia M
Hydrogen sulfide acts as a mediator of inflammation in acute pancreatitis: in vitro studies using isolated mouse pancreatic acinar cells.
J Cell Mol Med. 2007 Mar-Apr;11(2):315-26.
Hydrogen sulphide (H(2)S) is synthesized from L-cysteine via the action of cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS). We have earlier shown that H(2)S acts as a mediator of inflammation. However the mechanism remains unclear. In this study, we investigated the presence of H(2)S and the expression of H(2)S synthesizing enzymes, CSE and CBS, in isolated mouse pancreatic acini. Pancreatic acinar cells from mice were incubated with or without caerulein (10(-7) M for 30 and 60 min). Caerulein increased the levels of H(2)S and CSE mRNA expression while CBS mRNA expression was decreased. In addition, cells pre-treated with DL-propargylglycine (PAG, 3 mM), a CSE inhibitor, reduced the formation of H(2)S in caerulein treated cells, suggesting that CSE may be the main enzyme involved in H(2)S formation in mouse acinar cells. Furthermore, substance P (SP) concentration in the acini and expression of SP gene (preprotachykinin-A, PPT-A) and neurokinin-1 receptor (NK-1R), the primary receptor for SP, are increased in secretagogue caerulein-treated acinar cells. Inhibition of endogenous production of H(2)S by PAG significantly suppressed SP concentration, PPT-A expression and NK1-R expression in the acini. To determine whether H(2)S itself provoked inflammation in acinar cells, the cells were treated with H(2)S donor drug, sodium hydrosulphide (NaHS), (10, 50 and 100 muM), that resulted in a significant increase in SP concentration and expression of PPT-A and NK1-R in acinar cells. These results suggest that the pro-inflammatory effect of H(2)S may be mediated by SP-NK-1R related pathway in mouse pancreatic acinar cells. [Abstract/Link to Full Text]

Zhang Z, Zhang Z, Fauser U, Artelt M, Burnet M, Schluesener HJ
FTY720 attenuates accumulation of EMAP-II+ and MHC-II+ monocytes in early lesions of rat traumatic brain injury.
J Cell Mol Med. 2007 Mar-Apr;11(2):307-14.
FTY720 (Fingolimod) is a novel type of immunosuppressive agent inhibiting lymphocyte egress from secondary lymphoid tissues thereby causing peripheral lymphopenia. FTY720 can inhibit macrophage infiltration into inflammatory lesions under pathological conditions. FTY720 has been clinically evaluated for prophylaxis of allograft rejection and treatment of multiple sclerosis, showing promising immunosuppressive effects. A robust inflammatory response after traumatic brain injury (TBI) plays an important role in the secondary or delayed injuries of TBI. Here we have investigated by immunohistochemistry in a rat TBI model the effects of FTY720 on early cell accumulation into the inflammatory tissue response and on expression of major histo-compatibility complex class II (MHC-II) and endothelial-monocyte activating polypeptide II (EMAP-II). Accumulation of MHC-II(+) or EMAP-II(+) cells became significant 1 day after injury and continuously increased during the early time periods. Further, double-staining experiments confirmed that the major cellular sources of MHC-II were reactive macrophages, however MHC-II(+) cells only constituted a small subpopulation of reactive macrophages. Immediately after TBI, peripheral administration of FTY720 (1 mg/kg in 1 mL saline, every second day) significantly attenuated the accumulation of MHC-II(+) macrophages from Day 1 to 4 and significantly attenuated the accumulation of EMAP-II(+) macrophages/microglia at Day 4. Our findings show that FTY720 attenuates early accumulation of EMAP-II(+) and MHC-II(+) reactive monocytes following TBI, indicating that FTY720 might be a drug candidate to inhibit brain inflammatory reaction following TBI. [Abstract/Link to Full Text]

Zhang X, Hamada J, Nishimoto A, Takahashi Y, Murai T, Tada M, Moriuchi T
HOXC6 and HOXC11 increase transcription of S100beta gene in BrdU-induced in vitro differentiation of GOTO neuroblastoma cells into Schwannian cells.
J Cell Mol Med. 2007 Mar-Apr;11(2):299-306.
HOX genes encode transcription factors that play a key role in morphogenesis and cell differentiation during embryogenesis of animals. Human neuroblastoma cells are known to be chemically induced to differentiate into neuronal or Schwannian cells. In the present study, we investigated the roles of HOX genes in differentiation of GOTO neuroblastoma cells into Schwannian cells.When GOTO cells were grown in the presence of 5-bromo-2'-deoxyuridine (BrdU), they increased the expressions of two HOX genes (HOXC6 and HOXC11) and marker genes for Schwannian cells (S100beta and myelin basic protein). Forced expression of HOXC11 alone or both HOXC6 isoform 1 and HOXC11 induced the expression of S100beta in GOTO cells. In transient transfection experiments, the overexpression of HOXC6 and HOXC11 transactivated the S100beta promoter-reporter construct. Taken together, our results suggest that HOXC6 and HOXC11 are associated with differentiation of GOTO cells into Schwannian cells through the transcriptional activation of S100beta gene. [Abstract/Link to Full Text]

Shaw MM, Gurr WK, McCrimmon RJ, Schorderet DF, Sherwin RS
5'AMP-activated protein kinase alpha deficiency enhances stress-induced apoptosis in BHK and PC12 cells.
J Cell Mol Med. 2007 Mar-Apr;11(2):286-98.
5'AMP-activated protein kinase (AMPK) activation occurs under a variety of stress conditions but the role of this enzyme in the promotion or inhibition of stress-induced cell death is unclear. To address this issue, we transformed two different cell lines with shRNA-expressing plasmids, targeting the alpha subunit of AMPK, and verified AMPKalpha downregulation. The cell lines were then stressed by exposure to medium without glucose (PC12 cells) or with the viral thymidine kinase-specific DNA replication inhibitors: acyclovir, penciclovir and ganciclovir (herpes simplex virus thymidine kinase-expressing Baby Hamster Kidney cells). In non-AMPK-downregulated cells, these stress treatments induced AMPK upregulation and phosphorylation, leaving open the question whether the association of AMPK activation with stress-induced cell death reflects a successful death-promoting or an ineffective death-inhibiting activity. In AMPKalpha-deficient cells (expressing AMPKalpha-specific shRNAs or treated with Compound C) exposure to low glucose medium or DNA replication inhibitors led to an enhancement of cell death, indicating that, under the conditions examined, the role of activated AMPK is not to promote, but to protect from or delay stress-induced cell death. [Abstract/Link to Full Text]

Voutsadakis IA
Pathogenesis of colorectal carcinoma and therapeutic implications: the roles of the ubiquitin-proteasome system and Cox-2.
J Cell Mol Med. 2007 Mar-Apr;11(2):252-85.
Pathways of the molecular pathogenesis of colorectal carcinoma have been extensively studied and molecular lesions during the development of the disease have been revealed. High up in the list of colorectal cancer lesions are APC (adenomatous polyposis coli), K-ras, Smad4 (or DPC4-deleted in pancreatic cancer 4) and p53 genes. All these molecules are part of important pathways for the regulation of cell proliferation and apoptosis and as a result perturbation of these processes lead to carcinogenesis. The ubiquitin-proteasome system (UPS) is comprised of a multi-unit cellular protease system that regulates several dozens of cell proteins after their ligation with the protein ubiquitin. Given that among these proteins are regulators of the cell cycle, apoptosis, angiogenesis, adhesion and cell signalling, this system plays a significant role in cell fate and carcinogenesis. UPS inhibition has been found to be a pre-requisite for apoptosis and is already clinically exploited with the proteasome inhibitor bortezomib in multiple myeloma. Cyclooxygenase-2 (Cox-2) is the inducible form of the enzyme that metabolizes the lipid arachidonic acid to prostaglandin H2, the first step of prostaglandins production. This enzyme is up-regulated in colorectal cancer and in several other cancers. Inhibition of Cox-2 by aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) has been found to inhibit proliferation of colorectal cancer cells and in epidemiologic studies has been shown to reduce colon polyp formation in genetically predisposed populations and in the general population. NSAIDs have also Cox-independent anti-proliferative effects. Targeted therapies, the result of increasingly understanding carcinogenesis in the molecular level, have entered the field of anti-neoplastic treatment and are used by themselves and in combination with chemotherapy drugs. Combinations of targeted drugs have started also to be investigated. This article reviews the molecular pathogenesis of colorectal cancer, the roles of UPS and Cox-2 in it and puts forward a rational for their combined inhibition in colorectal cancer treatment. [Abstract/Link to Full Text]

Pützer BM
E2F1 death pathways as targets for cancer therapy.
J Cell Mol Med. 2007 Mar-Apr;11(2):239-51.
Defects in apoptotic programs contribute to a number of human diseases, ranging from neurodegenerative disorders to malignancy, and treatment failure. The genetic basis for apoptosis implies that cell death can be disrupted by mutations, raising the intriguing possibility that cell numbers can be regulated by factors that influence cell survival. It is well documented that the E2F1 transcription factor is a key regulator of apoptotic programs. E2F1-induced cell death occurs via multiple pathways, some of which involve the tumour suppressor p53, and autonomous of p53. This has led to the opinion that E2F1 functions as a tumour surveillance factor, detecting aberrant proliferation and engaging apoptotic pathways to protect the organism from developing tumours. Frequently, novel players are discovered that expand the interpretation of apoptosis control by E2F1. This information will help to produce new strategies to exploit E2F1-induced apoptosis for therapeutic benefit. [Abstract/Link to Full Text]

Lundstrom K
Structural genomics and drug discovery.
J Cell Mol Med. 2007 Mar-Apr;11(2):224-38.
Structure determination has already proven useful for lead optimization and direct drug design. The number of high-resolution structures available in public databases today exceeds 30,000 and will definitely aid in structure-based drug design. Structural genomics approaches covering whole genomes, topologically similar proteins or gene families are great assets for further progress in the development of new drugs. However, membrane proteins representing 70% of current drug targets are poorly characterized structurally. The problems have been related to difficulties in obtaining large amount of recombinant membrane proteins as well as their purification and structure determination. Structural genomics has proven successful in developing new methods in areas from expression to structure determination by studying a large number of target proteins in parallel. [Abstract/Link to Full Text]

Bleiziffer O, Eriksson E, Yao F, Horch RE, Kneser U
Gene transfer strategies in tissue engineering.
J Cell Mol Med. 2007 Mar-Apr;11(2):206-23.
Aiming for regeneration of severed or lost parts of the body, the combined application of gene therapy and tissue engineering has received much attention by regenerative medicine. Techniques of molecular biology can enhance the regenerative potential of a biomaterial by co-delivery of therapeutic genes, and several different strategies have been used to achieve that goal. Possibilities for application are many-fold and have been investigated to regenerate tissues such as skin, cartilage, bone, nerve, liver, pancreas and blood vessels. This review discusses advantages and problems encountered with the different gene delivery strategies as far as they relate to tissue engineering, analyses the positive aspects of polymeric gene delivery from matrices and discusses advances and future challenges of gene transfer strategies in selected tissues. [Abstract/Link to Full Text]

Rhodes JM, Simons M
The extracellular matrix and blood vessel formation: not just a scaffold.
J Cell Mol Med. 2007 Mar-Apr;11(2):176-205.
The extracellular matrix plays a number of important roles, among them providing structural support and information to cellular structures such as blood vessels imbedded within it. As more complex organisms have evolved, the matrix ability to direct signalling towards the vasculature and remodel in response to signalling from the vasculature has assumed progressively greater importance. This review will focus on the molecules of the extracellular matrix, specifically relating to vessel formation and their ability to signal to the surrounding cells to initiate or terminate processes involved in blood vessel formation. [Abstract/Link to Full Text]

Ranieri G, Grammatica L, Patruno R, Zito AF, Valerio P, Iacobellis S, Gadaleta C, Gasparini G, Ribatti D
A possible role of thymidine phosphorylase expression and 5-fluorouracil increased sensitivity in oropharyngeal cancer patients.
J Cell Mol Med. 2007 Mar-Apr;11(2):362-8.
Thymidine Pi deoxyribosyltransferase (TP) is an enzyme involved in DNA synthesis up-regulated in tumours and it is also a pro-angiogenic factor. TP cannot activate capecitabine, because capecitabine first needs conversion by carboxylesterase and cytidine deaminase into 5-deoxy-fluorouridine. This compound can be activated by TP to 5-fluorouracil (5-FU). Although TP is not necessary for 5-FU toxicity, experimental data suggest that high levels of TP correlate with an enhanced response to 5-FU therapy. In this study, we have analysed by immunohistochemistry CD34, CD68 and TP positive cells in bioptic samples from 53 patients with T(1-3) N(0-1) M(0) oropharyngeal squamous cell carcinoma (OSC) and from 24 patients with non-dysplastic oropharyngeal leukoplakia (NDOLP). Results showed that the mean of TP-positive cells, CD68 positive macrophages and CD34 positive endothelial cells eval-uated as microvessel density (MVD) was significantly higher in OSC than in NDOLP. Moreover, at a median follow-up of 19 months, patients with TP expression and higher MVD showed a better survival rate as compared to those with low MVD, probably as a consequence of 5-FU-based therapy.We hypothesized a role for TP in oropharyngeal tumourigenesis and 5-FU activation in the adjuvant setting of OSC patients. [Abstract/Link to Full Text]

Okamoto K, Ocker M, Neureiter D, Dietze O, Zopf S, Hahn EG, Herold C
bcl-2-specific siRNAs restore gemcitabine sensitivity in human pancreatic cancer cells.
J Cell Mol Med. 2007 Mar-Apr;11(2):349-61.
Gemcitabine has been shown to ameliorate disease related symptoms and to prolong overall survival in pancreatic cancer.Yet, resistance to Gemcitabine is commonly observed in this tumour entity and has been linked to increased expression of anti-apoptotic bcl-2. We therefore investigated if and to what extend silencing of bcl-2 by specific siRNAs (siBCL2) might enhance Gemcitabine effects in human pancreatic carcinoma cells. siBCL2 was transfected into the pancreatic cancer cell line YAP C alone and 72 hrs before co-incubation with different concentrations of Gemcitabine. Total protein and RNA were extracted for Western-blot analysis and quantitative polymerase chain reaction. Pancreatic cancer xenografts in male nude mice were treated intraperitoneally with siBCL2 alone, Gemcitabine and control siRNA or Gemcitabine and siBCL2 for 21 days. Combination of both methods lead to a synergistic induction of apoptosis at otherwise ineffective concentrations of Gemcitabine. Tumour growth suppression was also potentiated by the combined treatment with siBCL2 and Gemcitabine in vivo and lead to increased TUNEL positivity. In contrast, non-transformed human foreskin fibroblasts showed only minor responses to this treatment. Our results demonstrate that siRNA-mediated silencing of anti-apoptotic bcl-2 enhances chemotherapy sensitivity in human pancreatic cancer cells in vitro and might lead to improved therapy responses in advanced stages of this disease. [Abstract/Link to Full Text]

Manoach M, Tribulova N, Vogelezang D, Thomas S, Podzuweit T
Transient ventricular fibrillation and myosin heavy chain isoform profile.
J Cell Mol Med. 2007 Jan-Feb;11(1):171-4.
The present paper deals with spontaneous ventricular defibrillation in mammals and the possibility to facilitate its occurrence. Clinical and experimental evidence suggest that in the majority of cases, ventricular fibrillation (VF) is permanent, requiring defibrillation by electric shock. However, a growing number of reports show that VF can terminate spontaneously in various mammals, including human beings.The mechanisms involved in spontaneous ventricular defibrillation are controversial. Available reports imply that intracellular Ca2+ overload is the key event triggering VF and preventing its reversal. Since the sarcoplasmatic reticulum is the main intracellular Ca2+ regulating organelle and the activity of the cardiac SR Ca2+ ATPase (SERCA 2a) is its prime element of Ca2+ sequestration, spontaneous ventricular defibrillation likely requires high level of SERCA 2a activity. We suggest that mammalian hearts with high SERCA 2a activity defibrillate spontaneously and those with low activity only after its enhancement. Since high SERCA 2a activity is co-expressed with the myosin heavy chain (MHC) isoform V1, we assumed that those hearts preferentially expressing V1 MHC are able to defibrillate spontaneously. Hearts with small amounts of V1 MHC and correspondingly lower level of SERCA 2a activity can only defibrillate following administration of compounds that augment SERCA 2a activity and prevent intracellular Ca2+ overload. [Abstract/Link to Full Text]

Neri M, Cerretani D, Fiaschi AI, Laghi PF, Lazzerini PE, Maffione AB, Micheli L, Bruni G, Nencini C, Giorgi G, D'Errico S, Fiore C, Pomara C, Riezzo I, Turillazzi E, Fineschi V
Correlation between cardiac oxidative stress and myocardial pathology due to acute and chronic norepinephrine administration in rats.
J Cell Mol Med. 2007 Jan-Feb;11(1):156-70.
BACKGROUND: To investigate the cardiotoxic role of reactive oxygen species (ROS) and of products derived from catecholamines auto-oxidation, we studied: (1) the response of antioxidant cardiac cellular defence systems to oxidative stress induced by norepinephrine (NE) administration, (2) the effect of NE administration on cardiac beta1-adrenergic receptors by means of receptor binding assay, (3) the cellular morphological alterations related to the biologically cross-talk between the NE administration and cytokines [tumor necrosis factor-alpha (TNF-alpha), monocyte chemotactic protein-1 (MCP-1), interleukins IL6, IL8, IL10]. METHODS AND RESULTS: A total of 195 male rats was used in the experiment. All animals underwent electrocardiogram (EKG) before being sacrificed. The results obtained show that NE administration influences the antioxidant cellular defence system significantly increasing glutathione peroxidase (GPx) activity, glutathione reductase (GR) and superoxide dismutase (SOD). The oxidized glutathione (GSH/GSSG) ratio significantly decreases and malondialdehyde (MDA) levels increase showing a state of lipoperoxidation of cardiac tissue. We describe a significant apoptotic process randomly sparse in the damaged myocardium and the effect of ROS on the NE-mediated TNF-alpha, MCP-1, and IL6, IL8, IL10 production. CONCLUSIONS: Our results support the hypothesis that catecholamines may induce oxidative damage through reactive intermediates resulting from their auto-oxidation, irrespective of their interaction with adrenergic receptors, thus representing an important factor in the pathogenesis of catecholamines-induced cardiotoxicity. The rise of the cardioinhibitory cytokines may be interpreted as the adaptive response of jeopardized myocardium with respect to the cardiac dysfunction resulting from NE injection. [Abstract/Link to Full Text]

Kriebardis AG, Antonelou MH, Stamoulis KE, Economou-Petersen E, Margaritis LH, Papassideri IS
Progressive oxidation of cytoskeletal proteins and accumulation of denatured hemoglobin in stored red cells.
J Cell Mol Med. 2007 Jan-Feb;11(1):148-55.
Red blood cell (RBC) membrane proteins undergo progressive pathological alterations during storage. In conditions of increased cellular stress, the cytoskeleton also sustains certain modifications. The hemoglobin (Hb) content and oxidative status of the RBC cytoskeletons as a function of the storage period remain unclear. The possible Hb content and oxidative alterations occurring in the cytoskeletons in the course of storage were monitored in six units, by means of electrophoresis, immunoblotting and protein carbonylation assays. A proportion of the ghost-bound Hb consists of non-reducible crosslinkings of probably oxidized(denatured Hb or hemichromes.The defective Hb-membrane association was strongly affected by the prolonged storage. A progressive accumulation of Hb monomers, multimers and high molecular weight aggregates to corresponding cytoskeletons were also evident. The oxidative index of the cytoskeletal proteins was found increased, signalizing oxidative modifications in spectrin and possibly other cytoskeletal proteins. The reported data corroborate the evidence for oxidative damage in membrane proteins with emphasis to the cytoskeletal components. They partially address the pathophysiological mechanisms underlying the RBC storage lesion, add some new insight in the field of RBC storage as a hemoglobin- and cytoskeleton-associated pathology and suggest the possible use of antioxidants in the units intended for transfusion. [Abstract/Link to Full Text]

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Recent Articles in Molecules and Cells

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Jin EJ, Lee SY, Choi YA, Jung JC, Bang OS, Kang SS
BMP-2-enhanced chondrogenesis involves p38 MAPK-mediated down-regulation of Wnt-7a pathway.
Mol Cells. 2006 Dec 31;22(3):353-9.
The bone morphogenetic protein (BMP) family has been implicated in control of cartilage development. Here, we demonstrate that BMP-2 promotes chondrogenesis by activating p38 mitogen-activated protein kinase (MAPK), which in turn downregulates Wnt-7a/b-catenin signaling responsible for proteasomal degradation of Sox9. Exposure of mesenchymal cells to BMP-2 resulted in upregulation of Sox9 protein and a concomitant decrease in the level of b-catenin protein and Wnt-7a signaling. In agreement with this, the interaction of Sox9 with b-catenin was inhibited in the presence of BMP-2. Inhibition of the p38 MAPK pathway using a dominant negative mutant led to sustained Wnt-7a signaling and decreased Sox9 expression, with consequent inhibition of precartilage condensation and chondrogenic differentiation. Moreover, overexpression of b-catenin caused degradation of Sox9 via the ubiquitin/26S proteasome pathway. Our results collectively indicate that the increase in Sox9 protein resulting from downregulation of b-catenin/Wnt-7a signaling is mediated by p38 MAPK during BMP-2 induced chondrogenesis in chick wing bud mesenchymal cells. [Abstract/Link to Full Text]

Shin DJ, Kang JY, Kim YU, Yoon JS, Choy HE, Maeda Y, Kinoshita T, Hong Y
Isolation of new CHO cell mutants defective in CMP-sialic acid biosynthesis and transport.
Mol Cells. 2006 Dec 31;22(3):343-52.
Sialic acid is a sugar typically found at the N-glycan termini of glycoproteins in mammalian cells. Lec3 CHO cell mutants are deficient in epimerase activity, due to a defect in the gene that encodes a bifunctional UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). Sialic acid modification on the cell surface is partially affected in these cells. We have mutagenized Lec3 CHO cells and isolated six mutants (termed C2m) deficient in the cell surface expression of polysialic acid (PSA). Mutant C2m9 was partially defective in expression of cell-surface PSA and wheat germ agglutinin (WGA) binding, while in the other five mutants, both cell-surface PSA and WGA binding were undetectable. PSA expression was restored by complementation with the gene encoding the CMP-sialic acid transporter (CST), indicating that CST mutations were responsible for the phenotypes of the C2m cells. We characterized the CST mutations in these cells by Northern blotting and RT-PCR. C2m9 and C2m45 carried missense mutations resulting in glycine to glutamate substitutions at amino acids 217 (G217E) and 256 (G256E), respectively. C2m13, C2m39 and C2m31 had nonsense mutations that resulted in decreased CST mRNA stability, and C2m34 carried a putative splice site mutation. PSA and CD15s expression in CST-deficient Lec2 cells were partially rescued by G217E CST, but not by G256E CST, although both proteins were expressed at similar levels, and localized to the Golgi. These results indicate that the novel missense mutations isolated in this study affect CST activity. [Abstract/Link to Full Text]

Shin NR, Shin JH, Chun JH, Yoon SY, Kim BS, Oh HB, Rhie GE
Determination of neurotoxin gene expression in Clostridium botulinum type A by quantitative RT-PCR.
Mol Cells. 2006 Dec 31;22(3):336-42.
Real time reverse transcription (RT)-PCR was used to quantify the expression of the botulinum neurotoxin type A (BoNT/A) gene (cntA) by normalization with the expression of 16S rRNA. The method were confirmed by monitoring the mRNA levels of cntA during growth in five type A strains. In all but one of the strains the expression of cntA mRNA was maximal in the late exponential phase, and approximately 35-fold greater than in the early exponential phase. The concentration of the extracellular BoNT/A complex detected by ELISA was highest in stationary phase. Sodium nitrite and sorbic acid completely inhibited growth at 20 ppm and 4 mg ml-1, respectively. CntA expression became lower in proportion to the concentration of sorbic acid, and this reduction was confirmed by mouse bioassay. Our results show that real time RT-PCR can be used to quantify levels of C. botulinum type A neurotoxin transcripts and to assess the effects of food additives on botulinal risk. [Abstract/Link to Full Text]

Seo IR, Choi MR, Park CS, Kim do H
Effects of recombinant imperatoxin A (IpTxa) mutants on the rabbit ryanodine receptor.
Mol Cells. 2006 Dec 31;22(3):328-35.
Imperatoxin A (IpTxa), a 3.7 kDa peptide from the African scorpion Pandinus imperator, is an agonist of the skeletal muscle ryanodine receptor (RyR1). In order to study the structure of the toxin and its effect on RyR1, IpTxa cDNA was PCR-amplified using 3 pairs of primers, and the toxin was expressed in E. coli. The toxin was further purified by chromatography, and various point mutants in which basic amino acids were substituted by alanine were prepared by site-directed mutagenesis. Studies of single channel properties by the planar lipid bilayer method showed that the recombinant IpTxa was identical to the synthetic IpTxa with respect to high-performance liquid chromatography mobility, amino acid composition and specific effects on RyR1. Mutations of certain basic amino acids (Lys19, Arg23, and Arg33) dramatically reduced the capacity of the peptide to activate RyRs. A subconductance state predominated when Lys8 was substituted with alanine. These results suggest that some basic amino acid residues in IpTxa are important for activation of RyR1, and that Lys8 plays an important role in regulating the gating mode of RyR1. [Abstract/Link to Full Text]

Yoo HS, Eah JY, Kim JS, Kim YJ, Min MS, Paek WK, Lee H, Kim CB
DNA barcoding Korean birds.
Mol Cells. 2006 Dec 31;22(3):323-7.
DNA barcoding, an inventory of DNA sequences from a standardized genomic region, provides a bio-barcode for identifying and discovering species. Several recent studies suggest that the sequence diversity in a 648 bp region of the mitochondrial gene for cytochrome c oxi- dase I (COI) might serve as a DNA barcode for identify- ing animal species such as North American birds, in- sects and fishes. The present study tested the effective- ness of a COI barcode in discriminating Korean bird species. We determined the 5' terminus of the COI bar- code for 92 species of Korean birds and found that spe- cies identification was unambiguous; the genetic differ- ences between closely related species were, on average, 25 times higher than the differences within species. We identified only one misidentified species out of 239 specimens in a genetic resource bank, so confirming the accuracy of species identification in the banking system. We also identified two potential composite species, calling for further investigation using more samples. The finding of large COI sequence differences between species confirms the effectiveness of COI barcodes for identifying Korean bird species. To bring greater reliability to the identification of species, increased in- tra- and interspecies sampling, as well as supplementa- tion of the mitochondrial barcodes with nuclear ones, is needed. [Abstract/Link to Full Text]

Lim JT, Hwang UW
The complete mitochondrial genome of Pollicipes mitella (Crustacea, Maxillopoda, Cirripedia): non-monophylies of maxillopoda and crustacea.
Mol Cells. 2006 Dec 31;22(3):314-22.
The whole mitochondrial genome (14,915 nt) of Pollicipes mitella (Crustacea, Maxillopoda, Cirripedia, Thoracica) was sequenced and characterized. It is the shortest of the 31 completely sequenced crustacean mitochondrial genomes, with the exception of a copepod Tigriopus japonicus (14,628 nt). It consists of the usual 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 relatively short non-coding region (294 nt). The thoracican cirripeds apart from Megabalanus volcano have the same arrangement of protein-coding genes as Limulus polypemus, but there are frequent tRNA gene translocations (at least 8). Some interesting translocation features that may be specific to the thoracican cirriped lineage are as follows: 1) trnK-trnQ lies between the control region and trnI, 2) trnA-trnE lies between trnN and trnS1, 3) trnP lies between ND4L and trnT, and 4) trnY-trnC lies between trnS2 and ND1. In P. mitella there are two trnL genes (L1 and L2) in the typical crustacean positions (ND1-L1-LrRNA and CO1-L2-CO2). The present result is compared and discussed with the other three cirriped mitochondrial genomes from one pedunculate (Pollicipes polymerus) and two sessiles (Tetraclita japonica and M. volcano) published so far. Mitochondrial protein phylogenies reconstructed by the BI and ML algorithms show that the thoracican Cirripedia is monophyletic (BPP 100/BP 100) and associated with Remipedia (BPP 98/BP 35). In addition, Oligostraca, including Ostracoda, Branchiura, and Pentastomida, is a monophyletic group (BPP 99/BP 68), and is basal to all the other examined arthropods. Remipedia + Cirripedia appears as an independent lineage within Arthropoda, apart from Thoracopoda (Malacostraca, Branchiopda, and Cephalocarida). The Thoracopoda is paraphyletic to Hexapoda. The present result suggests that the monophylies of Crustacea and Maxillopoda should be reconsidered. [Abstract/Link to Full Text]

Lili J, Yuchen S, Zhengtao W
The chemokine SDF-1alpha suppresses fibronectin-mediated in vitro lymphocytes adhesion.
Mol Cells. 2006 Dec 31;22(3):308-13.
Stromal cell-derived factor (SDF-1) is a CXC chemokine that selectively activates the CXCR4 chemokine receptor. Fibronectin is an intracellular matrix component that binds integrin and mediates cell-matrix adhesion. Activation of the integrin receptor can occur in two ways: by ligand binding (outside-in signaling), and in response to intracellular events (inside-out signaling). In the current study we showed that SDF-1a inhibited adhesion of T lymphocyte Jurkat cells resulting from binding high concentrations of fibronectin as well as that of THP-1 monocytes. The effect of SDF-1a on fibronectin-mediated adhesion was partly reversed by the CXCR4 receptor antagonist T140. Our results suggest that an SDF-1/ CXCR4 signal pathway modulates fibronectin-mediated lymphocytes adhesion. [Abstract/Link to Full Text]

Hong CP, Plaha P, Koo DH, Yang TJ, Choi SR, Lee YK, Uhm T, Bang JW, Edwards D, Bancroft I, Park BS, Lee J, Lim YP
A Survey of the Brassica rapa genome by BAC-end sequence analysis and comparison with Arabidopsis thaliana.
Mol Cells. 2006 Dec 31;22(3):300-7.
Brassica rapa ssp. pekinensis (Chinese cabbage) is an economically important crop and a model plant for studies on polyploidization and phenotypic evolution. To gain an insight into the structure of the B. rapa genome we analyzed 12,017 BAC-end sequences for the presence of transposable elements (TEs), SSRs, centromeric satellite repeats and genes, and similarity to the closely related genome of Arabidopsis thaliana. TEs were estimated to occupy 14% of the genome, with 12.3% of the genome represented by retrotransposons. It was estimated that the B. rapa genome contains 43,000 genes, 1.6 times greater than the genome of A. thaliana. A number of centromeric satellite sequences, representing variations of a 176-bp consensus sequence, were identified. This sequence has undergone rapid evolution within the B. rapa genome and has diverged among the related species of Brassicaceae. A study of SSRs demonstrated a non-random distribution with a greater abundance within predicted intergenic regions. Our results provide an initial characterization of the genome of B. rapa and provide the basis for detailed analysis through whole-genome sequencing. [Abstract/Link to Full Text]

Choi HJ, Eun JS, Kim BG, Kim SY, Jeon H, Soh Y
Vitexin, an HIF-1alpha inhibitor, has anti-metastatic potential in PC12 cells.
Mol Cells. 2006 Dec 31;22(3):291-9.
Vitexin, a natural flavonoid compound identified as apigenin-8-C-b-D-glucopyranoside, has been reported to exhibit antioxidative and anti-inflammatory properties. In this study, we investigated its effect on hypoxia-inducible factor-1a (HIF-1a) in rat pheochromacytoma (PC12), human osteosarcoma (HOS) and human hepatoma (HepG2) cells. Vitexin inhibited HIF-1a in PC12 cells, but not in HOS or HepG2 cells. In addition, it diminished the mRNA levels of hypoxia-inducible genes such as vascular endothelial growth factor (VEGF), smad3, aldolase A, enolase 1, and collagen type III in the PC12 cells. We found that vitexin inhibited the migration of PC12 cells as well as their invasion rates, and it also inhibited tube formation by human umbilical vein endothelium cells (HUVECs). Interestingly, vitexin inhibited the hypoxia-induced activation of c-jun N-terminal kinase (JNK), but not of extracellular-signal regulated protein kinase (ERK), implying that it acts in part via the JNK pathway. Overall, these results suggest the potential use of vitexin as a treatment for diseases such as cancer. [Abstract/Link to Full Text]

Park K, Kang HM
Circadian expression of clock genes in the rat eye and brain.
Mol Cells. 2006 Dec 31;22(3):285-90.
The light sensing system in the eye directly affects the circadian oscillator in the mammalian suprachiasmatic nucleus (SCN). To investigate this relationship in the rat, we examined the circadian expression of clock genes in the SCN and eye tissue during a 24 h day/night cycle. In the SCN, rPer1 and rPer2 mRNAs were expressed in a clear circadian rhythm like rCry1 and rCry2 mRNAs, whereas the level of BMAL1 and CLOCK mRNAs decreased during the day and increased during the night with a relatively low amplitude. It seems that the clock genes of the SCN may function in response to a master clock oscillation in the rat. In the eye, the rCry1 and rCry2 were expressed in a circadian rhythm with an increase during subjective day and a decrease during subjective night. However, the expression of Opn4 mRNA did not exhibit a clear circadian pattern, although its expression was higher in daytime than at night. This suggests that cryptochromes located in the eye, rather than melanopsin, are the major photoreceptive system for synchronizing the circadian rhythm of the SCN in the rat. [Abstract/Link to Full Text]

Bae K, Lee K, Seo Y, Lee H, Kim D, Choi I
Differential effects of two period genes on the physiology and proteomic profiles of mouse anterior tibialis muscles.
Mol Cells. 2006 Dec 31;22(3):275-84.
The molecular components that generate and maintain circadian rhythms of physiology and behavior in mammals are present both in the brain (suprachiasmatic nucleus; SCN) and in peripheral tissues. Examination of mice with targeted disruptions of either mPer1 or mPer2 has shown that these two genes have key roles in the SCN circadian clock. Here we show that loss of the clock gene mPer2 affects forced locomotor performance in mice without altering muscle contractility. A proteomic analysis revealed that the anterior tibialis muscles of the mPer2 knockout mice had higher levels of glycolytic enzymes such as triose phosphate isomerase and enolase than those of either the wild type or mPer1 knockout mice. In addition, the level of expression of HSP90 in the mPer2 mutant mice was also significantly higher than in wildtype mice. These results suggest that the reduced locomotor endurance of the mPer2 knockout mice reflects a greater dependence on anaerobic metabolism under stress conditions, and that the two canonical clock genes, mPer1 and mPer2, play distinct roles in the physiology of skeletal muscle. [Abstract/Link to Full Text]

Kim YS, Cho JH, Ahn J, Hwang B
Upregulation of isoprenoid pathway genes during enhanced saikosaponin biosynthesis in the hairy roots of Bupleurum falcatum.
Mol Cells. 2006 Dec 31;22(3):269-74.
In order to characterize saikosaponin biosynthesis in Bupleurum falcatum, the expression of five isoprenoid pathway genes and their relationship to saikosaponin accumulation in the hairy roots were analyzed. The hairy roots exhibited a rapid accumulation of saikosaponins when incubated in a root culture medium (3XRCM). Homology-based RT-PCR was used to isolate core fragments of five genes, HMGR, IPPI, FPS, SS, and OSC, from the hairy roots. The deduced amino acid sequences exhibited amino acid identities of more than 85% to previously reported genes. Using the fragments as probes, the expression of these five genes in the hairy roots during incubation in 3XRCM medium was examined. Expression of all five genes in the hairy roots increased soon after incubation. In particular, the SS and OSC genes were coordinately induced at 8 days of incubation, and their expression persisted throughout the incubation period. A quantitative HPLC analysis showed that the saikosaponin content of the hairy root culture also began to increase at 8 days of culture. The correlation between SS transcript level and saikosaponin content in the hairy roots suggests that transcriptional regulation plays a regulatory role in saikosaponin biosynthesis. [Abstract/Link to Full Text]

Kim HS, Cheon YP
Spatio-temporal expression and regulation of dermatopontin in the early pregnant mouse uterus.
Mol Cells. 2006 Dec 31;22(3):262-8.
During endometrial differentiation the extracellular matrix (ECM) changes dramatically to prepare for implantation of the embryo. However, the genes regulating the ECM build-up in the uterine endometrium during early pregnancy are not well known. Using the PCR-select cDNA subtraction method, dermatopontin was identified in the uterus of a pregnant mouse on day 4 of gestation. Dermatopontin mRNA increased dramatically on day 3, and was at its highest level at the time of implantation. Administration of RU 486 significantly inhibited mRNA expression by day 4 of gestation, but ICI 182,780 did not. Progesterone markedly induced dermatopontin expression in ovariectomized uteri within 4 h of administration, whereas estrogen had little effect. In silico analysis revealed progesterone receptor binding sites in the dermatopontin promoter region. Decidualization did not induce expression of dermatopontin; instead dermatopontin mRNA became strongly localized at the interimplantation site. In situ hybridization revealed that expression gradually decreased in the luminal epithelial cells as pregnancy progressed, whereas it increased in the stromal cells. The pattern of localization and the changes of intensity of dermatopontin mRNA coincided with those of collagen. Collectively, these results strongly suggest that dermatopontin expression is steroid-dependent. They also suggest that, at the time of implantation, dermatopontin expression is primarily regulated spatio-temporally by progesterone via progesterone receptors, and is modulated by the decidual response during implantation. Dermatopontin may be one of the regulators used to remodel the uterine ECM for pregnancy. [Abstract/Link to Full Text]

Do JH, Choi DK
Normalization of microarray data: single-labeled and dual-labeled arrays.
Mol Cells. 2006 Dec 31;22(3):254-61.
DNA microarray is a powerful tool for high-throughput analysis of biological systems. Various computational tools have been created to facilitate the analysis of the large volume of data produced in DNA microarray experiments. Normalization is a critical step for obtaining data that are reliable and usable for subsequent analysis such as identification of differentially expressed genes and clustering. A variety of normalization methods have been proposed over the past few years, but no methods are still perfect. Various assumptions are often taken in the process of normalization. Therefore, the knowledge of underlying assumption and principle of normalization would be helpful for the correct analysis of microarray data. We present a review of normalization techniques from single-labeled platforms such as the Affymetrix GeneChip array to dual-labeled platforms like spotted array focusing on their principles and assumptions. [Abstract/Link to Full Text]

Kim KI, Baek SH
SUMOylation code in cancer development and metastasis.
Mol Cells. 2006 Dec 31;22(3):247-53.
Protein modification by small ubiquitin-like modifier (SUMO) controls diverse cellular functions of protein targets including transcription factors and coregulators mainly in the nucleus and participates in maintaining cellular homeostasis. In addition, SUMO system plays important roles in DNA damage repair and maintaining genome integrity. Thus, in some cases, the loss of control on SUMOylation or deSUMOylation processes causes a defect in maintaining homeostasis and hence gives a cue to cancer development. Furthermore, recent study showed that SUMO system is also involved in cancer metastasis. In this review, we will summarize and discuss the possible role of SUMO system in cancer development and metastasis. [Abstract/Link to Full Text]

Kook SH, Choi KC, Son YO, Lee KY, Hwang IH, Lee HJ, Chang JS, Choi IH, Lee JC
Satellite cells isolated from adult Hanwoo muscle can proliferate and differentiate into myoblasts and adipose-like cells.
Mol Cells. 2006 Oct 31;22(2):239-45.
This study examined whether adult bovine muscle satellite cells from 30-month-old Hanwoo cattle are multipotential. The satellite cells were found to have the potential to proliferate and differentiate into myoblasts with the formation of multinucleated cells. In addition, treatment with the peroxisome proliferator activating receptor-gamma (PPARgamma) agonist, rosiglitazone, promoted their trans-differentiation into adipocytes with significant increases in glycerol accumulation and glycerol-3-phosphate dehydrogenase activity. Western blot analysis revealed that increased levels of the adipocyte fatty acid-binding protein, PPARgamma and of CCAAT/enhancer-binding protein were closely related to rosiglitazone-induced differentiation of the cells. These findings demonstrate that satellite cells from adult Hanwoo cattle are multipotent, and that their trans-differentiation into adipocytes can be induced by rosiglitazone. [Abstract/Link to Full Text]

Song JT
Induction of a salicylic acid glucosyltransferase, AtSGT1, is an early disease response in Arabidopsis thaliana.
Mol Cells. 2006 Oct 31;22(2):233-8.
Endogenous salicylic acid (SA) and its predominant conjugates, SA 2-O-beta-D-glucoside (SAG) and the glucose ester of SA (SGE), increase dramatically during plant defense responses. Here I report the isolation and characterization of an Arabidopsis thaliana UDP-glucose:SA glucosyltransferase1 (AtSGT1) gene using a tobacco SGT gene previously reported, whose product catalyzes the formation of both SAG and SGE. The recombinant AtSGT1 protein had significant activities with SA and benzoic acid, and synthesized SAG and SGE. Northern blot analysis showed that AtSGT1 was rapidly induced both by exogenous SA and infection with the bacterial pathogen Pseudomonas syringae, indicating that pathogen-inducible AtSGT1 expression is an early disease response and may be involved in the accumulation of glucosyl SA during pathogenesis. [Abstract/Link to Full Text]

Kim J, Kim-Ha J
Ovarian tumors in Rbp9 mutants of Drosophila induce an immune response.
Mol Cells. 2006 Oct 31;22(2):228-32.
The Drosophila protein, Rbp9, is homologous to human Hu, which is reported to be involved in small cell lung cancer. Rbp9 functions in cystocyte differentiation, and mutations in Rbp9 cause ovarian tumors. Here we show that the antimicrobial peptide, Attacin, is upregulated in Rbp9 mutants, especially in ovaries where tumors form. Upregulation seems to result from activation of the NF-kappaB pathway since we detected nuclear localization of Relish in Rbp9 mutant ovaries but not in wild type ovaries. Inactivation of NF-kappaB in the Rbp9 mutant allows prolonged survival of malformed egg chambers. We conclude that Drosophila initiates an anti-tumor defense response via activation of NF-kappaB. [Abstract/Link to Full Text]

Kim NH, Jeong MS, Choi SY, Kang JH
Oxidative modification of cytochrome c by hydrogen peroxide.
Mol Cells. 2006 Oct 31;22(2):220-7.
Oxidative alteration of mitochondrial cytochrome c has been linked to disease and is one of the causes of pro-apoptotic events. We have investigated the modification of cytochrome c by H2O2. When cytochrome c was incubated with H2O2, oligomerization of the protein increased and the formation of carbonyl derivatives and dityrosine was stimulated. Radical scavengers prevented these effects suggesting that free radicals are implicated in the H2O2-mediated oligomerization. Oligomerization was significantly inhibited by the iron chelator, deferoxamine. During incubation of deoxyribose with cytochrome c and H2O2, damage to the deoxyribose occurred in parallel with the release of iron from cytochrome c. When cytochrome c that had been exposed to H2O2 was analyzed by amino acid analysis, the tyrosine, histidine and methionine residues proved to be particularly sensitive. These results suggest that H2O2-mediated cytochrome c oligomerization is due to oxidative damage resulting from free radicals generated by a combination of the peroxidase activity of cytochrome c and the Fenton reaction of free iron released from the oxidatively-damaged protein. [Abstract/Link to Full Text]

Lee MH, Lee SH, Kim H, Jin JB, Kim DH, Hwang I
A WD40 repeat protein, Arabidopsis Sec13 homolog 1, may play a role in vacuolar trafficking by controlling the membrane association of AtDRP2A.
Mol Cells. 2006 Oct 31;22(2):210-9.
Dynamin-related protein 2A (AtDRP2A, formally ADL6), a member of the dynamin family, is critical for protein trafficking from the TGN to the central vacuole. However, the mechanism controlling its activity is not well understood in plant cells. We isolated Arabidopsis sec13 homolog1 (AtSeh1) that interacts with AtDRP2A by a yeast two-hybrid screening. AtSeh1 has four WD40 motifs and amino acid sequence homology to Sec13, a component of COPII vesicles. Coimmunoprecipitation and protein pull-down experiments demonstrated specific interaction between AtSeh1 and AtDRP2A. AtSeh1 bound to the pleckstrin homology domain of AtDRP2A in competition with the C-terminal domain of the latter, and this resulted in inhibition of the interaction between AtDRP2A and PtdIns3P in vitro. AtSeh1 localized to multiple locations: the nucleus, the prevacuolar compartment and the Golgi complex. Based on these results we propose that AtSeh1 plays a role in regulating cycling of AtDRP2A between membrane-bound and soluble forms. [Abstract/Link to Full Text]

Park KA, Tanaka Y, Suenaga Y, Tamura TA
TATA-binding protein-related factor 2 is localized in the cytoplasm of mammalian cells and much of it migrates to the nucleus in response to genotoxic agents.
Mol Cells. 2006 Oct 31;22(2):203-9.
TBP (TATA-binding protein)-related factor 2 (TRF2) regulates transcription during a nuber of cellular processes. We previously demonstrated that it is localized in the cytoplasm and is translocated to the nucleus by DNA-damaging agents. However, the cytoplasmic localization of TRF2 is controversial. In this study, we reconfirmed its cytoplasmic localization in various ways and examined its nuclear migration. Stresses such as heat shock, redox agents, heavy metals, and osmotic shock did not affect localization whereas genotoxins such as methyl methanesulfonate (MMS), cisplatin, etoposide, and hydroxyurea caused it to migrate to the nucleus. Adriamycin, mitomycin C and gamma-rays had no obvious effect. We determined optimal conditions for the nuclear migration. The proportions of cells with nuclei enriched for TRF2 were 25-60% and 5-10% for stressed cells and control cells, respectively. Nuclear translocation was observed after 1 h, 4 h and 12 h for cisplatin, etoposide and MMS and hydroxyurea, respectively. The association of TRF2 with the chromatin and promoter region of the proliferating cell nuclear antigen (PCNA) gene, a putative target of TRF2, was increased by MMS treatment. Thus TRF2 may be involved in genotoxin-induced transcriptional regulation. [Abstract/Link to Full Text]

Lee SY, Jo S, Lee GE, Jeong LS, Kim YC, Park CS
Establishment of an assay for P2X7 receptor-mediated cell death.
Mol Cells. 2006 Oct 31;22(2):198-202.
The P2X7 receptor, an ATP-gated cation channel, induces cell death in immune cells and is involved in neurodegenerative diseases. Although the receptor plays various roles in these diseases, the cellular mechanisms involved are poorly understood and antagonists are limited. Here, the development of a cell-based assay for human P2X7 receptor is reported. We established permanent lines of HEK 293 cells expressing a high level of hP2X7 receptor. Functional activity of the hP2X7 receptor was confirmed by whole-cell patch recording of ATP-induced ion currents. Prolonged exposure to ATP resulted in death of the hP2X7-expressing HEK 293 cells and this cell death could be quantified. Two known P2X7 antagonists, PPADS and KN-62, blocked ATP-induced death in a concentration-dependent manner. Thus, this assay can be used to screen for new antagonists of hP2X7 receptors. [Abstract/Link to Full Text]

Lee JS, Yun BY, Kim SS, Cho C, Yoon YD, Cho BN
Changes in reproductive function and white blood cell proliferation induced in mice by injection of a prolactin-expressing plasmid into muscle.
Mol Cells. 2006 Oct 31;22(2):189-97.
Prolactin (PRL) is a pituitary hormone involved in various physiological processes, including lactation, mammary development, and immune function. To further investigate the in vivo and comparative endocrine roles of PRL, mouse PRL cDNA fused to the cytomegalovirus promoter, was introduced into muscle by direct injection. Previously we studied the function of rat PRL using the same protocol. PRL mRNA was detected in the muscle following injection by RT-PCR and subsequent Southern blot analysis. PRL was also detected and Western blot analysis revealed a relatively high level of serum PRL. In the pCMV-mPRL-injected female mice, the estrous cycle was extended, especially in diestrus stage and the uterus thickening that was shown in normal estrous stage was not observed. In the pCMV-mPRL-injected male mice, new blood vessels were first found at 5 weeks of age and fully developed blood vessels were found after 8 weeks in the testis. The number of Leydig cells increased within the testis and the testosterone level in serum was observed high. Finally, the number of white blood cells (WBCs) increased in the pCMV-mPRL-injected mice. The augmentation of WBCs persisted for at least 20 days after injection. When injection was combined with adrenalectomy, there was an even greater increase in number of WBCs, especially lymphocytes. This increase was returned normal by treatment with dexamethansone. Taken together, our data reveal that intramuscularly expressed mouse PRL influences reproductive functions in female, induces formation of new blood vessels in the testis, and augments WBC numbers. Of notice is that the Leydig cell proliferation with increased testosterone was conspicuously observed in the pCMV-mPRL-injected mice. These results also suggest subtle difference in function of PRL between mouse and rat species. [Abstract/Link to Full Text]

Lee JY, Lee YM, Kim MJ, Choi JY, Park EK, Kim SY, Lee SP, Yang JS, Kim DS
Methylation of the mouse DIx5 and Osx gene promoters regulates cell type-specific gene expression.
Mol Cells. 2006 Oct 31;22(2):182-8.
Dlx5 and Osx are master regulatory proteins essential for initiating the cascade leading to osteoblast differentiation in mammals, but the mechanism of osteoblast-specific expression is not fully understood. DNA methylation at CpG sequences is involved in tissue and cell type-specific gene expression. We investigated the methylation status of Dlx5 and Osx in osteogenic and nonosteogenic cell lines by methylation-specific PCR (MSP). The CpG dinucleotides of the Dlx5 and Osx promoter regions were unmethylated in osteogenic cell lines transcribing these genes but methylated in nonosteogenic cell lines. Treatment of C2C12 cells with 5-AzadC induced dose- and time-dependent expression of Dlx5 and Osx mRNA by demethylating the corresponding promoters. Furthermore the mRNAs for the osteoblast markers ALP and OC, which were undetectable in untreated cells, gradually increased after 5-AzadC treatment. In addition, BMP-2 stimulation induced Dlx5 expression by hypomethylating its promoter. These findings suggest that DNA methylation plays an important role in cell type-specific expression of Dlx5 and Osx. [Abstract/Link to Full Text]

Kim TW, Chung H, Kwon IC, Sung HC, Kang TH, Han HD, Jeong SY
Induction of immunity against hepatitis B virus surface antigen by intranasal DNA vaccination using a cationic emulsion as a mucosal gene carrier.
Mol Cells. 2006 Oct 31;22(2):175-81.
Delivery of DNA vaccines to airway mucosa would be an ideal method for mucosal immunization. However, there have been few reports of a suitable gene delivery system. In this study we used a cationic emulsion to immunize mice via the intranasal route with pCMV-S coding for Hepatitis B virus surface antigen (HBsAg). Complexing pCMV-S with a cationic emulsion dramatically enhanced HBsAg expression in both nasal tissue and lung, and was associated with increases in the levels of HBs-specific Abs in serum and mucosal fluids, of cytotoxic T lymphocytes (CTL) in the spleen and cervical and iliac lymph nodes, and of delayed-type hypersensitivity (DTH) against HBsAg. In contrast, very weak humoral and cellular immunities were observed following immunization with naked DNA. In support of these observations, a higher proliferative response of spleenocytes was detected in the group immunized with the emulsion/pCMV-S complex than in the group immunized with naked pCMV-S. These findings may facilitate development of an emulsion-mediated gene vaccination technique for use against intracellular pathogens that invade mucosal surfaces. [Abstract/Link to Full Text]

Han JY, Kim YS, Cho GJ, Roh GS, Kim HJ, Choi WJ, Paik WY, Rho GJ, Kang SS, Choi WS
Altered gene expression of caspase-10, death receptor-3 and IGFBP-3 in preeclamptic placentas.
Mol Cells. 2006 Oct 31;22(2):168-74.
Enhanced apoptosis has been observed in the placentas of women with preeclampsia, but few studies have examined changes at the molecular level. This study was designed to detect genes specifically expressed in full-term preeclamptic placentas. Tissue samples were collected immediately after cesarean delivery from 11 normal and 8 preeclamptic placentas at 35-40 weeks of gestation. Total RNAs were extracted and hybridized to a cDNA microarray. Results were confirmed by reverse-transcription polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. Hematoxylin and eosin and TUNEL staining were also performed to confirm apoptosis in preeclamptic placentas. Among 205 genes, three were up- or down-regulated in preeclamptic placentas. The expression of caspase-10 and death receptor 3 (DR-3) was significantly increased, whereas insulin-like growth factor binding protein-3 (IGFBP-3) was strongly down-regulated. RT-PCR analysis and Western blotting confirmed these effects. Immunohistochemical analysis showed that the DR-3, caspase-10 and IGFBP-3 proteins were localized in the syncytial membrane. Apoptosis in the trophoblast was also increased in term placentas from women with pregnancies complicated by preeclampsia. These results suggest that caspase-10, DR-3 and IGFBP-3 are involved in apoptosis in the preeclamptic placenta. [Abstract/Link to Full Text]

Guo JJ, Li QL, Zhang J, Huang AL
Histone deacetylation is involved in activation of CXCL10 upon IFNgamma stimulation.
Mol Cells. 2006 Oct 31;22(2):163-7.
Histone deacetylase (HDAC) activity is commonly associated with transcriptional repression. However, there is also evidence for a function in transcriptional activation. Previous studies have demonstrated a fundamental role of deacetylase activity in IFNa-responsive gene transcription. In the case of type II IFN (IFNgamma) results are controversial: some genes require HDAC activity, while transcription of others is repressed by HDAC. To investigate the effect of HDAC on transcription of an IFNgamma-activated gene, real-time PCR was used to measure CXCL10 mRNA in Hela cells stimulated with IFNgamma in the presence or absence of the HDAC inhibitor TSA. Chromatin imunoprecipitation combined with real-time PCR was used to check acetylation of histone H4 and recruitment of the STAT1 complex to the ISRE locus of the CXCL10 gene. Activation of CXCL10 transcription in response to IFNgamma was paralleled by a decrease in histone H4 acetylation and an increase in recruitment of the STAT1 complex to the CXCL10 ISRE locus. The transcription of CXCL10 and histone H4 deacetylation were blocked by TSA, but the latter had no obvious affect on recruitment of the STAT1 complex. Our data indicate that IFNgamma and STAT-dependent gene transcription requires the participation of HDAC, as does the IFNalpha-STAT pathway. [Abstract/Link to Full Text]

Basnet DB, Oh TJ, Vu TT, Sthapit B, Liou K, Lee HC, Yoo JC, Sohng JK
Angucyclines Sch 47554 and Sch 47555 from Streptomyces sp. SCC-2136: cloning, sequencing, and characterization.
Mol Cells. 2006 Oct 31;22(2):154-62.
The entire gene cluster involved in the biosynthesis of angucyclines Sch 47554 and Sch 47555 was cloned, sequenced, and characterized. Analysis of the nucleotide sequence of genomic DNA spanning 77.5-kb revealed a total of 55 open reading frames, and the deduced products exhibited strong sequence similarities to type II polyketide synthases, deoxysugar biosynthetic enzymes, and a variety of accessory enzymes. The involvement of this gene cluster in the pathway of Sch 47554 and Sch 47555 was confirmed by genetic inactivation of the aromatase, including a portion of the ketoreductase, which was disrupted by inserting the thiostrepton gene. [Abstract/Link to Full Text]

Choi E, Lee K, Song K
Function of rax2p in the polarized growth of fission yeast.
Mol Cells. 2006 Oct 31;22(2):146-53.
Cell polarity is critical for the division, differentiation, migration, and signaling of eukaryotic cells. RAX2 of budding yeast encodes a membrane protein localized at the cell cortex that helps maintain the polarity of the bipolar pattern. Here, we designate SPAC6f6.06c as rax2+ of Schizosaccharomyces pombe, based on its sequence homology with RAX2, and examine its function in cell polarity. S. pombe rax2+ is not essential, but Deltarax2 cells are slightly smaller and grow slower than wild type cells. During vegetative growth or arrest at G1 by mutation of cdc10, deletion of rax2+ increases the number of cells failing old end growth just after division. In addition, this failure of old end growth is dramatically increased in Deltatea1Deltarax2, pointing to genetic interaction of rax2+ with tea1+. Deltarax2 cells contain normal actin and microtubule cytoskeletons, but lack actin cables, and the polarity factor for3p is not properly localized at the growing tip. In Deltarax2 cells, and endogenous rax2p is localized at the cell cortex of growing cell tips in an actin- and microtubule-dependent manner. However, Deltarax2 cells show no defects in cell polarity during shmoo formation and conjugation. Taken together, these observations suggest that rax2p controls the cell polarity of fission yeast during vegetative growth by regulating for3p localization. [Abstract/Link to Full Text]

Lee Y, Park BC, Lee do H, Bae KH, Cho S, Lee CH, Lee JS, Myung PK, Park SG
Mouse transthyretin-related protein is a hydrolase which degrades 5-hydroxyisourate, the end product of the uricase reaction.
Mol Cells. 2006 Oct 31;22(2):141-5.
Uric acid is the end product of the purine degradation pathway in humans. It is catabolized to allantoin by urate oxidase or uricase (E.C. 1.7.3.3.) in most vertebrates except humans, some primates, birds, and certain species of reptiles. Here we provide evidence that mouse transthyretin-related protein facilitates the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction. Mutagenesis experiments showed that the residues that are absolutely conserved across the TRP family, including His11, Arg51, His102, and the C-terminal Tyr-Arg-Gly-Ser, may constitute the active site of mTRP. Based on these results, we propose that the transthyretin-related proteins present in diverse organisms are not functionally related to transthyretin but actually function as hydroxyisourate hydrolases. [Abstract/Link to Full Text]

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Recent Articles in Cell Research

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Ma YH, Hu JH, Zhou XG, Zeng RW, Mei ZT, Fei J, Guo LH
Transgenic mice overexpressing gamma-aminobutyric acid transporter subtype I develop obesity.
Cell Res. 2000 Dec;10(4):303-10.
Transgenic mice ubiquitously overexpressing murine gamma-aminobutyric acid transporter subtype I were created. Unexpectedly, these mice markedly exhibited heritable obesity, which features significantly increased body weight and fat deposition. Behavioral examination revealed that transgenic mice have slightly reduced spontaneous locomotive capacity and altered feeding pattern. This preliminary finding indicates that the inappropriate level of gamma-aminobutyric acid transporters may be directly or indirectly involved in the pathogenic mechanism underlying certain types of obesity. [Abstract/Link to Full Text]

Kong WH, Zheng G, LU JN, Tso JK
Temperature dependent expression of cdc2 and cyclin B1 in spermatogenic cells during spermatogenesis.
Cell Res. 2000 Dec;10(4):289-302.
p34cdc2 and Cyclin B1 are key components of cell cycle controlling machine and are believed to play a fundamental role in gametogenesis. It is also well known that, in scrotal mammals, spermatogenesis depends greatly on the maintenance of comparatively low temperature in the scrotum. To investigate whether the expression of cdc2 and cyclin B1 in spermatogenic cells during spermatogenesis is actually a temperature dependent event, in situ hybridization, Western blotting and immunohistochemistry analysis were used to study the expression of cdc2 and cyclin B1 in normal and cryptorchid testis. Results showed that the abdominal temperature had no significant influence on the transcription of cdc2 and cyclin B1 in the spermatogonia and pachytene/diplotene primary spermatocytes, but it blocked the translation of them. Due to the deficiency of p34cdc2 and Cyclin B1, the spermatogonia and pachytene/diplotene primary spermatocytes were unable to form MPF, hence, they couldn't undergo karyokinesis. The development of primary spermatocytes was arrested at the G2 to M phase transition. We also found that testosterone could regulate the Cyclin B1 expression in spermatogenic cells. Muscular injection of testosterone could recover spermatogenesis in the unilateral scrotal testis which was influenced by the contralateral cryptorchid testis, but it could not salvage the spermatogenesis block in the cryptorchid testis. [Abstract/Link to Full Text]

Qu CK
The SHP-2 tyrosine phosphatase: signaling mechanisms and biological functions.
Cell Res. 2000 Dec;10(4):279-88.
Cellular biological activities are tightly controlled by intracellular signaling processes initiated by extracellular signals. Protein tyrosine phosphatases, which remove phosphate groups from phosphorylated signaling molecules, play equally important tyrosine roles as protein tyrosine kinases in signal transduction. SHP-2, a cytoplasmic SH2 domain containing protein tyrosine phosphatase, is involved in the signaling pathways of a variety of growth factors and cytokines. Recent studies have clearly demonstrated that this phosphatase plays an important role in transducing signal relay from the cell surface to the nucleus, and is a critical intracellular regulator in mediating cell proliferation and differentiation. [Abstract/Link to Full Text]

Woo M, Hakem R, Mak TW
Executionary pathway for apoptosis: lessons from mutant mice.
Cell Res. 2000 Dec;10(4):267-78.
Apoptosis or programmed cell death (PCD) is an evolutionarily conserved cellular process that is essential for normal development and homeostasis of multicellular organisms. Defects in the apoptosis signaling result in many diseases including autoimmune diseases and cancer. The apoptosis signaling pathway was first described genetically in the nematode Caenorhabditis elegans which serves as a framework for the more complex apoptotic pathways that exist in mammals. In this review, we will discuss the apoptotic pathways that are emerging in mammals as elucidated by studies of gene-targeted mutant mice. [Abstract/Link to Full Text]

Rothstein TL
Inducible resistance to Fas-mediated apoptosis in B cells.
Cell Res. 2000 Dec;10(4):245-66.
Apoptosis produced in B cells through Fas (APO-1, CD95) triggering is regulated by signals derived from other surface receptors: CD40 engagement produces upregulation of Fas expression and marked susceptibility to Fas-induced cell death, whereas antigen receptor engagement, or IL-4R engagement, inhibits Fas killing and in so doing induces a state of Fas-resistance, even in otherwise sensitive, CD40-stimulated targets. Surface immunoglobulin and IL-4R utilize at least partially distinct pathways to produce Fas-resistance that differentially depend on PKC and STAT6, respectively. Further, surface immunoglobulin signaling for inducible Fas-resistance bypasses Btk, requires NF-kappaB, and entails new macromolecular synthesis. Terminal effectors of B cell Fas-resistance include the known anti-apoptotic gene products, Bcl-xL and FLIP, and a novel anti-apoptotic gene that encodes FAIM (Fas Apoptosis Inhibitory Molecule). faim was identified by differential display and exists in two alternatively spliced forms; faim-S is broadly expressed, but faim-L expression is tissue-specific. The FAIM sequence is highly evolu- tionarily conserved, suggesting an important role for this molecule throughout phylogeny. Inducible resistance to Fas killing is hypothesized to protect foreign antigen-specific B cells during potentially hazardous interactions with FasL-bearing T cells, whereas autoreactive B cells fail to become Fas-resistant and are deleted via Fas-dependent cytotoxicity. Inadvertent or aberrant acquisition of Fas-resistance may permit autoreactive B cells to escape Fas deletion, and malignant lymphocytes to impede anti-tumor immunity. [Abstract/Link to Full Text]

Chang YC, Xu YH
Expression of Bcl-2 inhibited Fas-mediated apoptosis in human hepatocellular carcinoma BEL-7404 cells.
Cell Res. 2000 Sep;10(3):233-42.
Apoptosis plays an important role in embryonic development, tissue remodeling, immune regulation and tumor regression. Two groups of molecules (Bcl-2 family and "Death factor" family) are involved in regulating apoptosis. In order to know about the effect of Bcl-2 on apoptosis induced by Fas, a typical member of "Death factor" family, the transfection experiments with expression vectors pcDNA3-fl and pcDNA3-bcl-2 were performed in BEL-7404 cells, a human hepatocellular carcinoma cell line which expresses endogenous Fas, but not FasL and Bcl-2. The data showed that the expression of FasL in pcDNA3-fl transfected hepatoma cells obviously induced the apoptosis of the cells. However, the overexpression of Bcl-2 in pcDNA3-bcl-2 transfected 7404/b-16 cells counteracted pcDNA3-fl transient transfection mediated apoptosis. Further study by co-transfection experiments indicated that Bid but not Bax (both were pro-apoptotic proteins of Bcl-2 family) blocked the inhibitory effect of Bcl-2 on Fas-mediated apoptosis. These results suggested that Fas-mediated apoptosis in human hepatoma cells is possibly regulated by Bcl-2 family proteins via mitochondria pathway. [Abstract/Link to Full Text]

Jiang ZF, Zhao Y, Hong X, Zhai ZH
Nuclear apoptosis induced by isolated mitochondria.
Cell Res. 2000 Sep;10(3):221-32.
We isolated and purified mitochondria from mouse livers and spinach leaves. When added into egg extracts of Xenopus laevis, they caused nuclei of mouse liver to undergo apoptotic changes. Chromatin condensation, margination and DNA ladder were observed. After incubating isolated mitochondria in some hypotonic solutions, and centrifuging these mixtures at high speed, we got mitochondrial supernatants. It was found that in the absence of cytosolic factor, the supernatant alone was able to induce apoptotic changes in nuclei. The effective components were partly of protein. DNA fragmentation was partly inhibited by caspase inhibitors AC-DEVD-CHO and AC-YVAD-CHO. Meanwhile, caspase inhibitors fully blocked chromatin condensation. Primary characterization of the nuclear endonuclease(s) induced by mitochondrial supernatants was also conducted. It was found that this endonuclease is different from endonuclease G, cytochrome c-induced nuclease, or Ca2+-activated endonuclease. [Abstract/Link to Full Text]

Zhang M, Zhang HQ, Xue SB
Effect of Bcl-2 and caspase-3 on calcium distribution in apoptosis of HL-60 cells.
Cell Res. 2000 Sep;10(3):213-20.
Apoptosis manifests in two major execution programs downstream of the death signal: the caspase pathway and organelle dysfunction. An important antiapoptosis factor, Bcl-2 protein, contributes in caspase pathway of apoptosis. Calcium, an important intracellular signal element in cells, is also observed to have changes during apoptosis, which maybe affected by Bcl-2 protein. We have previously reported that in Harringtonine (HT) induced apoptosis of HL-60 cells, there's a change of intracellular calcium distribution, moving from cytoplast especially Golgi's apparatus to nucleus and accumulating there with the highest concentration. We report here that caspase-3 becomes activated in HT-induced apoptosis of HL-60 cells, which can be inhibited by overexpression of Bcl-2 protein. No sign of apoptosis or intracellular calcium movement from Golgi's apparatus to nucleus in HL-60 cells overexpressing Bcl-2 or treated with Ac-DEVD-CHO, a specific inhibitor of caspase-3. The results indicate that activated caspase-3 can promote the movement of intracellular calcium from Golgi's apparatus to nucleus, and the process is inhibited by Ac-DEVD-CHO (inhibitor of caspase-3), and that Bcl-2 can inhibit the movement and accumulation of intracellular calcium in nucleus through its inhibition on caspase-3. Calcium relocalization in apoptosis seems to be irreversible, which is different from the intracellular calcium changes caused by growth factor. [Abstract/Link to Full Text]

Zhang JH, Xu M
DNA fragmentation in apoptosis.
Cell Res. 2000 Sep;10(3):205-11.
Cleavage of chromosomal DNA into oligonucleosomal size fragments is an integral part of apoptosis. Elegant biochemical work identified the DNA fragmentation factor (DFF) as a major apoptotic endonuclease for DNA fragmentation in vitro. Genetic studies in mice support the importance of DFF in DNA fragmentation and possibly in apoptosis in vivo. Recent work also suggests the existence of additional endonucleases for DNA degradation. Understanding the roles of individual endonucleases in apoptosis, and how they might coordinate to degrade DNA in different tissues during normal development and homeostasis, as well as in various diseased states, will be a major research focus in the near future. [Abstract/Link to Full Text]

Lee CY, Baehrecke EH
Genetic regulation of programmed cell death in Drosophila.
Cell Res. 2000 Sep;10(3):193-204.
Programmed cell death plays an important role in maintaining homeostasis during animal development, and has been conserved in animals as different as nematodes and humans. Recent studies of Drosophila have provided valuable information toward our understanding of genetic regulation of death. Different signals trigger the novel death regulators rpr, hid, and grim, that utilize the evolutionarily conserved iap and ark genes to modulate caspase function. Subsequent removal of dying cells also appears to be accomplished by conserved mechanisms. The similarity between Drosophila and human in cell death signaling pathways illustrate the promise of fruit flies as a model system to elucidate the mechanisms underlying regulation of programmed cell death. [Abstract/Link to Full Text]

Donjerkovi? D, Scott DW
Activation-induced cell death in B lymphocytes.
Cell Res. 2000 Sep;10(3):179-92.
Upon encountering the antigen (Ag), the immune system can either develop a specific immune response or enter a specific state of unresponsiveness, tolerance. The response of B cells to their specific Ag can be activation and proliferation, leading to the immune response, or anergy and activation-induced cell death (AICD), leading to tolerance. AICD in B lymphocytes is a highly regulated event initiated by crosslinking of the B cell receptor (BCR). BCR engagement initiates several signaling events such as activation of PLCgamma, Ras, and PI3K, which generally speaking, lead to survival. However, in the absence of survival signals (CD40 or IL-4R engagement), BCR crosslinking can also promote apoptotic signal transduction pathways such as activation of effector caspases, expression of pro-apoptotic genes, and inhibition of pro-survival genes. The complex interplay between survival and death signals determines the B cell fate and, consequently, the immune response. [Abstract/Link to Full Text]

Yang YL, Li XM
The IAP family: endogenous caspase inhibitors with multiple biological activities.
Cell Res. 2000 Sep;10(3):169-77.
IAPs (inhibitors of apoptosis) are a family of proteins containing one or more characteristic BIR domains. These proteins have multiple biological activities that include binding and inhibiting caspases, regulating cell cycle progression, and modulating receptor-mediated signal transduction. Our recent studies found the IAP family members XIAP and c-IAP1 are ubiquitinated and degraded in proteasomes in response to apoptotic stimuli in T cells, and their degradation appears to be important for T cells to commit to death. In addition to three BIR domains, each of these IAPs also contains a RING finger domain. We found this region confers ubiquitin protease ligase (E3) activity to IAPs, and is responsible for the auto-ubiquitination and degradation of IAPs after an apoptotic stimulus. Given the fact that IAPs can bind a variety of proteins, such as caspases and TRAFs, it will be of interest to characterize potential substrates of the E3 activity of IAPs and the effects of ubiquitination by IAPs on signal transduction, cell cycle, and apoptosis. [Abstract/Link to Full Text]

Yin XM
Signal transduction mediated by Bid, a pro-death Bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways.
Cell Res. 2000 Sep;10(3):161-7.
Two major apoptosis pathways have been defined in mammalian cells, the Fas/TNF-R1 death receptor pathway and the mitochondria pathway. The Bcl-2 family proteins consist of both anti-apoptosis and pro-apoptosis members that regulate apoptosis, mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events. However, death signals mediated by Fas/TNF-R1 receptors can usually activate caspases directly, bypassing the need for mitochondria and escaping the regulation by Bcl-2 family proteins. Bid is a novel pro-apoptosis Bcl-2 family protein that is activated by caspase 8 in response to Fas/TNF-R1 death receptor signals. Activated Bid is translocated to mitochondria and induces cytochrome c release, which in turn activates downstream caspases. Such a connection between the two apoptosis pathways could be important for induction of apoptosis in certain types of cells and responsible for the pathogenesis of a number of human diseases. [Abstract/Link to Full Text]

He YK, Xue WX, Sun YD, Yu XH, Liu PL
Leafy head formation of the progenies of transgenic plants of Chinese cabbage with exogenous auxin genes.
Cell Res. 2000 Jun;10(2):151-60.
The experiment was performed to evaluate the progenies of plant lines transgenic for auxin synthesis genes derived from Ri T-DNA. Four lines of the transgenic plants were self-crossed and the foreign auxin genes in plants of T5 generation were confirmed by Southern hybridization. Two lines, D1232 and D1653, showed earlier folding of expanding leaves than untransformed line and therefore had early initiation of leafy head. Leaf cuttings derived from plant of transgenic line D1653 produced more adventitious roots than the control whereas the cuttings from folding leaves had much more roots than rosette leaves at folding stage, and the cuttings from head leaves had more roots than rosette leaves at heading stage. It is demonstrated that early folding of transgenic leaf may be caused by the relatively higher concentration of auxin. These plant lines with auxin transgenes can be used for the study of hormonal regulation in differentiation and development of plant organs and for the breeding of new variety with rapid growth trait. [Abstract/Link to Full Text]

Zhou BH, Chen JS, Chai MQ, Zhao S, Liang J, Chen HH, Song JG
Activation of phospholipase D activity in transforming growth factor-beta-induced cell growth inhibition.
Cell Res. 2000 Jun;10(2):139-49.
Cells regulate phospholipase D (PLD) activity in response to numerous extracellular signals. Here, we investigated the involvement of PLD activity in transforming growth factor-beta (TGF-beta1)-mediated growth inhibition of epithelial cells. TGF-beta1 inhibits the growth of MDCK, Mv1Lu, and A-549 cells. In the presence of 0.4% butanol, TGF-beta1 induces an increase in the formation of phosphatidylbutanol, a unique product catalyzed by PLD. TGF-beta1 also induces an increase in phosphatidic acid (PA) level in A-549 and MDCK cells. TGF-beta1 induces an increase in the levels of DAG labeled with [3H]-myristic acid in A-549 and MDCK cells but not in Mv1Lu cells. No increase of DAG was observed in cells prelabeled with [3H]-arachidonic acid. The data presented suggest that PLD activation is involved in the TGF-beta1-induced cell growth inhibition. [Abstract/Link to Full Text]

Liu XL, Shen Y, Chen EJ, Zhai ZH
Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell-free extracts of Xenopus laevis eggs.
Cell Res. 2000 Jun;10(2):127-37.
Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation, nuclear envelope assembly, and nuclear reconstitution. Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle. The assembled nuclei, being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii. However, incubation of dinoflagellate Crythecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinium cohnii cells does not induce nuclear reconstitution. [Abstract/Link to Full Text]

Wu YD, Song JD
The expression of glucose regulated protein-94 in colorectal carcinoma cells treated by sodium butyrate.
Cell Res. 2000 Jun;10(2):115-25.
The expression of glucose regulated protein 94 (GRP94) during the treatment of human colorectal carcinoma cell line-Clone A cells with sodium butyrate was studied. Sodium butyrate (SB) can cause functional and morphological effects on Clone A cells including growth arrest at G0/G1 stage and cell differentiation as observed by morphological changes, MTT and flow cytometry assays, as well as reduced Grp94 gene expression as shown by Northern blot and Western blot assays. The possible mechanism of the correlation between Grp94 gene expression and tumor growth inhibition and cell differentiation is briefly discussed. [Abstract/Link to Full Text]

Jian LC, Li JH, Li PH, Chen TH
An electron microscopic-cytochemical localization of plasma membrane Ca(2+)-ATPase activity in poplar apical bud cells during the induction of dormancy by short-day photoperiods.
Cell Res. 2000 Jun;10(2):103-14.
Plasma membrane (PM) Ca2+-ATPase activity in poplar apical bud meristematic cells during short-day (SD)-induced dormancy development was examined by a cerium precipitation EM-cytochemical method. Ca2+-ATPase activity, indicated by the status of cerium phosphate precipitated grains, was localized mainly on the interior face (cytoplasmic side) of the PM when plants were grown under long days and reached a deep dormancy. A few reaction products were also observed on the nuclear envelope. When plant buds were developing dormancy after 28 to 42 d of SD exposure, almost no reaction products were present on the interior face of the PM. In contrast, a large number of cerium phosphate precipitated grains were distributed on the exterior face of the PM. After 70 d of SD exposure, when buds had developed a deep dormancy, the reaction products of Ca2+-ATPase activity again appeared on the interior face of the PM. The results seemed suggesting that two kinds of Ca2+-ATPases may be present on the PM during the SD-induced dormancy in poplar. One is the Ca2+-pumping ATPase, which is located on the interior face of the PM, for maintaining and restoring the Ca2+ homeostasis. The other might be an ecto-Ca2+-ATPase, which is located on the exterior face of the PM, for the exocytosis of cell wall materials as suggested by the fact of the cell wall thickening during the dormancy development in poplar. [Abstract/Link to Full Text]

Wei Q, Zhou DH, Shen QX, Chen J, Chen LW, Wang TL, Pei G, Chi ZQ
Human mu-opioid receptor overexpressed in Sf9 insect cells functionally coupled to endogenous Gi/o proteins.
Cell Res. 2000 Jun;10(2):93-102.
Human mu-opioid receptor (HmuOR) with a tag of six consecutive histidines at its carboxyl terminus had been expressed in recombinant baculovirus infected Sf9 insect cells. The maximal binding capacity for the [3H] diprenorphine and [3H]ohmefentanyl (Ohm) were 9.1 +/- 0.7 and 6.52 +/- 0.23 nmol/g protein, respectively. The [3H] diprenorphine or [3H] Ohm binding to the receptor expressed in Sf9 cells was strongly inhibited by mu-selective agonists [D-Ala2, N-methyl-Phe4, glyol5]enkephalin (DAGO), Ohm, and morphine, but neither by delta nor by kappa selective agonist. Na+ (100 mM) and GTP (50 microM) could reduce HmuOR agonists etorphine and Ohm affinity binding to the overexpressed HmuOR. mu-selective agonists DAGO and Ohm effectively stimulated [35S]GTP-gammaS binding (EC50 = 2.7 nM and 6.9 nM) and inhibited forskolin- stimulated cAMP accumulation (IC50 = 0.9 nM and 0.3 nM). The agonist-dependent effects could be blocked by opioid antagonist naloxone or by pretreatment of cells with pertussis toxin (PTX). These results demonstrated that HmuOR overexpressed in Sf9 insect cells functionally coupled to endogenous G(i/o) proteins. [Abstract/Link to Full Text]

Huang L, Li C
Leptin: a multifunctional hormone.
Cell Res. 2000 Jun;10(2):81-92.
Leptin is the protein product encoded by the obese (ob) gene. It is a circulating hormone produced primarily by the adipose tissue. ob/ob mice with mutations of the gene encoding leptin become morbidly obese, infertile, hyperphagic, hypothermic, and diabetic. Since the cloning of leptin in 1994, our knowledge in body weight regulation and the role played by leptin has increased substantially. We now know that leptin signals through its receptor, OB-R, which is a member of the cytokine receptor superfamily. Leptin serves as an adiposity signal to inform the brain the adipose tissue mass in a negative feedback loop regulating food intake and energy expenditure. Leptin also plays important roles in angiogenesis, immune function, fertility, and bone formation. Humans with mutations in the gene encoding leptin are also morbidly obese and respond to leptin treatment, demonstrating that enhancing or inhibiting leptin's activities in vivo may have potential therapeutic benefits. [Abstract/Link to Full Text]

Zhang RG, Wang XW, Yuan JH, Guo LX, Xie H
Using a non-radioisotopic, quantitative TRAP-based method detecting telomerase activities in human hepatoma cells.
Cell Res. 2000 Mar;10(1):71-7.
A non-radioisotopic, quantitative TRAP-based telomerase activity assay was established mainly by using SYBR Green-I staining instead of radioisotope. Comparing with conventional radioisotope based method, it was better in reproducibility and accuracy. Using this method, we found telomerase activities were absent in normal human liver cells, while detected in all of four human hepatoma cell lines (BEL-7404, SMMC-7721, QGY-7903 and HCCM) without significant differences. [Abstract/Link to Full Text]

Ma YH, Hu JH, Zhou XG, Mei ZT, Fei J, Guo LH
Gamma-aminobutyric acid transporter (GAT1) overexpression in mouse affects the testicular morphology.
Cell Res. 2000 Mar;10(1):59-69.
Gamma-aminobutyric acid and GABAergic receptors were previously reported to be distributed in reproductive systems besides CNS and predicted to participate in the modulation of testicular function. Gamma-aminobutyric acid transporter was implicated to be involved in this process. However, the potential role of gamma-aminobutyric transporter in testis has not been explored. In this study, we investigated the existence of mouse gamma-aminobutyric acid transporter subtype I (mGAT1) in testis. Wild-type and transgenic mice, which overexpressing mGAT1 in a variety of tissues, especially in testis, were primarily studied to approach the profile of mGAT1 in testis. Mice with overexpressed mGAT1 develop normally but with reduced mass and size of testis as compared with wild-type. Testicular morphology of transgenic mice exhibited overt abnormalities including focal damage of the spermatogenic epithelium accompanied by capillaries proliferation and increased diameter of seminiferous tubules lumen. Reduced number of spermatids was also found in some seminiferous tubules. Our results clearly demonstrate the presence of GAT1 in mouse testis and imply that GAT1 is possibly involved in testicular function. [Abstract/Link to Full Text]

Hu JH, He XB, Yan YC
Identification of gamma-aminobutyric acid transporter (GAT1) on the rat sperm.
Cell Res. 2000 Mar;10(1):51-8.
Some recent studies indicated that GABAergic system is involved in mammalian sperm acrosome reaction (AR), but direct evidence pertaining to the expression of gat1 in mammalian sperm is not yet demonstrated. In this study, we evaluated the presence of 67kDa GAT1 protein and mRNA in rat testis by Western blotting and reverse transcription-polymerase chain reaction. Meanwhile, immunohistochemical and immunofluorescent analyses also identified GAT1 protein on the elongated spermatid and sperm. These results indicated that rat testis is a novel site of gat1 expression. Further studies should be taken to explore the role of GAT1 protein on sperm acrosome reaction. [Abstract/Link to Full Text]

Li NL, Zhang DQ, Zhou KY, Cartman A, Leroux JY, Poole AR, Zhang YP
Isolation and characteristics of autoreactive T cells specific to aggrecan G1 domain from rheumatoid arthritis patients.
Cell Res. 2000 Mar;10(1):39-49.
Our previous work showed that the cartilage proteoglycan aggrecan could induce an erosive polyarthritis and spondylitis in BALB/c mice and the G1 globular domain of the aggrecan (G1) contained the arthritogenic region. To elucidate whether autoreactive T cells to G1 are expressed in rheumatoid arthritis patients, we analyzed the frequency of human G1-specific T cells in the peripheral blood of five rheumatoid arthritis patients and tried to establish G1-reactive T cell lines from these rheumatoid arthritis patients. The results showed that the G1-specific T cells in PBL were detectable at the range of 4.97 +/- 0.5 x 10(-6) in peripheral blood lymphocytes. We have also generated 15 G1-specific T lymphocyte lines from these patients with a standard split-well method. All these cells expressed fine specificity to human recombinant G1, but not to unrelated antigen. All the 15 lines expressed a pan-T cell marker and 13 of them selectively used the alphabeta T cell receptor. Two of them used gammadelta T cell receptor. The 13 of these T cell lines was CD4 positive. One line expressed CD8. One line expressed both CD4 and CD8. Moreover, 14 out of 15 lines expressed the Th-1 cytokine profile, characterized by interferon-gamma positivity and IL-4 negativity. No Th-2 type cell line was generated. These data provide strong evidence in favor of the presence of autoreactive T cells in the rheumatoid arthritis patients. What is the mechanism(s) that these autoreactive T cells attack self-target and whether these G1-specific, Th-1 type T cell lines can induce arthritis in immune deficiency mice are currently under investigation. [Abstract/Link to Full Text]

Li XF, Shen RJ, Liu PL, Tang ZC, He YK
Molecular characters and morphological genetics of CAL gene in Chinese cabbage.
Cell Res. 2000 Mar;10(1):29-38.
BcpCAL, the homologous gene of CAL, was isolated from Chinese cabbage. Unlike BobCAL of cauliflower, BcpCAL did not hold the terminating mutation in the fifth exon. After crosses of cauliflower with Chinese cabbage, the resultant hybrids failed to form curd, which implicates the genetic complement of BcpCAL to the mutated BobCAL in the function of curd formation. One of CAL gene isolated from the hybrid apparently comes from the female parent (Chinese cabbage) even though there are a few of the bases substituted and deleted. The result offers the molecular and genetic evidences for the study of biological function of CAL in morphological genetics of curd. [Abstract/Link to Full Text]

Zhu ZY, Sun YH
Embryonic and genetic manipulation in fish.
Cell Res. 2000 Mar;10(1):17-27.
Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been thoroughly studied in China since 1960s. Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults. Most importantly, nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish. This was the first case of cloned fish with somatic cells. Based on the technique of microinjection, recombinant MThGH gene has been transferred into fish eggs and the first batch of transgenic fish were produced in 1984. The behavior of foreign gene was characterized and the onset of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis. This eventually led to the transgenic mosaicism. The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults. The transgenic common carp were more efficient in utilizing dietary protein than the controls. An "all-fish" gene construct CAgcGH has been made by splicing the common carp beta-actin gene (CA) promoter onto the grass carp growth hormone gene (gcGH) coding sequence. The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait. Combination of techniques of fish cell culture, gene transformation with cultured cells and nuclear transplantation should be able to generate homogeneous strain of valuable transgenic fish to fulfil human requirement in 21st century. [Abstract/Link to Full Text]

Donjerkovic D, Scott DW
Regulation of the G1 phase of the mammalian cell cycle.
Cell Res. 2000 Mar;10(1):1-16.
In any multi-cellular organism, the balance between cell division and cell death maintains a constant cell number. Both cell division cycle and cell death are highly regulated events. Whether the cell will proceed through the cycle or not, depends upon whether the conditions required at the checkpoints during the cycle are fulfilled. In higher eucaryotic cells, such as mammalian cells, signals that arrest the cycle usually act at a G1 checkpoint. Cells that pass this restriction point are committed to complete the cycle. Regulation of the G1 phase of the cell cycle is extremely complex and involves many different families of proteins such as retinoblastoma family, cyclin dependent kinases, cyclins, and cyclin kinase inhibitors. [Abstract/Link to Full Text]

Kong LW, Ding XY, Kitani H, Shiurba R, Jing NH
Evidence for a mouse brain-specific variant of alpha-tubulin.
Cell Res. 1999 Dec;9(4):315-25.
While studying the neural precursor cell intermediate filament protein known as nestin in the developing mouse brain, we observed a strong cross-reaction of our nestin antibody with a 50 kDa protein that appeared on embryonic day 10 and continued to accumulate until postnatal day 1. Here we report evidence that this protein is a brain-specific variant form of alpha-tubulin and discuss its implications. [Abstract/Link to Full Text]

Fang CM, Shi C, Xu YH
Deregulated c-myc expression in quiescent CHO cells induces target gene transcription and subsequent apoptotic phenotype.
Cell Res. 1999 Dec;9(4):305-14.
Human c-myc cDNA was fused with the hormone-binding domain (HBD) cDNA of murine estrogen receptor gene and the chimeric gene was introduced into the CHO cells. The fusion protein, c-MycER, becomes activated when the synthetic steroid, 4-hydroxy-tamoxifen (OHT), binds HBD. Activated c-MycER, likely c-Myc, can induce quiescent CHO cells reentry into S phase and subsequent cell death under serum-free condition. In addition, the expression of some proposed c-myc target genes such as ODC, MrDb, cad, rcc1 and rcl were found to increase upon OHT induction before S phase entry and apoptosis, indicating that these target genes are involved in cell cycle regulation and/or apoptosis control. However, the mutant D106-143c-MycER protein does not have above activities. [Abstract/Link to Full Text]

Pei D
Leukolysin/MMP25/MT6-MMP: a novel matrix metalloproteinase specifically expressed in the leukocyte lineage.
Cell Res. 1999 Dec;9(4):291-303.
A novel matrix metalloproteinase (MMP) was identified from leukocytes and found to be specifically expressed by peripheral blood leukocytes among 29 different tissues examined. Named leukolysin, it encodes for 562 residues with a conserved MMP structure, i.e., pre-, pro-, catalytic, hinge- and hemopexin-like domains, but also a RXK/RR motif, known for its role in MMP zymogen activation, and a C-terminal hydrophobic segment. Overall, leukolysin displays the strongest homology to the newly identified MT-MMP subgroup with 45% and 39% identities to MT4- and MT1-MMPs vs 30% and 31.5% to MMP1 and 3 respectively. Unlike MT4-MMP whose proteolytic activity remains undefined, a C-terminally truncated leukolysin is expressed as a strong gelatinolytic species at 28 kDa which is derived from a cell-associated 34 kDa proenzyme, presumably by furin or proprotein convertase mediated removal of the propeptide (approximately 6 kDa). By green fluorescent protein (GFP) tagging, the intracellular proenzyme is localized to granules throughout the cell, suggesting that activation occur immediately prior to secretion. Taken together, leukolysin may be part of the proteolytic arsenal deployed by leukocytes during inflammatory responses. Molecular cloning of a novel MMP: MMP:25 [Abstract/Link to Full Text]

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Recent Articles in Cellular & Molecular Biology Letters

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Mila-Kierzenkowska C, Kedziora-Kornatowska K, Wo?niak A, Drewa T, Wo?niak B, Drewa S, Krzyzy?ska-Malinowska E, Makarewicz R
The effect of brachytherapy on antioxidant status and lipid peroxidation in patients with cancer of the uterine cervix.
Cell Mol Biol Lett. 2004;9(3):511-8.
The aim of this study was to investigate the effect of brachytherapy on lipid peroxidation and antioxidant status in patients with uterine cervix cancer. The study was conducted on 84 uterine cervix cancer patients from the Brachytherapy Department of the Regional Centre of Oncology in Bydgoszcz. Patients with uterine cervix cancer were found to have elevated levels of lipid peroxidation and antioxidant defence system impairment relative to healthy females. The results of the study indicate that brachytherapy has no direct effect on the antioxidant system of patients with uterine cervical carcinoma. However, the normalisation of catalase and glutathione peroxidase activity and erythrocyte TBARS level observed six months after the end of therapy may be due to the arrest of the progression of the disease. [Abstract/Link to Full Text]

Narbutt J, Lesiak A, Zak-Prelich M, Wo?niacka A, Sysa-Jedrzejowska A, Tybura M, Robak T, Smolewski P
The distribution of peripheral blood dendritic cells assayed by a new panel of anti-BDCA monoclonal antibodies in healthy representatives of the polish population.
Cell Mol Biol Lett. 2004;9(3):497-509.
A growing number of studies are being performed on the role of dendritic cells (DCs) in the etiopathogenesis of various conditions. Therefore, it is extremely important to establish the best comparable methods for the determination of the absolute count of blood dendritic cells (BDCs) or their subsets, and the reference normal values for comparisons. The aim of our study was to assess a normal profile of BDCs in the non-cultured human blood of healthy Polish volunteers. BDCs were detected among peripheral blood mononuclear cells (PBMC) from 99 healthy people, aged 18-56. Based on the panel of novel anti-BDCA1, BDCA2 and BDCA3 monoclonal antibodies (MoAbs), three main subpopulations of BDCs were distinguished: two myeloid types of BDCs, MDC1(BDCA-1+/ CD11c+ /HLA-DR+) or MDC2 (BDCA-3+/CD32-/CD64-/HLA-DR+), and a plasmacytoid subtype, PDC (BDCA-2+/CD123+/HLA-DR+). The number and percentage of BDCs were correlated with the age, gender, photosensitivity (phototype, minimal erythemal dose -- MED) and morphological parameters of the healthy volunteers. BDCs represented 0.83% of the PBMC and the median total BDC number was 44.0 cell/microl. The total BDC number correlated with the WBC count (rho=0.40, p=0.001) as well as with the lymphocyte and monocyte counts (rho=0.20, p=0.045 and rho=0.26, p=0.009, respectively). The median percentage of the MDC1 count (0.20%) was twice as high as the MDC2 count (0.10%). The median PDC count was 28.2 cell/microl, and these cells represented 0.50% of the PBMC. There was a positive correlation between PDC and skin photosensitivity (rho=0.28, p=0.005). An inverse correlation between the PDC count and the age of the examined volunteers was also found (rho=-0.22, p=0.029). Our study provides the first referential data on normal rates and counts of BDCs and their subpopulations, assessed by the new panel of anti-BDCA MoAbs, in healthy Polish subjects. The method used in the study allowed the determination of BDCs and their subset numbers in a relatively small blood volume. [Abstract/Link to Full Text]

Dzwonek A, Mikula M, Woszczy?ski M, Hennig E, Ostrowski J
Protective effect of vaccination with DNA of the H. Pylori genomic library in experimentally infected mice.
Cell Mol Biol Lett. 2004;9(3):483-95.
Immunologically mediated protection against H. pylori infection is an attractive alternative to antibiotic treatment. We compared the efficacy of conventional protein vaccination with that of genetic vaccination against experimental infection with H. pylori in mice. For oral immunization, we used the recombinant peptide of an antigenic fragment of UreB (rUreB) or H. pylori-whole cell lysate antigens, and for genetic immunization, we used recombinant pcDNA and pSec plasmids inserted with the fragment of ureB or DNA of the H. pylori genomic library. Mice were challenged with the mouse stomach-adapted H. pylori Sidney Strain. The detection of gastric bacterial colonization was performed by real-time PCR of a 26-kDa Helicobacter-specific gene, and the presence of serum H. pylori-specific antibodies was determined using direct ELISA assay. The most effective treatment appeared to be oral vaccination with rUreB and either intramuscular or intradermal vaccination with DNA of the H. pylori genomic library. Intradermal genetic vaccination with genomic library DNA significantly increased the IgG antibody response. Our study revealed acceptable efficacies of genetic vaccination with DNA of the H. pylori genomic library. [Abstract/Link to Full Text]

Götte M, Sofeu Feugaing DD, Kresse H
Biglycan is internalized via a chlorpromazine-sensitive route.
Cell Mol Biol Lett. 2004;9(3):475-81.
The small leucine-rich proteoglycan biglycan (BGN) is abundantly expressed in mesenchymal tissues. Its expression level is related to the phenotypic differentiation of cells. A dysregulation in BGN expression occurs under several pathological conditions, including glomerulonephritis, mesothelioma, pancreatic cancer and a mouse model of osteoporosis. Since the extracellular concentration of BGN is regulated both by secretion and endocytosis, we performed mechanistic studies on BGN endocytosis in human skin fibroblasts in vitro, using inhibitors of different endocytic routes. Chlorpromazine, an inhibitor of the clathrin-coated pit-pathway reduced endocytosis of BGN in human skin fibroblasts by 40%, and decreased degradation of BGN by 66% Filipin, an inhibitor of the caveolae pathway, and Tyrphostin AG 1478, a specific inhibitor of EGF-receptor phosphorylation that partially inhibits endocytosis of the structurally related proteoglycan decorin, had no influence on BGN internalization and degradation. Our data indicates that the classical clathrin-mediated endocytic pathway is a major route for the internalization of BGN. Based on the differential susceptibility to pharmacological inhibition, it appears that BGN endocytosis seems to be at least in part mechanistically different from decorin uptake. [Abstract/Link to Full Text]

Wang J, Wu W, Zuo K, Fei J, Sun X, Lin J, Li X, Tang K
Isolation and characterization of a serine/threonine protein kinase SOS2 gene from Brassica napus.
Cell Mol Biol Lett. 2004;9(3):465-73.
A full-length cDNA of a new serine/threonine (Ser/Thr) protein kinase gene, designated as BnSOS2 (GenBank Acc. No.AY310413), was cloned from Brassica napus by rapid amplification of cDNA ends (RACE). The full-length cDNA of BnSOS2 was 1779 bp and contained a 1539-bp open reading frame encoding a protein of 512 amino acids. Homology analysis shows that BnSOS2 strongly resembles other Ser/Thr protein kinase genes, and that its putative protein belongs to a typical Ser/Thr kinase family. Northern blot analysis reveals that BnSOS2 is salt-inducible. Our results indicate that BnSOS2 is a new member of the plant SOS2 gene family, which may play an important role in salt tolerance of plants. [Abstract/Link to Full Text]

Szemraj J, Kawecka I, Bartkowiak J, Zyli?ska L
The effect of antisense oligonucleotide treatment of plasma membrane Ca(+2)-ATPase in PC12 cells.
Cell Mol Biol Lett. 2004;9(3):451-64.
Plasma membrane Ca(+2)-ATPase (PMCA), encoded by four separate genes, constitutes a high affinity system extruding Ca(+2) outside the cell. The nerve growth factor-treated PC12 cell line possesses all four main PMCA isoforms. To evaluate the potential role of PMCA isoforms in the differentiation process, we transiently suppressed the expression of PMCA2 and 3 using the antisense oligonucleotides. In the transfected PC12 cells, we observed morphological changes, slowed neurite extension and diminished survival of the cells. The apparent transport activity and affinity of the calcium pump to Ca(+2) were lower in the cells with suppressed PMCA2 and 3 isoforms than in the control cells. Moreover, in the transfected PC12 plasma membranes, the calcium pump was insensitive to stimulation by calmodulin. These findings suggest that PMCA2 and 3 isoforms may be involved in developmental and differentiation processes. [Abstract/Link to Full Text]

Moghaieb RE, Saneoka H, Fujita K
Shoot regeneration from GUS-transformed tomato (Lycopersicon esculentum) hairy root.
Cell Mol Biol Lett. 2004;9(3):439-49.
To study the influence of genetic background on the transformation and regeneration of cultivated tomato plants, hairy root lines of tomato (Lycopersicon esculentum) were obtained by inoculating the hypocotyl explants of three tomato cultivars with the Agrobacterium rhizogenes strain DCAR-2, which harbors the pBI-121 binary vector. The Ri-T-DNA transformation into the plant DNA was confirmed by both of mikimopine and GUS assay analyses. The regeneration efficiency from hairy root explants was assessed. The data indicated that white embryonic calli were formed within two weeks in the presence of 2 mgl(-1) 2, 4-D plus 0.25 mgl(-1) kinetin. Adventitious shoots emerged from the embryonic callus in the presence of 1 mgl(-1) GA3 along with 0.5 mgl(-1) NAA. The regeneration frequency was higher in the cultivar UC-97, followed by Momotaro and then Edkawi. Molecular confirmation of the integration of the GUS gene into the hairy root-derived plants genomes was done via PCR using GUS-specific primers and also using Southern blotting analysis. Our data shows that regeneration is possible from hairy roots of the cultivated tomato and this system could be used to produce transgenic tomato plants expressing the genes present in Agrobacterium rhizogenes binary vectors. [Abstract/Link to Full Text]

Sjakste N
Site-specific excision or protection of an alpha A globin gene genomic site in apoptotic transformed chicken erythroblasts.
Cell Mol Biol Lett. 2004;9(3):429-37.
There is still no clarity on whether the endonuclease incisions in apoptotic cells are induced randomly in the genome or induced in some preferable sites. In order to evaluate the intensity of DNA fragmentation in the chicken alpha-globin domain, AEV-virus transformed chicken erythroblasts (HD3) were incubated in a serum free medium, and their DNA was Southern blotted and hybridised with probes representing different fragments of the domain. Probes corresponding to the upstream areas of the domain mostly hybridised with high molecular weight DNA. Unlike these, the probe corresponding to the 2 Kb BamHI-BamHI fragment, containing the alphaA globin gene (B18), revealed a 5 Kb band on the hybridisation autoradiographs. The probe to the neighbouring upstream fragment did not reveal this band, but it was clearly seen on hybridisations with a downstream 1 Kb BamHI-BamHI fragment. The intensity of the band increased with overall apoptotic DNA degradation, hence its appearance should be coupled to apoptosis. Hybridisation of BamHI-digested DNA with B18 probe revealed a shortening of the 2 Kb band in preparations of DNA from apoptotic cells. The presumable positions of the cuts correspond to the formerly described DNase hypersensitive sites in the domain. Slot-blot and Northern hybridisation of RNA extracted from apoptotic HD3 cells revealed that the excision of the area of the B18 gene is coupled to a decrease in the intensity of alphaA globin gene transcription. Transcription of the non-erythroid NIK gene, transcribed in the upstream part of the domain, did not depend on the level of apoptotic DNA fragmentation. [Abstract/Link to Full Text]

Takahashi H, Yamaguchi T, Koga M, Kageura H, Terada S
DNA replication reaction in Xenopus cell-free system is suppressed by high pressure.
Cell Mol Biol Lett. 2004;9(3):423-7.
Previously, we demonstrated that when mouse erythroleukemia cells are exposed to a pressure of 80 MPa, the cell-cycle progression of S-phase cells is retarded. To examine the effects of high pressure on DNA replication, we used a Xenopus cell-free system. From cell-cycle progression of sperm nuclei, it was found that sperm nuclei are stable to a pressure of 80 MPa, whereas egg extracts are susceptible to high pressure. Similarly, biotin-16-dUTP was incorporated into 80 MPa-treated sperm nuclei in pressure-untreated extracts, but not into naive sperm nuclei in 80 MPa-treated extracts. These results indicate that DNA replication in Xenopus cell-free system is suppressed by the susceptibility of the extracts to a pressure of 80 MPa. [Abstract/Link to Full Text]

Sun X, Liu Y, Hu G, Wang H
Protective effects of nicotine against glutamate-induced neurotoxicity in PC12 cells.
Cell Mol Biol Lett. 2004;9(3):409-22.
This study aimed to assess whether nicotine prevented glutamate neurotoxicity in PC12 cells, and to identify the molecular mechanisms of any effects. The results showed that glutamate neurotoxicity in PC12 cells could be prevented by treatment with nicotine at concentrations of 10 nmol x l(-1) - 1 mmol x l(-1). This effect was in turn found to be inhibited by the application of the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine. Nicotine significantly decreased the basal level of intracellular free Ca(+2) and enhanced the buffering action on Ca(+2) overload induced by high concentrations of glutamate (5 mmol x l(-1)). In addition, nicotine treatment up-regulated the mRNA and protein expression of apoptosis-related factors including bcl-2 mRNA and protein, but down-regulated the expression of bax mRNA and protein. It is concluded that the protective effects of nicotine against the neurotoxicity induced by glutamate are mediated by nAChRs, due to the increased buffering action on Ca(+2)and the modulation of apoptotic processes. [Abstract/Link to Full Text]

Gasiorowski K, Steciwko A, Grata-Borkowska U, Drobnik J
The impact of neuraminidase on apoptosis in cultures of blood lymphocytes isolated from rats bearing morris hepatoma.
Cell Mol Biol Lett. 2004;9(2):389-99.
Lymphocytes were obtained by heart-punction from rats bearing Morris hepatoma. In the short term, 18-hour cultures of these lymphocytes exhibited a significantly higher amount of apoptotic cells than lymphocyte cultures from the healthy, control animals. Neuraminidase, injected into the caudal vein of the rats with Morris hepatoma, caused a marked lowering in the amount of apoptotic blood-lymphocytes and an elevation of the amount of viable cells. The possible mechanism of neuraminidase preventing the apoptosis of blood-circulating lymphocytes in tumour hosts is discussed herein. [Abstract/Link to Full Text]

Kargol A
Application of the ensemble nonequilibrium response spectroscopy to shaker potassium channel gating.
Cell Mol Biol Lett. 2004;9(2):375-88.
Standard electrophysiology techniques study relaxation transients in voltage-gated ion channels generated by discrete voltage steps. The nonequilibrium response spectroscopy involves analyzing responses to fluctuating potentials. We apply the ensemble NRS method to gating kinetics of Shaker potassium ion channels. We evaluate various proposed Markov models of channel gating from the nonequilibrium response viewpoint. These new NRS protocols can be used to test otherwise indistinguishable models or improve estimates for parameters of channel kinetics models. [Abstract/Link to Full Text]

Gorensek M, Jaksimovi? C, Kregar-Velikonja N, Gorensek M, Knezevic M, Jeras M, Pavlovcic V, Cör A
Nucleus pulposus repair with cultured autologous elastic cartilage derived chondrocytes.
Cell Mol Biol Lett. 2004;9(2):363-73.
Low back pain is one of the most common medical conditions in the Western world. Disc degeneration, an inevitable process of ageing, is one of the major causes of low back pain. Autologous chondrocyte transplantation (ACT) is an increasingly popular method of addressing pathological disorders of cartilage. The purpose of our study was to determine whether autologous chondrocytes from elastic cartilage could survive and synthesise a cartilage specific matrix in the intervertebral disc of rabbits. Sixteen lumbar intervertebral discs (IVD) of New Zealand White rabbits were analysed. In 6 IVD, the nucleus pulposus was evacuated and replaced with tissue engineered autologous chondrocytes from auricular cartilage. In the second group, only the nucleus pulposus was evacuated from 6 IVD, with no chondrocytes implantation. Four non-operated IVD were used as a control. Six months after the operation, the animals were euthanized and the IVD were analysed histologically. Autologous cartilage implants were well tolerated by the host for up to six months in vivo. There was only hyaline-like cartilage in the place of the nucleus pulposus. We could not detect any elastic fibres in the new cartilage matrix. In IVD from which only the nucleus pulposus was evacuated and no chondrocytes were implanted, just fibrous tissue was found instead of nucleus pulposus. The overall histological analysis of new cartilage produced after implantation in our study confirmed the hypothesis that ACT from auricular cartilage can be implanted into the IVD instead of the nucleus pulposus and that a significant percentage of implanted chondrocytes survive and produce hyaline-like cartilage. [Abstract/Link to Full Text]

Birsin MA, Ozgen M
A comparison of callus induction and plant regeneration from different embryo explants of triticale (x Triticosecale wittmack).
Cell Mol Biol Lett. 2004;9(2):353-61.
Immature, mature and endosperm-supported mature embryos of six triticale cultivars (BDMT-98-8S, Melez-2001, Mikham-2002, Presto, Tacettin Bey and Tatlicak-97) were cultured in vitro to compare the levels of callus induction and plant regeneration. Immature embryos, 15-18 days after anthesis, were aseptically excised and placed with the scutellum upwards on a callus culture medium consisting of Murashige and Skoog (MS) mineral salts supplemented with 2 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were aseptically excised from the imbibed seeds and placed scutellum up on MS medium supplement with 2 mg l(-1) 2,4-D. Endosperm-supported mature embryos were moved slightly in the imbibed mature seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg l(-1) 2,4-D for callus induction. The developed calli and regenerated plants were maintained on hormone-free MS medium. Variability among the genotypes was observed for all the types of embryo culture. Immature embryos from "Presto" and endosperm-supported mature embryos from "Mikham 2002" had excellent regeneration capacities (92.0% and 97.3%, respectively) and the highest number of plants regenerated growing in soil (9 and 13, respectively). A comparison of the responses of the three explants used indicated that the endosperm-supported mature embryo was the most useful explant for plant regeneration in triticale. [Abstract/Link to Full Text]

Ramalho-Santos J, Pedroso De Lima MC
The role of target membrane sialic acid residues in the fusion activity of the influenza virus: the effect of two types of ganglioside on the kinetics of membrane merging.
Cell Mol Biol Lett. 2004;9(2):337-51.
The influenza virus enters target cells via the action of hemagglutinin proteins (HA) inserted into the viral envelope. HA promotes membrane fusion between the viral envelope and endosomal membrane at low pH, following viral binding to sialic acid-containing receptors on target cells, and internalization by endocytosis. The effect of target membrane sialic acid residues on the fusion activity of the influenza virus towards model membranes was evaluated by both reduction, (i.e. treating somatic cells with neuraminidase- (NA-) prior to virus-cell interactions), and by supplementing liposomes with the gangliosides GD1a and GT1b. The harshness of the neuraminidase pretreatment of target cells required to affect virus-induced membrane merging was found to greatly depend on the assay conditions, i.e. whether a virus-cell prebinding step at neutral pH was included prior to acidification. Minor concentrations of neuraminidase were found to greatly reduce virus fusion, but only in the absence of a prebinding step; they had no effect if this step was included. Although membrane merging was greatly reduced following cell neuraminidase pretreatment, virus-cell association at low pH was not disturbed proportionately. This probably reflects unspecific virus-cell binding under these conditions, probably of inactivated or aggregated virus particles, which does not translate into membrane merging. This seems to suggest both that target membrane sialic acid can protect the virus from losing its activity before triggering membrane merging, and that the importance of this interaction is not merely to ensure virus-target proximity. With liposomes, we found that both types of ganglioside supported efficient fusion, with GD1a promoting a slightly faster initial rate. However, in this case, virus-target proximity closely mirrored fusion activity, thus pointing to differential specificity between targets routinely used to assay influenza virus fusion activity. [Abstract/Link to Full Text]

Hawrylak B, Szyma?ska M
Selenium as a sulphydrylic group inductor in plants.
Cell Mol Biol Lett. 2004;9(2):329-36.
We investigated the effect of selenium form and dose on the total glutathione and non-protein -SH group contents in the edible spinach (Spinacia oleracea L.) and ground tomato (Lycopersicon esculentum Mill.) plants. Our experiments were carried out in a hydroponic culture. Selenium was added to the culture medium in its selenite (Na2SeO3 x 5H2O) and selenate (Na2SeO4) forms. Regardless of the selenium form, we observed an increase in the non-protein thiol content. The non-protein -SH group content depended on the form and dose of selenium as well as on the organ and plant species. Regardless of the selenium form, a higher content of non-protein -SH groups were found in the spinach biomass than in the tomato biomass. Selenite contributed to a larger accumulation of non-protein -SH groups in the roots, whereas selenate contributed to their accumulation in the shoots [Abstract/Link to Full Text]

Krze?lak A, Lipi?ska A
Galectin-3 as a multifunctional protein.
Cell Mol Biol Lett. 2004;9(2):305-28.
Galectin-3 is a 31 kDa member of a growing family of beta-galactoside-binding animal lectins. This protein is expressed in a variety of tissues and cell types and is mainly found in the cytoplasm, although, depending on cell type and proliferative state, a significant amount of this lectin can also be detected in the nucleus, on the cell surface or in the extracellular environment. Galectin-3 is secreted from cells by a novel and incompletely understood mechanism that is independent of the classical secretory pathway through the endoplasmic reticulum/Golgi network. Galectin-3 exhibits pleiotropic biological function, playing a key role in many physiological and pathological processes. [Abstract/Link to Full Text]

Lenfant F, Lahet JJ, Chaillot B, Freysz M
The protective effects of lidocaine on human erythrocytes stored for seven days at 04 degrees C.
Cell Mol Biol Lett. 2004;9(2):301-4.
Erythrocyte storage may result in cell damage due to an alteration of membrane integrity, which results in potassium efflux and hemolysis. Lidocaine has been shown to protect erythrocytes from oxidative stress by a possible membrane effect. We conducted this study to examine the effects of lidocaine on human erythrocyte storage. Erythrocytes were kept for seven days at 04 degrees C in the absence or in presence of plasma, and of lidocaine at 36.9 and 221.6 microM. Cell damage was assessed by measuring potassium efflux in the supernatant after seven days, and studying potassium efflux and hemolysis induced by oxidative stress. As expected, erythrocyte storage in the presence of plasma was less deleterious. Lidocaine decreased potassium efflux after 7 days' storage. Resistance toward oxidative stress was greater when the erythrocytes had been kept in the presence of plasma. Considering that lidocaine is widely used in various clinical situations, this data may be of clinical relevance. [Abstract/Link to Full Text]

Al-Forkan M, Power JB, Anthony P, Lowe KC, Davey MR
Agrobacterium-mediated transformation of Bangladeshi Indica rices.
Cell Mol Biol Lett. 2004;9(2):287-300.
Morphologically normal, fertile transgenic plants were obtained by co-culturing embryogenic calli of the Bangladeshi Indica rice cultivars BR26 and Binni with Agrobacterium tumefaciens strain LBA4404 carrying the super binary vector pTOK233. Acetosyringone (100 microM) in the medium during co-culture (25-28 degrees C) and selection on hygromycin B (50 mg l(-1)) were essential for efficient transformation. Stable integration and expression of beta-glucuronidase, neomycin phosphotransferase and hygromycin phosphotransferase genes in regenerated plants were confirmed by histochemical and fluorometric assays, ELISA and Southern analysis. Two to 3 copies of T-DNA were integrated into regenerated plants; transgene expression did not correlate with gene copy number. Mendelian segregation of transgenes occurred in T1 seed progeny. [Abstract/Link to Full Text]

Smaczy?ska-de Rooij I, Migdalski A, Rytka J
Alpha-Ketoglutarate dehydrogenase and lipoic acid synthase are important for the functioning of peroxisomes of Saccharomyces cerevisiae.
Cell Mol Biol Lett. 2004;9(2):271-86.
A method was devised to search for yeast mutants impaired in peroxisome functioning, indicating cross-talk between metabolic pathways. Two mutants were isolated; they are impaired in oleate utilisation and carry mutations in the KGD1 and LIP5 genes encoding the E1 component of the mitochondrial alpha-ketoglutarate dehydrogenase complex and lipoic acid synthase, respectively. The results presented indicate that the Kgd1 and Lip5 proteins are important for the expression of genes encoding peroxisomal matrix proteins, although they are not necessary for the biogenesis of this cellular compartment. [Abstract/Link to Full Text]

Bukowska B
Damage to erythrocytes caused by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (in vitro).
Cell Mol Biol Lett. 2004;9(2):261-70.
The effects of the exposure of human erythrocytes to different concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin were studied. Particular attention was paid to lipid peroxidation, haemoglobin oxidation, and changes in the activity of catalase and glutathione peroxidase. Human erythrocytes at a 5% haematocrit were incubated with 2,3,7,8-TCDD at concentrations of 0.2 ppm to 1.6 ppm (ng-microg/ml erythrocytes) for 1 hour. The results obtained show that 2,3,7,8-TCDD induces the generation of lipid peroxides and the oxidation of Hb, and decreases the activity of catalase and glutathione peroxidase. This supports the thesis that TCDD causes oxidative stress in erythrocytes. [Abstract/Link to Full Text]

Boraty?ski J, Syper D, Weber-Dabrowska B, ?usiak-Szelachowska M, Po?niak G, Górski A
Preparation of endotoxin-free bacteriophages.
Cell Mol Biol Lett. 2004;9(2):253-9.
Bacteriophages (phages) are bacterial viruses that interact with bacterial walls and invade bacterial cells. Moreover, they disturb bacterial metabolism and lead to bacteria lysis. In the case of Gram-negative bacteria crude phage cultures, apart from the phages themselves, the bacterial debris, bacterial proteins and nucleic acids contain endotoxins. These endotoxins (lipopolysaccharides) posses a high degree of toxicity in vitro and in vivo, and their removal is essential for safety in antibacterial bacteriophage therapy. An effective, scaleable purification of bacteriophages from endotoxins was accomplished by sequential ultrafiltration through polysulfone membrane (30 nm) followed by chromatography on sepharose 4B and Matrex Cellulofine Sulfate. The phage fraction after gel filtration chromatography routinely contained endotoxins in the 150-2500 EU/ml range. The procedure yielded bacteriophages contaminated with as little as 0.4-7 EU/ml (Limulus assay). This value lies within the permitted level for intravenous applications (5 EU/kg/h by European Pharmacopoeia, 1997). [Abstract/Link to Full Text]

Jankowiak K, Lesicka J, Pacak A, Rybarczyk A, Szweykowska-Kuli?ska Z
A comparison of group II introns of plastid tRNALysUUU genes encoding maturase protein.
Cell Mol Biol Lett. 2004;9(2):239-51.
All higher plant plastid genomes have six classes of tRNA genes containing introns. One of those is the tRNALysUUU gene, which encodes maturase protein. In the case of liverwort species from the genus Porella and mosses from the genus Plagiomnium, the maturase coding gene (matK) represents a truncated form of other plant matK genes: several subdomains of the reverse transcriptase-like domain and so-called domain X are not present in these ORFs. These ORFs probably represent pseudogenes of the matK gene. The analysis of codon usage within the matK gene revealed the presence of strong A/T pressure. The use of codons with the third letter being U or A varies from 71-93%. The comparison of maturase amino acid sequences at the family level shows a high identity between species. However, when liverwort and angiosperm maturase sequences are compared, the percentage of identity drops dramatically. The calculated values of the number of nucleotide substitutions vary considerably, even when liverwort species are compared pairwise. The phenetic tree of relationships between plant species on the basis of tRNALysUUU intron sequences concur with the generally accepted plant phylogeny. [Abstract/Link to Full Text]

Rakoczy-Trojanowska M, Bolibok H
Characteristics and a comparison of three classes of microsatellite-based markers and their application in plants.
Cell Mol Biol Lett. 2004;9(2):221-38.
Microsatellites (SSR--simple sequence repeats, STR--short tandem repeats, SSLP--simple sequence length polymorphism, VNTR--variable number of tandem repeats) are the class of repetitive DNA sequences present in all living organisms. Particular characteristics of microsatellites, such as their presence in the genomes of all living organisms, high level of allelic variation, co-dominant mode of inheritance and potential for automated analysis make them an excellent tool for a number of approaches like genotyping, mapping and positional cloning of genes. The three most popular types of markers containing microsatellite sequences that are presently used are: (1) SSR (simple sequence repeats), generated by amplifying in a PCR reaction with the use of primers complementary to flanking regions; (2) ISSR (inter-simple sequence repeats), based on the amplification of regions between inversely oriented closely spaced microsatellites; and (3) SAMPL (selective amplification of microsatellite polymorphic loci), which utilises AFLP (amplified fragment-length polymorphism) methodology, with one exception--for the second amplification, one of the starters is complementary to the microsatellite sequence. The usefulness of the three above-mentioned markers for numerous purposes has been well documented for plants. [Abstract/Link to Full Text]

Krajewska WM, Mas?owska I
Caveolins: structure and function in signal transduction.
Cell Mol Biol Lett. 2004;9(2):195-220.
The caveolin family proteins are typically associated with microdomains that are found in the plasma membrane of numerous cells. These microdomains are referred to as/called caveolae. Caveolins are small proteins (18-24 kDa) that have a hairpin loop conformation with both the N and C termini exposed to the cytoplasm. Apart from having a structural function within caveolae, these proteins have the capacity to bind cholesterol as well as a variety of proteins, such as receptors, Src-like kinases, G-proteins, H-Ras, MEK/ERK kinases and nitric oxide synthases, which are involved in signal transduction processes. Considerable data allow the assumption to be made that the majority of the interactions with signaling molecules hold them in an inactive or repressed state. The activity of caveolins seems to be dependent on its specific post-translation modifications. It is suggested that caveolins fulfill a role in the modulation of cellular signaling cascades. [Abstract/Link to Full Text]

Buevich AV, Lundberg S, Sethson I, Edlund U, Backman L
NMR studies of calcium-binding to mutant alpha-spectrin EF-hands.
Cell Mol Biol Lett. 2004;9(1):167-86.
The co-operative calcium binding mechanism of the two C-terminal EF-hands of human alphaII-spectrin has been investigated by site-specific mutagenesis and multi-dimensional NMR spectroscopy. To analyse the calcium binding of each EF-hand independently, two mutant structures (E33A and D69S) of wild type alpha-spectrin were prepared. According to NMR analysis both E33A and D69S were properly folded. The unmutated EF-hand in these mutants remained nearly intact and active in calcium binding, whereas the mutated EF-hand lost its affinity for calcium completely. The apparent calcium binding affinity of the E33A mutant was much lower compared to the D39S mutant (approximately 2470 microM and approximately 240 microM, respectively). When the chemical shift perturbations were followed upon calcium titration, a positive correlation between the D69S mutant and the binding of the first calcium ion to the wild type was revealed. These observations showed that the first EF-hand in spectrin binds the first calcium ion and thereby triggers a conformational change that allows the second calcium ion to bind to the other EF-hand. [Abstract/Link to Full Text]

Fedorov A, Lukyanov D, Rogoli?ski J, Wid?ak P, Podgornaya O, Rzeszowska-Wolny J
The nuclear protein p30 specifically interacts with a nuclear matrix attachment region from the rat genome.
Cell Mol Biol Lett. 2004;9(1):153-65.
In our previous study, a 454 bp DNA fragment was isolated from rat genomic DNA as an element which interacts with nuclear matrix proteins, i.e. a Matrix Associated Region (MAR). Computer analyses revealed that the right half of this fragment, named RME (Rat MAR Element), possesses a high matrix association potential and is likely to be responsible for the matrix association of the whole sequence. RME was used as a probe in an electrophoretic mobility shift assay (EMSA), and with the use of Southwestern blotting, a rat liver nuclear protein which binds specifically to it was identified. Its molecular mass was estimated by SDS-PAGE as 30 kDa (p30). Polyclonal antibodies raised against protein-RME complexes caused a super-shift of specific complexes in EMSA, and bound to p30 in nuclear extracts of rat liver in Western blotting. The immunofluorescence labelling of a rat embryonic fibroblast cell monolayer with anti-p30 antibody revealed a mainly intranuclear pattern of staining. [Abstract/Link to Full Text]

Bany-?aszewicz U, Kami?ska J, Klimczak-Jajor E, Ko?cielak J
The activity of alpha1,6-fucosyltransferase during human megakaryocytic differentiation.
Cell Mol Biol Lett. 2004;9(1):145-52.
alpha1,6-Fucosyltransferase (6FucT, E.C. 2.4.1.68) is one of the enzymes involved in the synthesis of N-linked glycans of the GpIIb/IIIa complex (CD41a) which is present on megakaryocytes (MKs) and platelets. In this study, we examined 6FucT activity in ex vivo cultures of immunoselected cord blood CD34(+) cells grown in a medium promoting megakaryocytopoiesis. Our results show that the activity of 6FucT increased ahead of, and thereafter concomitantly with, cells expressing the CD41a antigen. When the CD41a(+) subpopulation of cells was immunoselected (using anti-CD61 i.e. anti-GpIIIa antibodies), its 6FucT activity increased proportionally to the yield of CD61(+)(+)(+) cells. Taking into account the heavy load of 6FucT in platelets and megakaryocytes, we regard this enzyme as a candidate for the earliest marker of MK-commitment in cultured hematopoietic stem cells. Such a marker should allow an earlier detection and earlier transplantation of patients' own, ex vivo expanded, Mk progenitors. [Abstract/Link to Full Text]

Barg E, Gasiorowski K, Brokos B, Swiedrych A, Skórkowska K
A high frequency of apoptosis was found in cultures of lymphocytes isolated from the venous blood of children born with a low birth weight.
Cell Mol Biol Lett. 2004;9(1):135-43.
Children born with a low birth weight (below 2500 g) exhibit a slower rate of development, and a greater tendency towards morbidity and mortality, together with a deficit of weight and height. One reason could be an increase in the level of cell elimination by apoptosis. The aim of this study was to evaluate and compare the incidence of apoptotic and necrotic (dead) cells in cultures of peripheral blood lymphocytes obtained from children born with a low birth weight and from children with a normal birth weight. Peripheral blood lymphocytes were obtained by venipuncture (10 ml) and isolated using the density gradient centrifugation method. The lymphocytes were cultured for 48 h in a culture medium containing low concentrations of fetal calf serum. A comparison study was performed between low birth weight children and normal birth weight children and the susceptibility of their lymphocytes to apoptosis and to necrosis in serum-deficient feeding culture conditions. The amount of apoptotic cells and the percentage of dead cells were significantly higher in cultures of lymphocytes obtained from low birth weight children than in cultures from normal birth weight children. The two estimated parameters inversely correlated with the concentration of fetal calf serum in the culture medium. Pulsed field gel electrophoresis showed increased DNA degradation patterns in the cultures of lymphocytes obtained from low birth weight children. Our results should be perceived as an indication that, under worse feeding conditions, the elimination of cells by apoptosis and by necrosis is significantly higher for lymphocytes of low birth weight children than for those of normal birth weight children. The enhanced elimination of lymphocytes is related to a greater susceptibility to infections, especially of the respiratory tract, as established in the retrospective analysis of the anamneses of the examined group of low birth weight children. [Abstract/Link to Full Text]

Wid?ak W, Wid?ak P
MAR/SAR elements flank the rat hst70 gene transcription unit.
Cell Mol Biol Lett. 2004;9(1):123-33.
The rat hst70 gene is specifically expressed in spermatocytes and spermatids. This tissue-specific expression of the gene is primarily mediated through cis-acting elements located within the 0.4 kb segment upstream of the coding region, including two transcription initiation sites. Here, we study the 5' and 3' distal elements flanking the hst70 gene and find that they possess structural motifs characteristic of MAR/SAR elements, and exhibit enhanced affinities for nuclear matrix binding in vitro. Such elements bind efficiently to matrices from either the testis or the liver, i.e. tissues where the gene is either fully active or repressed, although one subfragment in the 5' region was identified as exhibiting testis-specific interactions. Surprisingly, the activity of the CAT reporter gene was repressed in testis-transient transfection assays when the hst70 promoter sequences were extended into the 5' MAR/SAR. [Abstract/Link to Full Text]

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Recent Articles in BMC Cell Biology

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Ivanov AI, McCall IC, Babbin B, Samarin SN, Nusrat A, Parkos CA
Microtubules regulate disassembly of epithelial apical junctions.
BMC Cell Biol. 2006;712.
BACKGROUND: Epithelial tight junction (TJ) and adherens junction (AJ) form the apical junctional complex (AJC) which regulates cell-cell adhesion, paracellular permeability and cell polarity. The AJC is anchored on cytoskeletal structures including actin microfilaments and microtubules. Such cytoskeletal interactions are thought to be important for the assembly and remodeling of apical junctions. In the present study, we investigated the role of microtubules in disassembly of the AJC in intestinal epithelial cells using a model of extracellular calcium depletion. RESULTS: Calcium depletion resulted in disruption and internalization of epithelial TJs and AJs along with reorganization of perijunctional F-actin into contractile rings. Microtubules reorganized into dense plaques positioned inside such F-actin rings. Depolymerization of microtubules with nocodazole prevented junctional disassembly and F-actin ring formation. Stabilization of microtubules with either docetaxel or pacitaxel blocked contraction of F-actin rings and attenuated internalization of junctional proteins into a subapical cytosolic compartment. Likewise, pharmacological inhibition of microtubule motors, kinesins, prevented contraction of F-actin rings and attenuated disassembly of apical junctions. Kinesin-1 was enriched at the AJC in cultured epithelial cells and it also accumulated at epithelial cell-cell contacts in normal human colonic mucosa. Furthermore, immunoprecipitation experiments demonstrated association of kinesin-1 with the E-cadherin-catenin complex. CONCLUSION: Our data suggest that microtubules play a role in disassembly of the AJC during calcium depletion by regulating formation of contractile F-actin rings and internalization of AJ/TJ proteins. [Abstract/Link to Full Text]

McKenzie G, Ward G, Stallwood Y, Briend E, Papadia S, Lennard A, Turner M, Champion B, Hardingham GE
Cellular Notch responsiveness is defined by phosphoinositide 3-kinase-dependent signals.
BMC Cell Biol. 2006;710.
BACKGROUND: Notch plays a wide-ranging role in controlling cell fate, differentiation and development. The PI3K-Akt pathway is a similarly conserved signalling pathway which regulates processes such as differentiation, proliferation and survival. Mice with disrupted Notch and PI3K signalling show phenotypic similarities during haematopoietic cell development, suggesting functional interaction between these pathways. RESULTS: We show that cellular responsiveness to Notch signals depends on the activity of the PI3K-Akt pathway in cells as diverse as CHO cells, primary T-cells and hippocampal neurons. Induction of the endogenous PI3K-Akt pathway in CHO cells (by the insulin pathway), in T-cells (via TCR activation) or in neurons (via TrKB activation) potentiates Notch-dependent responses. We propose that the PI3K-Akt pathway exerts its influence on Notch primarily via inhibition of GSK3-beta, a kinase known to phosphorylate and regulate Notch signals. CONCLUSION: The PI3K-Akt pathway acts as a "gain control" for Notch signal responses. Since physiological levels of intracellular Notch are often low, coincidence with PI3K-activation may be crucial for induction of Notch-dependent responses. [Abstract/Link to Full Text]

Chang HH, Oh PY, Ingber DE, Huang S
Multistable and multistep dynamics in neutrophil differentiation.
BMC Cell Biol. 2006;711.
BACKGROUND: Cell differentiation has long been theorized to represent a switch in a bistable system, and recent experimental work in micro-organisms has revealed bistable dynamics in small gene regulatory circuits. However, the dynamics of mammalian cell differentiation has not been analyzed with respect to bistability. RESULTS: Here we studied how HL60 promyelocytic precursor cells transition to the neutrophil cell lineage after stimulation with the differentiation inducer, dimethyl sulfoxide (DMSO). Single cell analysis of the expression kinetics of the differentiation marker CD11b (Mac-1) revealed all-or-none switch-like behavior, in contrast to the seemingly graduated change of expression when measured as a population average. Progression from the precursor to the differentiated state was detected as a discrete transition between low (CD11bLow) and high (CD11bHigh) expressor subpopulations distinguishable in a bimodal distribution. Hysteresis in the dependence of CD11b expression on DMSO dose suggests that this bimodality may reflect a bistable dynamic. But when an "unswitched" (CD11bLow) subpopulation of cells in the bistable/bimodal regime was isolated and cultured, these cells were found to differ from undifferentiated precursor cells in that they were "primed" to differentiate. CONCLUSION: These findings indicate that differentiation of human HL60 cells into neutrophils does not result from a simple state transition of a bistable switch as traditionally modeled. Instead, mammalian differentiation appears to be a multi-step process in a high-dimensional system, a result which is consistent with the high connectivity of the cells' complex underlying gene regulatory network. [Abstract/Link to Full Text]

Machuca C, Mendoza-Milla C, Córdova E, Mejía S, Covarrubias L, Ventura J, Zentella A
Dexamethasone protection from TNF-alpha-induced cell death in MCF-7 cells requires NF-kappaB and is independent from AKT.
BMC Cell Biol. 2006;79.
BACKGROUND: The biochemical bases for hormone dependence in breast cancer have been recognized as an important element in tumor resistance, proliferation and metastasis. On this respect, dexamethasone (Dex) dependent protection against TNF-alpha-mediated cell death in the MCF-7 cell line has been demonstrated to be a useful model for the study of this type of cancer. Recently, cytoplasmic signaling induced by steroid receptors has been described, such as the activation of the PI3K/Akt and NF-kappaB pathways. We evaluated their possible participation in the Dex-dependent protection against TNF-alpha-mediated cell death. RESULTS: Cellular cultures of the MCF-7 cell line were exposed to either, TNF-alpha or TNF-alpha and Dex, and cell viability was evaluated. Next, negative dominants of PI3K and IkappaB-alpha, designed to block the PI3K/Akt and NF-kappaB pathways, respectively, were transfected and selection and evaluation of several clones overexpressing the mutants were examined. Also, correlation with inhibitor of apoptosis proteins (IAPs) expression was examined. Independent inhibition of these two pathways allowed us to test their participation in Dex-dependent protection against TNF-alpha-cytotoxicity in MCF-7 cells. Expression of the PI3K dominant negative mutant did not alter the protection conferred by Dex against TNF-alpha mediated cell death. Contrariwise, clones expressing the IkappaB-alpha dominant negative mutant lost the Dex-conferred protection against TNF-alpha. In these clones degradation of c-IAP was accelerated, while that of XIAP was remained unaffected. CONCLUSION: NF-kappaB, but not PI3K/Akt activation, is required for the Dex protective effect against TNF-alpha-mediated cell death, and correlates with lack of degradation of the anti-apoptotic protein c-IAP1. [Abstract/Link to Full Text]

Sawai H, Okada Y, Funahashi H, Matsuo Y, Takahashi H, Takeyama H, Manabe T
Interleukin-1alpha enhances the aggressive behavior of pancreatic cancer cells by regulating the alpha6beta1-integrin and urokinase plasminogen activator receptor expression.
BMC Cell Biol. 2006;78.
BACKGROUND: In human pancreatic cancer progression, the alpha6beta1-integrin is expressed on cancer cell surface during invasion and metastasis formation. In this study, we investigated whether interleukin (IL)-1alpha induces the alterations of integrin subunits and urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) expression in pancreatic cancer cells. We hypothesize that the alterations of integrin subunits and uPA/uPAR expression make an important role in signaling pathways responsible for biological behavior of pancreatic cancer cells. RESULTS: IL-1alpha upregulated the expression of alpha6 and beta1 integrins without any alterations of alpha5 and alphav integrins expression. IL-1alpha also induced enhancement in the expression of uPA/uPAR in pancreatic cancer cells. IL-1alpha enhanced the proliferation, adhesion, and migration in pancreatic cancer cells, and IL-1alpha-induced alterations of uPA/uPAR expression correlated with the increased the migration of pancreatic cancer cells. Upregulation of alpha6 integrin subunit and uPA/uPAR correlated with the activation of Ras and downstream extracellular signal-regulated kinase (ERK) pathways. IL-1alpha-induced activation of Ras and downstream ERK can be inhibited by using inhibitory antibodies against alpha6 and beta1 integrin and uPAR, consistent with the inhibition of proliferation, adhesion and migration of pancreatic cancer cells. Immunohistochemical analysis demonstrated a significant association between strong expressions of alpha6 integrin with uPAR in pancreatic cancer specimens. Furthermore, the strong expression of alpha6 integrin and uPAR was found to be independent prognosticator in pancreatic cancer patients. CONCLUSION: Based on these findings, we conclude that IL-1alpha can induce selective upregulation of alpha6beta1-integrin and uPA/uPAR in pancreatic cancer cells and these changes may modulate the aggressive functions of pancreatic cancer. [Abstract/Link to Full Text]

Ryabinina OP, Subbian E, Iordanov MS
D-MEKK1, the Drosophila orthologue of mammalian MEKK4/MTK1, and Hemipterous/D-MKK7 mediate the activation of D-JNK by cadmium and arsenite in Schneider cells.
BMC Cell Biol. 2006;77.
BACKGROUND: The family of c-Jun NH2-terminal kinases (JNK) plays important roles in embryonic development and in cellular responses to stress. Toxic metals and their compounds are potent activators of JNK in mammalian cells. The mechanism of mammalian JNK activation by cadmium and sodium arsenite involves toxicant-induced oxidative stress. The study of mammalian signaling pathways to JNK is complicated by the significant degree of redundancy among upstream JNK regulators, especially at the level of JNK kinase kinases (JNKKK). RESULTS: Using Drosophila melanogaster S2 cells, we demonstrate here that cadmium and arsenite activate Drosophila JNK (D-JNK) via oxidative stress as well, thus providing a simpler model system to study JNK signaling. To elucidate the signaling pathways that lead to activation of D-JNK in response to cadmium or arsenite, we employed RNA interference (RNAi) to knock down thirteen upstream regulators of D-JNK, either singly or in combinations of up to seven at a time. CONCLUSION: D-MEKK1, the fly orthologue of mammalian MEKK4/MTK1, and Hemipterous/D-MKK7 mediates the activation of D-JNK by cadmium and arsenite. [Abstract/Link to Full Text]

Fabbri F, Carloni S, Brigliadori G, Zoli W, Lapalombella R, Marini M
Sequential events of apoptosis involving docetaxel, a microtubule-interfering agent: a cytometric study.
BMC Cell Biol. 2006;76.
BACKGROUND: Despite the great advances in the understanding of programmed cell death, little attention has been paid to the sequence of the events that characterise it. In particular, the course of apoptotic events induced by microtubule-interfering agents such as taxanes is poorly understood. In order to increase such knowledge, we studied a number of independent biochemical and cytological modifications using cytometric methods in a bladder cancer cell line treated with the second generation taxane, docetaxel. RESULTS: Within a few hours, drug treatment had induced mitochondrial membrane transition, cell shrinkage and a decrease in granularity. Cell cycle was almost completely blocked in G2/M phase within 24 hours. The hypodiploid peak started to become prominent 48 hours after the treatment. At the same time, the appearance of a DNA ladder demonstrated caspase-dependent chromatin fragmentation. Concurrently, specific cell surface modifications took place, involving at first glycoprotein syalilation and later phospholipid asymmetry. DNA fragmentation was subsequently detected by TUNEL assay. Over time, cell membranes became permeable to propidium iodide. A very similar time-course of apoptotic events was found after treatment of a myelomonocytic cell line with the same drug. CONCLUSION: After discussing some characteristics of the methods employed and their limitations, a succession of apoptotic events over time is suggested, in which the collapse of mitochondrial transmembrane potential (Deltapsim) is the earliest sign of apoptosis. [Abstract/Link to Full Text]

Clotworthy M, Traynor D
On the effects of cycloheximide on cell motility and polarisation in Dictyostelium discoideum.
BMC Cell Biol. 2006;75.
BACKGROUND: Cycloheximide is a protein synthesis inhibitor that acts specifically on the 60S subunit of eukaryotic ribosomes. It has previously been shown that a short incubation of Dictyostelium discoideum amoebae in cycloheximide eliminates fluid phase endocytosis. RESULTS: We found that treatment with cycloheximide also causes the amoebae to retract their pseudopodia, round up and cease movement. Furthermore, fluid phase endocytosis, phagocytosis and capping cease in the presence of 2 mM cycloheximide, although membrane uptake, as measured using FM1-43, is unaffected. In the presence of cycloheximide, aggregation-competent amoebae sensitive to cAMP, although round, can still localise CRAC, ABP120, PI3K and actin polymerisation in response to a micropipette filled with cAMP. The behaviour of wild-type amoebae in the presence of cycloheximide is surprisingly similar to that of amoebae having a temperature-sensitive version of NSF at the restrictive temperature. CONCLUSION: Our results may suggest that, upon cycloheximide treatment, either a labile protein required for polarised membrane recycling is lost, or a control mechanism linking protein synthesis to membrane recycling is activated. [Abstract/Link to Full Text]

Fathke C, Wilson L, Shah K, Kim B, Hocking A, Moon R, Isik F
Wnt signaling induces epithelial differentiation during cutaneous wound healing.
BMC Cell Biol. 2006;74.
BACKGROUND: Cutaneous wound repair in adult mammals does not regenerate the original epithelial architecture and results in altered skin function. We propose that lack of regeneration may be due to the absence of appropriate molecular signals to promote regeneration. In this study, we investigated the regulation of Wnt signaling during cutaneous wound healing and the consequence of activating either the beta-catenin-dependent or beta-catenin-independent Wnt signaling on epidermal architecture during wound repair. RESULTS: We determined that the expression of Wnt ligands that typically signal via the beta-catenin-independent pathway is up-regulated in the wound while the beta-catenin-dependent Wnt signaling is activated in the hair follicles adjacent to the wound edge. Ectopic activation of beta-catenin-dependent Wnt signaling with lithium chloride in the wound resulted in epithelial cysts and occasional rudimentary hair follicle structures within the epidermis. In contrast, forced expression of Wnt-5a in the deeper wound induced changes in the interfollicular epithelium mimicking regeneration, including formation of epithelia-lined cysts in the wound dermis, rudimentary hair follicles and sebaceous glands, without formation of tumors. CONCLUSION: These findings suggest that adult interfollicular epithelium is capable of responding to Wnt morphogenic signals necessary for restoring epithelial tissue patterning in the skin during wound repair. [Abstract/Link to Full Text]

Langford KJ, Askham JM, Lee T, Adams M, Morrison EE
Examination of actin and microtubule dependent APC localisations in living mammalian cells.
BMC Cell Biol. 2006;73.
BACKGROUND: The trafficking of the adenomatous polyposis coli (APC) tumour suppressor protein in mammalian cells is a perennially controversial topic. Immunostaining evidence for an actin-associated APC localisation at intercellular junctions has been previously presented, though live imaging of mammalian junctional APC has not been documented. RESULTS: Using live imaging of transfected COS-7 cells we observed intercellular junction-associated pools of GFP-APC in addition to previously documented microtubule-associated GFP-APC and a variety of minor localisations. Although both microtubule and junction-associated populations could co-exist within individual cells, they differed in their subcellular location, dynamic behaviour and sensitivity to cytoskeletal poisons. GFP-APC deletion mutant analysis indicated that a protein truncated immediately after the APC armadillo repeat domain retained the ability to localise to adhesive membranes in transfected cells. Supporting this, we also observed junctional APC immunostaining in cultures of human colorectal cancer cell line that express truncated forms of APC. CONCLUSION: Our data indicate that APC can be found in two spatially separate populations at the cell periphery and these populations can co-exist in the same cell. The first localisation is highly dynamic and associated with microtubules near free edges and in cell vertices, while the second is comparatively static and is closely associated with actin at sites of cell-cell contact. Our imaging confirms that human GFP-APC possesses many of the localisations and behaviours previously seen by live imaging of Xenopus GFP-APC. However, we report the novel finding that GFP-APC puncta can remain associated with the ends of shrinking microtubules. Deletion analysis indicated that the N-terminal region of the APC protein mediated its junctional localisation, consistent with our observation that truncated APC proteins in colon cancer cell lines are still capable of localising to the cell cortex. This may have implications for the development of colorectal cancer. [Abstract/Link to Full Text]

Sikand K, Kaul D, Varma N
Receptor Ck-dependent signaling regulates hTERT gene transcription.
BMC Cell Biol. 2006;72.
BACKGROUND: Available evidence suggests that the regulation of telomerase activity primarily depends on the transcriptional control of the human telomerase reverse transcriptase (hTERT) gene. Although several activators and repressors of hTERT gene transcription have been identified, the exact mechanism by which hTERT transcription is repressed in normal cells and activated in cancer cells remains largely unknown. In an attempt to identify possible novel mechanisms involved in the regulation of hTERT transcription, the present study examined the role of Receptor Ck, a cell surface receptor specific for cholesterol, in the transcription of hTERT gene in normal human peripheral blood mononuclear cells. RESULTS: Activated Receptor Ck was found to down-regulate hTERT mRNA expression by repressing the transcription of c-myc gene. Receptor Ck-dependent signaling was also found to down-regulate the mRNA expression of the gene coding for the ligand inducible transcription factor, peroxisome proliferator-activated receptor gamma (PPARgamma). The ligand activation of PPARgamma resulted in the down-regulation of c-myc and hTERT mRNA expression. By using specific activator and inhibitor of protein kinase C (PKC), it was demonstrated that Receptor Ck dependent down-regulation of hTERT gene transcription involved inhibition of PKC. In addition, 25-hydroxycholesterol was found to contribute to the transcriptional regulation of hTERT gene. CONCLUSION: Taken together, the findings of this study present evidence for a molecular link between cholesterol-activated Receptor Ck and hTERT transcription, and provide new insights into the regulation of hTERT expression in normal human peripheral blood mononuclear cells. [Abstract/Link to Full Text]

Macdonald A, Campbell DG, Toth R, McLauchlan H, Hastie CJ, Arthur JS
Pim kinases phosphorylate multiple sites on Bad and promote 14-3-3 binding and dissociation from Bcl-XL.
BMC Cell Biol. 2006;71.
BACKGROUND: Pim-1, 2 and 3 are a group of enzymes related to the calcium calmodulin family of protein kinases. Over-expression of Pim-1 and Pim-2 in mice promotes the development of lymphomas, and up-regulation of Pim expression has been observed in several human cancers. RESULTS: Here we show that the pim kinases are constitutively active when expressed in HEK-293 cells and are able to phosphorylate the Bcl-2 family member Bad on three residues, Ser112, Ser136 and Ser155 in vitro and in cells. In vitro mapping showed that Pim-2 predominantly phosphorylated Ser112, while Pim-1 phosphorylated Ser112, but also Ser136 and Ser155 at a reduced rate compared to Ser112. Pim-3 was found to be the least specific for Ser112, and the most effective at phosphorylating Ser136 and Ser155. Pim-3 was also able to phosphorylate other sites in Bad in vitro, including Ser170, another potential in vivo site. Mutation of Ser136 to alanine prevented the phosphorylation of Ser112 and Ser155 by Pim kinases in HEK-293 cells, suggesting that this site must be phosphorylated first in order to make the other sites accessible. Pim phosphorylation of Bad was also found to promote the 14-3-3 binding of Bad and block its association with Bcl-XL. CONCLUSION: All three Pim kinase family members predominantly phosphorylate Bad on Ser112 and in addition are capable of phosphorylating Bad on multiple sites associated with the inhibition of the pro-apoptotic function of Bad in HEK-293 cells. This would be consistent with the proposed function of Pim kinases in promoting cell proliferation and preventing cell death. [Abstract/Link to Full Text]

Hajeri VA, Trejo J, Padilla PA
Characterization of sub-nuclear changes in Caenorhabditis elegans embryos exposed to brief, intermediate and long-term anoxia to analyze anoxia-induced cell cycle arrest.
BMC Cell Biol. 2005;647.
BACKGROUND: The soil nematode C. elegans survives oxygen-deprived conditions (anoxia; <.001 kPa O2) by entering into a state of suspended animation in which cell cycle progression reversibly arrests. The majority of blastomeres of embryos exposed to anoxia arrest at interphase, prophase and metaphase. The spindle checkpoint proteins SAN-1 and MDF-2 are required for embryos to survive 24 hours of anoxia. To further investigate the mechanism of cell-cycle arrest we examined and compared sub-nuclear changes such as chromatin localization pattern, post-translational modification of histone H3, spindle microtubules, and localization of the spindle checkpoint protein SAN-1 with respect to various anoxia exposure time points. To ensure analysis of embryos exposed to anoxia and not post-anoxic recovery we fixed all embryos in an anoxia glove box chamber. RESULTS: Embryos exposed to brief periods to anoxia (30 minutes) contain prophase blastomeres with chromosomes in close proximity to the nuclear membrane, condensation of interphase chromatin and metaphase blastomeres with reduced spindle microtubules density. Embryos exposed to longer periods of anoxia (1-3 days) display several characteristics including interphase chromatin that is further condensed and in close proximity to the nuclear membrane, reduction in spindle structure perimeter and reduced localization of SAN-1 at the kinetochore. Additionally, we show that the spindle checkpoint protein SAN-1 is required for brief periods of anoxia-induced cell cycle arrest, thus demonstrating that this gene product is vital for early anoxia responses. In this report we suggest that the events that occur as an immediate response to brief periods of anoxia directs cell cycle arrest. CONCLUSION: From our results we conclude that the sub-nuclear characteristics of embryos exposed to anoxia depends upon exposure time as assayed using brief (30 minutes), intermediate (6 or 12 hours) or long-term (24 or 72 hours) exposures. Analyzing these changes will lead to an understanding of the mechanisms required for initiation and maintenance of cell cycle arrest in respect to anoxia exposure time as well as order the events that occur to bring about anoxia-induced cell cycle arrest. [Abstract/Link to Full Text]

Damer CK, Bayeva M, Hahn ES, Rivera J, Socec CI
Copine A, a calcium-dependent membrane-binding protein, transiently localizes to the plasma membrane and intracellular vacuoles in Dictyostelium.
BMC Cell Biol. 2005;646.
BACKGROUND: Copines are soluble, calcium-dependent membrane binding proteins found in a variety of organisms. Copines are characterized as having two C2 domains at the N-terminal region followed by an "A domain" at the C-terminal region. The "A domain" is similar in sequence to the von Willebrand A (VWA) domain found in integrins. The presence of C2 domains suggests that copines may be involved in cell signaling and/or membrane trafficking pathways. RESULTS: We have identified six copines genes in the Dictyostelium discoideum genome, cpnA-cpnF, and have focused our studies on cpnA. CpnA is expressed throughout development and was shown to be capable of binding to membranes in a calcium-dependent manner in vitro. A GFP-tagged CpnA was also capable of binding to membranes in a calcium-dependent manner in vitro. In live wildtype Dictyostelium cells expressing GFP-CpnA, GFP-CpnA was typically found in the cytoplasm without any specific localization to membranes. However, in a small subset of starved cells, GFP-CpnA was observed to bind transiently (typically approximately 1-10 s) to the plasma membrane and intracellular vacuoles. In some cells, the transient membrane localization of GFP-CpnA was observed to occur multiple times in an oscillatory manner over several minutes. In plasma membrane disrupted cells, GFP-CpnA was observed to associate with the plasma membrane and intracellular vacuoles in a calcium-dependent manner. In fixed cells, GFP-CpnA labeled the plasma membrane and intracellular vacuoles, including contractile vacuoles, organelles of the endolysosomal pathway, and phagosomes. CONCLUSION: Our results show that Dictyostelium has multiple copine homologs and provides an excellent system in which to study copine function. The localization of a GFP-tagged CpnA to the plasma membrane, contractile vacuoles, organelles of the endolysosomal pathway, and phagosomes suggests that CpnA may have a role in the function of these organelles or the trafficking to and from them. In addition, we hypothesize that the observed transient oscillatory membrane localization of GFP-CpnA in a small subset of starved cells is caused by fast calcium waves and speculate that CpnA may have a role in development, particularly in the differentiation of stalk cells. [Abstract/Link to Full Text]

Tran TC, Singleton C, Fraley TS, Greenwood JA
Cysteine-rich protein 1 (CRP1) regulates actin filament bundling.
BMC Cell Biol. 2005;645.
BACKGROUND: Cysteine-rich protein 1 (CRP1) is a LIM domain containing protein localized to the nucleus and the actin cytoskeleton. CRP1 has been demonstrated to bind the actin-bundling protein alpha-actinin and proposed to modulate the actin cytoskeleton; however, specific regulatory mechanisms have not been identified. RESULTS: CRP1 expression increased actin bundling in rat embryonic fibroblasts. Although CRP1 did not affect the bundling activity of alpha-actinin, CRP1 was found to stabilize the interaction of alpha-actinin with actin bundles and to directly bundle actin microfilaments. Using confocal and photobleaching fluorescence resonance energy transfer (FRET) microscopy, we demonstrate that there are two populations of CRP1 localized along actin stress fibers, one associated through interaction with alpha-actinin and one that appears to bind the actin filaments directly. Consistent with a role in regulating actin filament cross-linking, CRP1 also localized to the membrane ruffles of spreading and PDGF treated fibroblasts. CONCLUSION: CRP1 regulates actin filament bundling by directly cross-linking actin filaments and stabilizing the interaction of alpha-actinin with actin filament bundles. [Abstract/Link to Full Text]

Mayer R, Brero A, von Hase J, Schroeder T, Cremer T, Dietzel S
Common themes and cell type specific variations of higher order chromatin arrangements in the mouse.
BMC Cell Biol. 2005;644.
BACKGROUND: Similarities as well as differences in higher order chromatin arrangements of human cell types were previously reported. For an evolutionary comparison, we now studied the arrangements of chromosome territories and centromere regions in six mouse cell types (lymphocytes, embryonic stem cells, macrophages, fibroblasts, myoblasts and myotubes) with fluorescence in situ hybridization and confocal laser scanning microscopy. Both species evolved pronounced differences in karyotypes after their last common ancestors lived about 87 million years ago and thus seem particularly suited to elucidate common and cell type specific themes of higher order chromatin arrangements in mammals. RESULTS: All mouse cell types showed non-random correlations of radial chromosome territory positions with gene density as well as with chromosome size. The distribution of chromosome territories and pericentromeric heterochromatin changed during differentiation, leading to distinct cell type specific distribution patterns. We exclude a strict dependence of these differences on nuclear shape. Positional differences in mouse cell nuclei were less pronounced compared to human cell nuclei in agreement with smaller differences in chromosome size and gene density. Notably, the position of chromosome territories relative to each other was very variable. CONCLUSION: Chromosome territory arrangements according to chromosome size and gene density provide common, evolutionary conserved themes in both, human and mouse cell types. Our findings are incompatible with a previously reported model of parental genome separation. [Abstract/Link to Full Text]

Arigoni M, Bracco E, Lusche DF, Kae H, Weeks G, Bozzaro S
A novel Dictyostelium RasGEF required for chemotaxis and development.
BMC Cell Biol. 2005;643.
BACKGROUND: Ras proteins are guanine-nucleotide-binding enzymes that couple cell surface receptors to intracellular signaling pathways controlling cell proliferation and differentiation, both in lower and higher eukaryotes. They act as molecular switches by cycling between active GTP and inactive GDP-bound states, through the action of two classes of regulatory proteins: a) guanine nucleotide exchange factor (GEFs) and b) GTP-ase activating proteins (GAPs). Genome wide analysis of the lower eukaryote Dictyostelium discoideum revealed a surprisingly large number of Ras Guanine Nucleotide Exchange Factors (RasGEFs). RasGEFs promote the activation of Ras proteins by catalyzing the exchange of GDP for GTP, thus conferring to RasGEFs the role of main activator of Ras proteins. Up to date only four RasGEFs, which are all non-redundant either for growth or development, have been characterized in Dictyostelium. We report here the identification and characterization of a fifth non-redundant GEF, RasGEFM. RESULTS: RasGEFM is a multi-domain protein containing six poly-proline stretches, a DEP, RasGEFN and RasGEF catalytic domain. The rasGEFM gene is differentially expressed during growth and development. Inactivation of the gene results in cells that form small, flat aggregates and fail to develop further. Expression of genes required for aggregation is delayed. Chemotaxis towards cAMP is impaired in the mutant, due to inability to inhibit lateral pseudopods. Endogenous cAMP accumulates during early development to a much lower extent than in wild type cells. Adenylyl cyclase activation in response to cAMP pulses is strongly reduced, by contrast guanylyl cyclase is stimulated to higher levels than in the wild type. The actin polymerization response to cAMP is also altered in the mutant. Cyclic AMP pulsing for several hours partially rescues the mutant. In vitro experiments suggest that RasGEFM acts downstream of the cAMP receptor but upstream of the G protein. CONCLUSION: The data indicate that RasGEFM is involved in the establishment of the cAMP relay system. We propose that RasGEFM is a component of a Ras regulated pathway, which integrate signals acting as positive regulator for adenylyl cyclase and negative regulator for guanylyl cyclase. Altered guanylyl cyclase, combined with defective regulation of actin polymerization, results in altered chemotaxis. [Abstract/Link to Full Text]

Rusu D, Loret S, Peulen O, Mainil J, Dandrifosse G
Immunochemical, biomolecular and biochemical characterization of bovine epithelial intestinal primocultures.
BMC Cell Biol. 2005;642.
BACKGROUND: Cultures of enterocytes and colonocytes represent valuable tools to study growth and differentiation of epithelial cells. In vitro models may be used to evaluate passage or toxicity of drugs, interactions of enteropathogenes bacteria strains with intestinal epithelium and other physiologic or pathologic phenomenon involving the digestive tract. RESULTS: Cultures of bovine colonocytes and jejunocytes were obtained from organoid-enriched preparations, using a combination of enzymatic and mechanical disruption of the intestine epithelium, followed by an isopicnic centrifugation discarding most single cells. Confluent cell monolayers arising from plated organoids exhibited epithelium typical features, such as the pavement-like structure, the presence of apical microvilli and tight junctions. Accordingly, cells expressed several markers of enterocyte brush border (i.e. maltase, alkaline phosphatase and fatty acid binding protein) as well as an epithelial cytoskeleton component (cytokeratin 18). However, enterocyte primocultures were also positive for the vimentin immunostaining (mesenchyme marker). Vimentin expression studies showed that this gene is constitutively expressed in bovine enterocytes. Comparison of the vimentin expression profile with the pattern of brush border enzymes activities, suggested that the decrease of cell differentiation level observed during the enterocyte isolation procedure and early passages of the primoculture could result from a post-transcriptional de-repression of vimentin synthesis. The low differentiation level of bovine enterocytes in vitro could partly be counteracted adding butyrate (1-2 mM) or using a glucose-deprived culture medium. CONCLUSION: The present study describes several complementary approaches to characterize bovine primary cultures of intestinal cells. Cultured cells kept their morphologic and functional characteristics during several generations. [Abstract/Link to Full Text]

Mayawala K, Vlachos DG, Edwards JS
Computational modeling reveals molecular details of epidermal growth factor binding.
BMC Cell Biol. 2005;641.
BACKGROUND: The ErbB family of receptors are dysregulated in a number of cancers, and the signaling pathway of this receptor family is a critical target for several anti-cancer drugs. Therefore a detailed understanding of the mechanisms of receptor activation is critical. However, despite a plethora of biochemical studies and recent single particle tracking experiments, the early molecular mechanisms involving epidermal growth factor (EGF) binding and EGF receptor (EGFR) dimerization are not as well understood. Herein, we describe a spatially distributed Monte Carlo based simulation framework to enable the simulation of in vivo receptor diffusion and dimerization. RESULTS: Our simulation results are in agreement with the data from single particle tracking and biochemical experiments on EGFR. Furthermore, the simulations reveal that the sequence of receptor-receptor and ligand-receptor reaction events depends on the ligand concentration, receptor density and receptor mobility. CONCLUSION: Our computer simulations reveal the mechanism of EGF binding on EGFR. Overall, we show that spatial simulation of receptor dynamics can be used to gain a mechanistic understanding of receptor activation which may in turn enable improved cancer treatments in the future. [Abstract/Link to Full Text]

Chuong SD, Park NI, Freeman MC, Mullen RT, Muench DG
The peroxisomal multifunctional protein interacts with cortical microtubules in plant cells.
BMC Cell Biol. 2005;640.
BACKGROUND: The plant peroxisomal multifunctional protein (MFP) possesses up to four enzymatic activities that are involved in catalyzing different reactions of fatty acid beta-oxidation in the peroxisome matrix. In addition to these peroxisomal activities, in vitro assays revealed that rice MFP possesses microtubule- and RNA-binding activities suggesting that this protein also has important functions in the cytosol. RESULTS: We demonstrate that MFP is an authentic microtubule-binding protein, as it localized to the cortical microtubule array in vivo, in addition to its expected targeting to the peroxisome matrix. MFP does not, however, interact with the three mitotic microtubule arrays. Microtubule co-sedimentation assays of truncated versions of MFP revealed that multiple microtubule-binding domains are present on the MFP polypeptide. This indicates that these regions function together to achieve high-affinity binding of the full-length protein. Real-time imaging of a transiently expressed green fluorescent protein-MFP chimera in living plant cells illustrated that a dynamic, spatial interaction exits between peroxisomes and cortical microtubules as peroxisomes move along actin filaments or oscillate at fixed locations. CONCLUSION: Plant MFP is associated with the cortical microtubule array, in addition to its expected localization in the peroxisome. This observation, coupled with apparent interactions that frequently occur between microtubules and peroxisomes in the cell cortex, supports the hypothesis that MFP is concentrated on microtubules in order to facilitate the regulated import of MFP into peroxisomes. [Abstract/Link to Full Text]

Olins DE, Olins AL
Granulocyte heterochromatin: defining the epigenome.
BMC Cell Biol. 2005;639.
BACKGROUND: Mammalian blood neutrophilic granulocytes are terminally differentiated cells, possessing extensive heterochromatin and lobulated (or ring-shaped) nuclei. Despite the extensive amount of heterochromatin, neutrophils are capable of increased gene expression, when activated by bacterial infection. Understanding the mechanisms of transcriptional repression and activation in neutrophils requires detailing the chromatin epigenetic markers, which are virtually undescribed in this cell type. Much is known about the heterochromatin epigenetic markers in other cell-types, permitting a basis for comparison with those of mature normal neutrophilic granulocytes. RESULTS: Immunostaining and immunoblotting procedures were employed to study the presence of repressive histone modifications and HP1 proteins in normal human and mouse blood neutrophils, and in vitro differentiated granulocytes of the mouse promyelocytic (MPRO) system. A variety of repressive histone methylation markers were detectable in these granulocytes (di- and trimethylated H3K9; mono-, di- and trimethyl H3K27; di- and trimethyl H4K20). However, a paucity of HP1 proteins was noted. These granulocytes revealed negligible amounts of HP1 alpha and beta, but exhibited detectable levels of HP1 gamma. Of particular interest, mouse blood and MPRO undifferentiated cells and granulocytes revealed clear co-localization of trimethylated H3K9, trimethylated H4K20 and HP1 gamma with pericentric heterochromatin. CONCLUSION: Mature blood neutrophils possess some epigenetic heterochromatin features that resemble those of well-studied cells, such as lymphocytes. However, the apparent paucity of HP1 proteins in neutrophils suggests that heterochromatin organization and binding to the nuclear envelope may differ in this cell-type. Future investigations should follow changes in epigenetic markers and levels of HP1 proteins during granulopoiesis and bacterial activation of neutrophils. [Abstract/Link to Full Text]

Addison CL, Nör JE, Zhao H, Linn SA, Polverini PJ, Delaney CE
The response of VEGF-stimulated endothelial cells to angiostatic molecules is substrate-dependent.
BMC Cell Biol. 2005;638.
BACKGROUND: The microenvironment surrounding cells can exert multiple effects on their biological responses. In particular the extracellular matrix surrounding cells can profoundly influence their behavior. It has been shown that the extracellular matrix composition in tumors is vastly different than that found in normal tissue with increased amounts of certain matrices such as collagen I. It has been previously demonstrated that VEGF stimulation of endothelial cells growing on type I collagen results in the induction of bcl-2 expression and enhanced endothelial cell survival. We sought to investigate whether this increased endothelial cell survival resulted in the failure of angiostatic molecules to inhibit angiogenesis. RESULTS: We now demonstrate that VEGF-induced survival on collagen I impairs the ability of three known angiostatic molecules, TSP-1, IP-10 and endostatin to inhibit endothelial cell proliferation. Apoptosis of endothelial cells, growing on collagen I, induced by TSP-1 and IP-10 was also inhibited following VEGF stimulation. In contrast, endostatin induced apoptosis in these same cells. Further analysis determined that endostatin did not decrease the expression of bcl-2 nor did it increase activation of caspase-3 in the presence of VEGF. Alternatively, it appeared that in the presence of VEGF, endostatin induced the activation of caspase-8 in endothelial cells grown on collagen I. Furthermore, only endostatin had the ability to inhibit VEGF-induced sprout formation in collagen I gels. CONCLUSION: These data suggest that TSP-1, IP-10 and endostatin inhibit endothelial cells via different mechanisms and that only endostatin is effective in inhibiting angiogenic activities in the presence of collagen I. Our results suggest that the efficacy of angiostatic treatments may be impaired depending on the context of the extracellular matrix within the tumor environment and thus could impede the efficacy of angiostatic therapies. [Abstract/Link to Full Text]

Place RF, Noonan EJ, Giardina C
HDACs and the senescent phenotype of WI-38 cells.
BMC Cell Biol. 2005;637.
BACKGROUND: Normal cells possess a limited proliferative life span after which they enter a state of irreversible growth arrest. This process, known as replicative senescence, is accompanied by changes in gene expression that give rise to a variety of senescence-associated phenotypes. It has been suggested that these gene expression changes result in part from alterations in the histone acetylation machinery. Here we examine the influence of HDAC inhibitors on the expression of senescent markers in pre- and post-senescent WI-38 cells. RESULTS: Pre- and post-senescent WI-38 cells were treated with the HDAC inhibitors butyrate or trichostatin A (TSA). Following HDAC inhibitor treatment, pre-senescent cells increased p21WAF1 and beta-galactosidase expression, assumed a flattened senescence-associated morphology, and maintained a lower level of proteasome activity. These alterations also occurred during normal replicative senescence of WI-38 cells, but were not accentuated further by HDAC inhibitors. We also found that HDAC1 levels decline during normal replicative senescence. CONCLUSION: Our findings indicate that HDACs impact numerous phenotypic changes associated with cellular senescence. Reduced HDAC1 expression levels in senescent cells may be an important event in mediating the transition to a senescent phenotype. [Abstract/Link to Full Text]

Roffers-Agarwal J, Xanthos JB, Miller JR
Regulation of actin cytoskeleton architecture by Eps8 and Abi1.
BMC Cell Biol. 2005;636.
BACKGROUND: The actin cytoskeleton participates in many fundamental processes including the regulation of cell shape, motility, and adhesion. The remodeling of the actin cytoskeleton is dependent on actin binding proteins, which organize actin filaments into specific structures that allow them to perform various specialized functions. The Eps8 family of proteins is implicated in the regulation of actin cytoskeleton remodeling during cell migration, yet the precise mechanism by which Eps8 regulates actin organization and remodeling remains elusive. RESULTS: Here, we show that Eps8 promotes the assembly of actin rich filopodia-like structures and actin cables in cultured mammalian cells and Xenopus embryos, respectively. The morphology of actin structures induced by Eps8 was modulated by interactions with Abi1, which stimulated formation of actin cables in cultured cells and star-like structures in Xenopus. The actin stars observed in Xenopus animal cap cells assembled at the apical surface of epithelial cells in a Rac-independent manner and their formation was accompanied by recruitment of N-WASP, suggesting that the Eps8/Abi1 complex is capable of regulating the localization and/or activity of actin nucleators. We also found that Eps8 recruits Dishevelled to the plasma membrane and actin filaments suggesting that Eps8 might participate in non-canonical Wnt/Polarity signaling. Consistent with this idea, mis-expression of Eps8 in dorsal regions of Xenopus embryos resulted in gastrulation defects. CONCLUSION: Together, these results suggest that Eps8 plays multiple roles in modulating actin filament organization, possibly through its interaction with distinct sets of actin regulatory complexes. Furthermore, the finding that Eps8 interacts with Dsh and induced gastrulation defects provides evidence that Eps8 might participate in non-canonical Wnt signaling to control cell movements during vertebrate development. [Abstract/Link to Full Text]

Wozniak MJ, Melzer M, Dorner C, Haring HU, Lammers R
The novel protein KBP regulates mitochondria localization by interaction with a kinesin-like protein.
BMC Cell Biol. 2005;635.
BACKGROUND: Members of the Kinesin-3 family of kinesin-like proteins mediate transport of axonal vesicles (KIF1A, KIF1Bbeta), distribution of mitochondria (KIF1Balpha) and anterograde Golgi to ER vesicle transport (KIF1C). Until now, little is known about the regulation of kinesin-like proteins. Several proteins interact with members of this protein family. Here we report on a novel, KIF1 binding protein (KBP) that was identified in yeast two-hybrid screens. RESULTS: KBP was identified by using the yeast-two-hybrid system with an amino-terminal fragment of KIF1C as a bait that is strongly homologous to KIF1B. Here we investigated the interaction of KBP and KIF1B. The full length proteins coimmunoprecipitated after overexpression and in untransfected 293 cells. Immunofluorescence experiments revealed that KBP was mainly localized to mitochondria, as has been described for KIF1Balpha. Overexpression of a deletion mutant or reduction of the KBP protein level using an anti-sense construct led to an aggregation of mitochondria. Such an effect is probably due to the lower activity of KIF1Balpha in the absence of KBP, as was revealed in motility assays. CONCLUSION: KBP is a new binding partner for KIF1Balpha that is a regulator of its transport function and thus represents a new type of kinesin interacting protein. [Abstract/Link to Full Text]

Petersén A, Stewénius Y, Björkqvist M, Gisselsson D
Euploidy in somatic cells from R6/2 transgenic Huntington's disease mice.
BMC Cell Biol. 2005;634.
BACKGROUND: Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by a CAG repeat expansion in the HD gene. The huntingtin protein expressed from HD has an unknown function but is suggested to interact with proteins involved in the cell division machinery. The R6/2 transgenic mouse is the most widely used model to study HD. In R6/2 fibroblast cultures, a reduced mitotic index and high frequencies of multiple centrosomes and aneuploid cells have recently been reported. Aneuploidy is normally a feature closely connected to neoplastic disease. To further explore this unexpected aspect of HD, we studied cultures derived from 6- and 12-week-old R6/2 fibroblasts, skeletal muscle cells, and liver cells. RESULTS: Cytogenetic analyses revealed a high frequency of polyploid cells in cultures from both R6/2 and wild-type mice with the greatest proportions of polyploid cells in cultures derived from skeletal muscle cells of both genotypes. The presence of polyploid cells in skeletal muscle in vivo was confirmed by fluorescence in situ hybridisation with centromeric probes. Enlarged and supernumerary centrosomes were found in cultures from both R6/2 and wild-type mice. However, no aneuploid cells could be found in any of the tissues. CONCLUSION: We conclude that polyploid cells are found in fibroblast and skeletal muscle cultures derived from both R6/2 and wild-type littermate mice and that aneuploidy is unlikely to be a hallmark of HD. [Abstract/Link to Full Text]

Helip-Wooley A, Westbroek W, Dorward H, Mommaas M, Boissy RE, Gahl WA, Huizing M
Association of the Hermansky-Pudlak syndrome type-3 protein with clathrin.
BMC Cell Biol. 2005;633.
BACKGROUND: Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelle biogenesis characterized by oculocutaneous albinism and prolonged bleeding. These clinical findings reflect defects in the formation of melanosomes in melanocytes and dense bodies in platelets. HPS type-3 (HPS-3) results from mutations in the HPS3 gene, which encodes a 1004 amino acid protein of unknown function that contains a predicted clathrin-binding motif (LLDFE) at residues 172-176. RESULTS: Clathrin was co-immunoprecipitated by HPS3 antibodies from normal but not HPS3 null melanocytes. Normal melanocytes expressing a GFP-HPS3 fusion protein demonstrated partial co-localization of GFP-HPS3 with clathrin following a 20 degrees C temperature block. GFP-HPS3 in which the predicted clathrin-binding domain of HPS3 was mutated (GFP-HPS3-delCBD) did not co-localize with clathrin under the same conditions. Immunoelectron microscopy of normal melanocytes expressing GFP-HPS3 showed co-localization of GFP-HPS3 with clathrin, predominantly on small vesicles in the perinuclear region. In contrast, GFP-HPS3-delCBD did not co-localize with clathrin and exhibited a largely cytoplasmic distribution. CONCLUSION: HPS3 associates with clathrin, predominantly on small clathrin-containing vesicles in the perinuclear region. This association most likely occurs directly via a functional clathrin-binding domain in HPS3. These results suggest a role for HPS3 and its protein complex, BLOC-2, in vesicle formation and trafficking. [Abstract/Link to Full Text]

Minakhina S, Myers R, Druzhinina M, Steward R
Crosstalk between the actin cytoskeleton and Ran-mediated nuclear transport.
BMC Cell Biol. 2005;632.
BACKGROUND: Transport of macromolecules into and out of the nucleus is a highly regulated process. The RanGTP/RanGDP gradient controls the trafficking of molecules exceeding the diffusion limit of the nuclear pore across the nuclear envelope. RESULTS: We found genetic interaction between genes establishing the Ran gradient, nuclear transport factor 2 (ntf-2), Ran GTPase activating protein (Sd), and the gene encoding Drosophila Profilin, chickadee (chic). The severe eye phenotype caused by reduction of NTF2 is suppressed by loss of function mutations in chic and gain of function mutations in Sd (RanGAP). We show that in chic mutants, as in Sd-RanGAP, nuclear export is impaired. CONCLUSION: Our data suggest that Profilin and the organization of the actin cytoskeleton play an important role in nuclear trafficking. [Abstract/Link to Full Text]

Aguiar CB, Lobăo-Soares B, Alvarez-Silva M, Trentin AG
Glycosaminoglycans modulate C6 glioma cell adhesion to extracellular matrix components and alter cell proliferation and cell migration.
BMC Cell Biol. 2005;631.
BACKGROUND: Adhesion to extracellular matrix (ECM) components has been implicated in the proliferative and invasive properties of tumor cells. We investigated the ability of C6 glioma cells to attach to ECM components in vitro and described the regulatory role of glycosaminoglycans (GAGs) on their adhesion to the substrate, proliferation and migration. RESULTS: ECM proteins (type IV collagen, laminin and fibronectin) stimulate rat C6 glioma cell line adhesion in vitro, in a dose-dependent manner. The higher adhesion values were achieved with type IV collagen. Exogenous heparin or chondroitin sulfate impaired, in a dose-dependent manner the attachment of C6 glioma cell line to laminin and fibronectin, but not to type IV collagen. Dextran sulfate did not affect C6 adhesion to any ECM protein analyzed, indicating a specific role of GAGs in mediating glioma adhesion to laminin and fibronectin. GAGs and dextran sulfate did not induce C6 glioma detachment from any tested substrate suggesting specific effect in the initial step of cell adhesion. Furthermore, heparin and chondroitin sulfate impaired C6 cells proliferation on fibronectin, but not on type IV collagen or laminin. In contrast, both GAGs stimulate the glioma migration on laminin without effect on type IV collagen or fibronectin. CONCLUSION: The results suggest that GAGs and proteoglycans regulate glioma cell adhesion to ECM proteins in specific manner leading to cell proliferation or cell migration, according to the ECM composition, thus modulating tumor cell properties. [Abstract/Link to Full Text]

Carey RM, Balcz BA, Lopez-Coviella I, Slack BE
Inhibition of dynamin-dependent endocytosis increases shedding of the amyloid precursor protein ectodomain and reduces generation of amyloid beta protein.
BMC Cell Biol. 2005;630.
BACKGROUND: The amyloid precursor protein (APP) is transported via the secretory pathway to the cell surface, where it may be cleaved within its ectodomain by alpha-secretase, or internalized within clathrin-coated vesicles. An alternative proteolytic pathway occurs within the endocytic compartment, where the sequential action of beta- and gamma-secretases generates the amyloid beta protein (Abeta). In this study, we investigated the effects of modulators of endocytosis on APP processing. RESULTS: Human embryonic kidney cells were transfected with a dominant negative mutant of dynamin I, an important mediator of clathrin-dependent endocytosis, and APP proteolysis was analyzed. Overexpression of the mutant dynamin (dyn I K44A) resulted in increased shedding of the APP ectodomain (sAPPalpha), accumulation of the C-terminal alpha-secretase product C83, and a reduction in the release of Abeta. Levels of mature APP on the cell surface were increased in cells expressing dyn I K44A, and internalization of surface-immunolabeled APP, assessed by fluorescence microscopy, was inhibited. Dynamin is a substrate for protein kinase C (PKC), and it was hypothesized that activators of PKC, which are known to stimulate alpha-secretase-mediated cleavage of APP, might exert their effects by inhibiting dynamin-dependent endocytosis. However, the internalization of surface-biotinylated APP was unaffected by treatment of cells with phorbol 12-myristate 13-acetate in the presence of the alpha-secretase inhibitor TAPI-1. CONCLUSION: The results indicate that APP is internalized by a dynamin-dependent process, and suggest that alterations in the activity of proteins that mediate endocytosis might lead to significant changes in Abeta production. [Abstract/Link to Full Text]

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Recent Articles in Eukaryotic Cell

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Damer CK, Bayeva M, Kim PS, Ho LK, Eberhardt ES, Socec CI, Lee JS, Bruce EA, Goldman-Yassen AE, Naliboff LC
Copine A is required for cytokinesis, contractile vacuole function, and development in Dictyostelium.
Eukaryot Cell. 2007 Mar;6(3):430-42.
Copines make up a family of soluble, calcium-dependent, membrane binding proteins found in a variety of eukaryotic organisms. In an earlier study, we identified six copine genes in the Dictyostelium discoideum genome and focused our studies on cpnA. Our previous localization studies of green fluorescent protein-tagged CpnA in Dictyostelium suggested that CpnA may have roles in contractile vacuole function, endolysosomal trafficking, and development. To test these hypotheses, we created a cpnA- knockout strain, and here we report the initial characterization of the mutant phenotype. The cpnA- cells exhibited normal growth rates and a slight cytokinesis defect. When placed in starvation conditions, cpnA- cells appeared to aggregate into mounds and form fingers with normal timing; however, they were delayed or arrested in the finger stage. When placed in water, cpnA- cells formed unusually large contractile vacuoles, indicating a defect in contractile vacuole function, while endocytosis and phagocytosis rates for the cpnA- cells were similar to those seen for wild-type cells. These studies indicate that CpnA plays a role in cytokinesis and contractile vacuole function and is required for normal development, specifically in the later stages prior to culmination. We also used real-time reverse transcription-PCR to determine the expression patterns of all six copine genes during development. The six copine genes were expressed in vegetative cells, with each gene exhibiting a distinct pattern of expression throughout development. All of the copine genes except cpnF showed an upregulation of mRNA expression at one or two developmental transitions, suggesting that copines may be important regulators of Dictyostelium development. [Abstract/Link to Full Text]

Zakrzewska A, Boorsma A, Delneri D, Brul S, Oliver SG, Klis FM
Cellular processes and pathways that protect Saccharomyces cerevisiae cells against the plasma membrane-perturbing compound chitosan.
Eukaryot Cell. 2007 Apr;6(4):600-8.
Global fitness analysis makes use of a genomic library of tagged deletion strains. We used this approach to study the effect of chitosan, which causes plasma membrane stress. The data were analyzed using T-profiler, which was based on determining the sensitivities of groups of deletion strains to chitosan, as defined by Gene Ontology (GO) and by genomic synthetic lethality screens, in combination with t statistics. The chitosan-hypersensitive groups included a group of deletion strains characterized by a defective HOG (high-osmolarity glycerol) signaling pathway, indicating that the HOG pathway is required for counteracting chitosan-induced stress. Consistent with this, activation of this pathway in wild-type cells by hypertonic conditions offered partial protection against chitosan, whereas hypotonic conditions sensitized the cells to chitosan. Other chitosan-hypersensitive groups were defective in RNA synthesis and processing, actin cytoskeleton organization, protein N-glycosylation, ergosterol synthesis, endocytosis, or cell wall formation, predicting that these cellular functions buffer the cell against the deleterious effect of chitosan. These predictions were supported by showing that tunicamycin, miconazole, and staurosporine (which target protein N-glycosylation, ergosterol synthesis, and the cell wall integrity pathway, respectively) sensitized Saccharomyces cerevisiae cells to chitosan. Intriguingly, the GO-defined group of deletion strains belonging to the "cytosolic large ribosomal subunit" was more resistant to chitosan. We propose that global fitness analysis of yeast in combination with T-profiler is a powerful tool to identify specific cellular processes and pathways that are required for survival under stress conditions. [Abstract/Link to Full Text]

Steinberg G
Hyphal growth: a tale of motors, lipids, and the Spitzenkörper.
Eukaryot Cell. 2007 Mar;6(3):351-60. [Abstract/Link to Full Text]

Rollenhagen C, Hodge CA, Cole CN
Following temperature stress, export of heat shock mRNA occurs efficiently in cells with mutations in genes normally important for mRNA export.
Eukaryot Cell. 2007 Mar;6(3):505-13.
Heat shock leads to accumulation of polyadenylated RNA in nuclei of Saccharomyces cerevisiae cells, transcriptional induction of heat shock genes, and efficient export of polyadenylated heat shock mRNAs. These studies were conducted to examine the requirements for export of mRNA following heat shock. We used in situ hybridization to detect SSA4 mRNA (encoding Hsp70) and flow cytometry to measure the amount of Ssa4p-green fluorescent protein (GFP) produced following heat shock. Npl3p and Yra1p are mRNA-binding proteins recruited to nascent mRNAs and are essential for proper mRNA biogenesis and export. Heat shock mRNA was exported efficiently in temperature-sensitive npl3, yra1, and npl3 yra1 mutant strains. Nevertheless, Yra1p was recruited to heat shock mRNA, as were Nab2p and Npl3p. Interestingly, Yra1p was not recruited to heat shock mRNA in yra1-1 cells, suggesting that Npl3p is required for recruitment of Yra1p. The THO complex, which functions in transcription elongation and in recruitment of Yra1p, was not required for heat shock mRNA export, although normal mRNA export is impaired in growing cells lacking THO complex proteins. Taken together, these studies indicate that export following heat shock depends upon fewer factors than does mRNA export in growing cells. Furthermore, even though some mRNA-binding proteins are dispensable for efficient export of heat shock mRNA, those that are present in nuclei of heat shocked cells were recruited to heat shock mRNA. [Abstract/Link to Full Text]

Dumitru R, Navarathna DH, Semighini CP, Elowsky CG, Dumitru RV, Dignard D, Whiteway M, Atkin AL, Nickerson KW
In vivo and in vitro anaerobic mating in Candida albicans.
Eukaryot Cell. 2007 Mar;6(3):465-72.
Candida albicans cells of opposite mating types are thought to conjugate during infection in mammalian hosts, but paradoxically, the mating-competent opaque state is not stable at mammalian body temperatures. We found that anaerobic conditions stabilize the opaque state at 37 degrees C, block production of farnesol, and permit in vitro mating at 37 degrees C at efficiencies of up to 84%. Aerobically, farnesol prevents mating because it kills the opaque cells necessary for mating, and as a corollary, farnesol production is turned off in opaque cells. These in vitro observations suggest that naturally anaerobic sites, such as the efficiently colonized gastrointestinal (GI) tract, could serve as niches for C. albicans mating. In a direct test of mating in the mouse GI tract, prototrophic cells were obtained from auxotrophic parent cells, confirming that mating will occur in this organ. These cells were true mating products because they were tetraploid, mononuclear, and prototrophic, and they contained the heterologous hisG marker from one of the parental strains. [Abstract/Link to Full Text]

Lee JH, Nguyen TN, Schimanski B, Günzl A
Spliced leader RNA gene transcription in Trypanosoma brucei requires transcription factor TFIIH.
Eukaryot Cell. 2007 Apr;6(4):641-9.
Trypanosomatid parasites share a gene expression mode which differs greatly from that of their human and insect hosts. In these unicellular eukaryotes, protein-coding genes are transcribed polycistronically and individual mRNAs are processed from precursors by spliced leader (SL) trans splicing and polyadenylation. In trans splicing, the SL RNA is consumed through a transfer of its 5'-terminal part to the 5' end of mRNAs. Since all mRNAs are trans spliced, the parasites depend on strong and continuous SL RNA synthesis mediated by RNA polymerase II. As essential factors for SL RNA gene transcription in Trypanosoma brucei, the general transcription factor (GTF) IIB and a complex, consisting of the TATA-binding protein-related protein 4, the small nuclear RNA-activating protein complex, and TFIIA, were recently identified. Although T. brucei TFIIA and TFIIB are extremely divergent to their counterparts in other eukaryotes, their characterization suggested that trypanosomatids do form a class II transcription preinitiation complex at the SL RNA gene promoter and harbor orthologues of other known GTFs. TFIIH is a GTF which functions in transcription initiation, DNA repair, and cell cycle control. Here, we investigated whether a T. brucei TFIIH is important for SL RNA gene transcription and found that silencing the expression of the highly conserved TFIIH subunit XPD in T. brucei affected SL RNA gene synthesis in vivo, and depletion of this protein from extract abolished SL RNA gene transcription in vitro. Since we also identified orthologues of the TFIIH subunits XPB, p52/TFB2, and p44/SSL1 copurifying with TbXPD, we concluded that the parasite harbors a TFIIH which is indispensable for SL RNA gene transcription. [Abstract/Link to Full Text]

Enke C, Zekert N, Veith D, Schaaf C, Konzack S, Fischer R
Aspergillus nidulans Dis1/XMAP215 protein AlpA localizes to spindle pole bodies and microtubule plus ends and contributes to growth directionality.
Eukaryot Cell. 2007 Mar;6(3):555-62.
The dynamics of cytoplasmic microtubules (MTs) is largely controlled by a protein complex at the MT plus end. In Schizosaccharomyces pombe and in filamentous fungi, MT plus end-associated proteins also determine growth directionality. We have characterized the Dis1/XMAP215 family protein AlpA from Aspergillus nidulans and show that it determines MT dynamics as well as hyphal morphology. Green fluorescent protein-tagged AlpA localized to MT-organizing centers (centrosomes) and to MT plus ends. The latter accumulation occurred independently of conventional kinesin or the Kip2-familiy kinesin KipA. alpA deletion strains were viable and only slightly temperature sensitive. Mitosis, nuclear migration, and nuclear positioning were not affected, but hyphae grew in curves rather than straight, which appeared to be an effect of reduced MT growth and dynamics. [Abstract/Link to Full Text]

Eliahu N, Igbaria A, Rose MS, Horwitz BA, Lev S
Melanin biosynthesis in the maize pathogen Cochliobolus heterostrophus depends on two mitogen-activated protein kinases, Chk1 and Mps1, and the transcription factor Cmr1.
Eukaryot Cell. 2007 Mar;6(3):421-9.
The maize pathogen Cochliobolus heterostrophus requires two mitogen-activated protein kinases (MAPKs), Chk1 and Mps1, to produce normal pigmentation. Young colonies of mps1 and chk1 deletion mutants have a white and autolytic appearance, which was partially rescued by a hyperosmotic environment. We isolated the transcription factor Cmr1, an ortholog of Colletotrichum lagenarium Cmr1 and Magnaporthe grisea Pig1, which regulates melanin biosynthesis in C. heterostrophus. Deletion of CMR1 in C. heterostrophus resulted in mutants that lacked dark pigmentation and acquired an orange-pink color. In cmr1 deletion strains the expression of putative scytalone dehydratase (SCD1) and hydroxynaphthalene reductase (BRN1 and BRN2) genes involved in melanin biosynthesis was undetectable, whereas expression of PKS18, encoding a polyketide synthase, was only moderately reduced. In chk1 and mps1 mutants expression of PKS18, SCD1, BRN1, BRN2, and the transcription factor CMR1 itself was very low in young colonies, slightly up-regulated in aging colonies, and significantly induced in hyperosmotic conditions, compared to invariably high expression in the wild type. These findings indicate that two MAPKs, Chk1 and Mps1, affect Cmr1 at the transcriptional level and this influence is partially overridden in stress conditions including aging culture and hyperosmotic environment. Surprisingly, we found that the CMR1 gene was transcribed in both sense and antisense directions, apparently producing mRNA as well as a long noncoding RNA transcript. Expression of the antisense CMR1 was also Chk1 and Mps1 dependent. Analysis of chromosomal location of the melanin biosynthesis genes in C. heterostrophus resulted in identification of a small gene cluster comprising BRN1, CMR1, and PKS18. Since expression of all three genes depends on Chk1 and Mps1 MAPKs, we suggest their possible epigenetic regulation. [Abstract/Link to Full Text]

Tsujioka M, Zhukovskaya N, Yamada Y, Fukuzawa M, Ross S, Williams JG
Dictyostelium Myb transcription factors function at culmination as activators of ancillary stalk differentiation.
Eukaryot Cell. 2007 Mar;6(3):568-70.
ecmB and mrrA are expressed in the cups that cradle Dictyostelium spore heads, and MybE is necessary for their expression in lower but not upper cup cells. A Myb site within the mrrA promoter is necessary for expression in both cups. Thus, multiple Myb proteins are required for ancillary stalk differentiation. [Abstract/Link to Full Text]

Hawle P, Horst D, Bebelman JP, Yang XX, Siderius M, van der Vies SM
Cdc37p is required for stress-induced high-osmolarity glycerol and protein kinase C mitogen-activated protein kinase pathway functionality by interaction with Hog1p and Slt2p (Mpk1p).
Eukaryot Cell. 2007 Mar;6(3):521-32.
The yeast Saccharomyces cerevisiae utilizes rapidly responding mitogen-activated protein kinase (MAPK) signaling cascades to adapt efficiently to a changing environment. Here we report that phosphorylation of Cdc37p, an Hsp90 cochaperone, by casein kinase 2 controls the functionality of two MAPK cascades in yeast. These pathways, the high-osmolarity glycerol (HOG) pathway and the cell integrity (protein kinase C) MAPK pathway, mediate adaptive responses to high osmotic and cell wall stresses, respectively. Mutation of the phosphorylation site Ser14 in Cdc37p renders cells sensitive to osmotic stress and cell wall perturbation by calcofluor white. We found that levels of the MAPKs Hog1p and Slt2p (Mpk1p) in cells are reduced in a cdc37-S14A mutant, and consequently downstream responses mediated by Hog1p and Slt2p are compromised. Furthermore, we present evidence that Hog1p and Slt2p both interact in a complex with Cdc37p in vivo, something that has not been reported previously. The interaction of Hsp90, Slt2p, and Hog1p with Cdc37p depends on the phosphorylation status of Cdc37p. In fact, our biochemical data show that the osmosensitive phenotype of the cdc37-S14A mutant is due to the loss of the interaction between Cdc37p, Hog1p, and Hsp90. Likewise, during cell wall stress, the interaction of Slt2p with Cdc37p and Hsp90 is crucial for Slt2p-dependent downstream responses, such as the activation of the transcription factor Rlm1p. Interestingly, phosphorylated Slt2p, but not phosphorylated Hog1p, has an increased affinity for Cdc37p. Together these observations suggest that Cdc37p acts as a regulator of MAPK signaling. [Abstract/Link to Full Text]

Sutterwala SS, Creswell CH, Sanyal S, Menon AK, Bangs JD
De novo sphingolipid synthesis is essential for viability, but not for transport of glycosylphosphatidylinositol-anchored proteins, in African trypanosomes.
Eukaryot Cell. 2007 Mar;6(3):454-64.
De novo sphingolipid synthesis is required for the exit of glycosylphosphatidylinositol (GPI)-anchored membrane proteins from the endoplasmic reticulum in yeast. Using a pharmacological approach, we test the generality of this phenomenon by analyzing the transport of GPI-anchored cargo in widely divergent eukaryotic systems represented by African trypanosomes and HeLa cells. Myriocin, which blocks the first step of sphingolipid synthesis (serine + palmitate --> 3-ketodihydrosphingosine), inhibited the growth of cultured bloodstream parasites, and growth was rescued with exogenous 3-ketodihydrosphingosine. Myriocin also blocked metabolic incorporation of [3H]serine into base-resistant sphingolipids. Biochemical analyses indicate that the radiolabeled lipids are not sphingomyelin or inositol phosphorylceramide, suggesting that bloodstream trypanosomes synthesize novel sphingolipids. Inhibition of de novo sphingolipid synthesis with myriocin had no adverse effect on either general secretory trafficking or GPI-dependent trafficking in trypanosomes, and similar results were obtained with HeLa cells. A mild effect on endocytosis was seen for bloodstream trypanosomes after prolonged incubation with myriocin. These results indicate that de novo synthesis of sphingolipids is not a general requirement for secretory trafficking in eukaryotic cells. However, in contrast to the closely related kinetoplastid Leishmania major, de novo sphingolipid synthesis is essential for the viability of bloodstream-stage African trypanosomes. [Abstract/Link to Full Text]

Cole KC, McLaughlin HW, Johnson DI
Use of bimolecular fluorescence complementation to study in vivo interactions between Cdc42p and Rdi1p of Saccharomyces cerevisiae.
Eukaryot Cell. 2007 Mar;6(3):378-87.
Saccharomyces cerevisiae Cdc42p functions as a GTPase molecular switch, activating multiple signaling pathways required to regulate cell cycle progression and the actin cytoskeleton. Regulatory proteins control its GTP binding and hydrolysis and its subcellular localization, ensuring that Cdc42p is appropriately activated and localized at sites of polarized growth during the cell cycle. One of these, the Rdi1p guanine nucleotide dissociation inhibitor, negatively regulates Cdc42p by extracting it from cellular membranes. In this study, the technique of bimolecular fluorescence complementation (BiFC) was used to study the dynamic in vivo interactions between Cdc42p and Rdi1p. The BiFC data indicated that Cdc42p and Rdi1p interacted in the cytoplasm and around the periphery of the cell at the plasma membrane and that this interaction was enhanced at sites of polarized cell growth during the cell cycle, i.e., incipient bud sites, tips and sides of small- and medium-sized buds, and the mother-bud neck region. In addition, a ring-like structure containing the Cdc42p-Rdi1p complex transiently appeared following release from G1-phase cell cycle arrest. A homology model of the Cdc42p-Rdi1p complex was used to introduce mutations that were predicted to affect complex formation. These mutations resulted in altered BiFC interactions, restricting the complex exclusively to either the plasma membrane or the cytoplasm. Data from these studies have facilitated the temporal and spatial modeling of Rdi1p-dependent extraction of Cdc42p from the plasma membrane during the cell cycle. [Abstract/Link to Full Text]

Dar MA, Sharma A, Mondal N, Dhar SK
Molecular cloning of apicoplast-targeted Plasmodium falciparum DNA gyrase genes: unique intrinsic ATPase activity and ATP-independent dimerization of PfGyrB subunit.
Eukaryot Cell. 2007 Mar;6(3):398-412.
DNA gyrase, a typical type II topoisomerase that can introduce negative supercoils in DNA, is essential for replication and transcription in prokaryotes. The apicomplexan parasite Plasmodium falciparum contains the genes for both gyrase A and gyrase B in its genome. Due to the large sizes of both proteins and the unusual codon usage of the highly AT-rich P. falciparum gyrA (PfgyrA) and PfgyrB genes, it has so far been impossible to characterize these proteins, which could be excellent drug targets. Here, we report the cloning, expression, and functional characterization of full-length PfGyrB and functional domains of PfGyrA. Unlike Escherichia coli GyrB, PfGyrB shows strong intrinsic ATPase activity and follows a linear pattern of ATP hydrolysis characteristic of dimer formation in the absence of ATP analogues. These unique features have not been reported for any known gyrase so far. The PfgyrB gene complemented the E. coli gyrase temperature-sensitive strain, and, together with the N-terminal domain of PfGyrA, it showed typical DNA cleavage activity. Furthermore, PfGyrA contains a unique leucine heptad repeat that might be responsible for dimerization. These results confirm the presence of DNA gyrase in eukaryotes and confer great potential for drug development and organelle DNA replication in the deadliest human malarial parasite, P. falciparum. [Abstract/Link to Full Text]

Kim S, Wolyniak MJ, Staab JF, Sundstrom P
A 368-base-pair cis-acting HWP1 promoter region, HCR, of Candida albicans confers hypha-specific gene regulation and binds architectural transcription factors Nhp6 and Gcf1p.
Eukaryot Cell. 2007 Apr;6(4):693-709.
To elucidate the molecular mechanisms controlling the expression of the hypha-specific adhesin gene HWP1 of Candida albicans, its promoter was dissected and analyzed using a green fluorescent protein reporter gene. A 368-bp region, the HWP1 control region (HCR), was critical for activation under hypha-inducing conditions and conferred developmental regulation to a heterologous ENO1 promoter. A more distal region of the promoter served to amplify the level of promoter activation. Using gel mobility shift assays, a 249-bp subregion of HCR, HCRa, was found to bind at least four proteins from crude extracts of yeasts and hyphae with differing binding patterns dependent on cell morphology. Four proteins with DNA binding activities were identified by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis after separation by anion-exchange and heparin-Sepharose chromatography. One protein with high similarity to Nhp6, an HMG1 family member in Saccharomyces cerevisiae, and another with weak similarity to an HMG-like condensation factor from Physarum polycephalum implicated changes in chromatin structure as a critical process in hypha-specific gene regulation. Proteins with strong homology to histones were also found. These studies are the first to identify proteins that bind to a DNA segment that confers developmental gene regulation in C. albicans and suggest a new model for hypha-specific gene regulation. [Abstract/Link to Full Text]

Dignard D, El-Naggar AL, Logue ME, Butler G, Whiteway M
Identification and characterization of MFA1, the gene encoding Candida albicans a-factor pheromone.
Eukaryot Cell. 2007 Mar;6(3):487-94.
In the opaque state, MTLa and MTLalpha strains of Candida albicans are able to mate, and this mating is directed by a pheromone-mediated signaling process. We have used comparisons of genome sequences to identify a C. albicans gene encoding a candidate a-specific mating factor. This gene is conserved in Candida dubliniensis and is similar to a three-gene family in the related fungus Candida parapsilosis but has extremely limited similarity to the Saccharomyces cerevisiae MFA1 (ScMFA1) and ScMFA2 genes. All these genes encode C-terminal CAAX box motifs characteristic of prenylated proteins. The C. albicans gene, designated CaMFA1, is found on chromosome 2 between ORF19.2165 and ORF19.2219. MFA1 encodes an open reading frame of 42 amino acids that is predicted to be processed to a 14-amino-acid prenylated mature pheromone. Microarray analysis shows that MFA1 is poorly expressed in opaque MTLa cells but is induced when the cells are treated with alpha-factor. Disruption of this C. albicans gene blocks the mating of MTLa cells but not MTLalpha cells, while the reintegration of the gene suppresses this cell-type-specific mating defect. [Abstract/Link to Full Text]

Pasrija R, Banerjee D, Prasad R
Structure and function analysis of CaMdr1p, a major facilitator superfamily antifungal efflux transporter protein of Candida albicans: identification of amino acid residues critical for drug/H+ transport.
Eukaryot Cell. 2007 Mar;6(3):443-53.
We have cloned and overexpressed multidrug transporter CaMdr1p as a green fluorescent protein-tagged protein to show its capability to extrude drug substrates. The drug extrusion was sensitive to pH and energy inhibitors and displayed selective substrate specificity. CaMdr1p has a unique and conserved antiporter motif, also called motif C [G(X6)G(X3)GP(X2)GP(X2)G], in its transmembrane segment 5 (TMS 5). Alanine scanning of all the amino acids of the TMS 5 by site-directed mutagenesis highlighted the importance of the motif, as well as that of other residues of TMS 5, in drug transport. The mutant variants of TMS 5 were placed in four different categories. The first category had four residues, G244, G251, G255, and G259, which are part of the conserved motif C, and their substitution with alanine resulted in increased sensitivity to drugs and displayed impaired efflux of drugs. Interestingly, first category mutants, when replaced with leucine, resulted in more dramatic loss of drug resistance and efflux. Notwithstanding the location in the core motif, the second category included residues which are part of the motif, such as P260, and those which were not part of the motif, such as L245, W248, P256, and F262, whose substitution with alanine resulted in a severe loss of drug resistance and efflux. The third category included G263, which is a part of motif C, but unlike other conserved glycines, its replacement with alanine or leucine showed no change in the phenotype. The replacement of the remaining 11 residues of the fourth category did not result in any change. The putative helical wheel projection showed clustering of functionally critical residues to one side and thus suggests an asymmetric nature of TMS 5. [Abstract/Link to Full Text]

Segmüller N, Ellendorf U, Tudzynski B, Tudzynski P
BcSAK1, a stress-activated mitogen-activated protein kinase, is involved in vegetative differentiation and pathogenicity in Botrytis cinerea.
Eukaryot Cell. 2007 Feb;6(2):211-21.
The gene bcsak1, encoding a mitogen-activated protein kinase (MAPK) of Botrytis cinerea, was cloned and characterized. The protein has high homology to the yeast Hog1 and to corresponding MAPKs from filamentous fungi, but it shows unique functional features. The protein is phosphorylated under osmotic stress, specific fungicides, and oxidative stress mediated by H(2)O(2) and menadione. Northern blot analyses indicate that only a subset of typical oxidative stress response genes is regulated by BcSAK1. In contrast to most other fungal systems, Deltabcsak1 mutants are significantly impaired in vegetative and pathogenic development: they are blocked in conidia formation, show increased sclerotial development, and are unable to penetrate unwounded plant tissue. These data indicate that in B. cinerea the stress-activated MAPK cascade is involved in essential differentiation programs. [Abstract/Link to Full Text]

Liang XH, Hury A, Hoze E, Uliel S, Myslyuk I, Apatoff A, Unger R, Michaeli S
Genome-wide analysis of C/D and H/ACA-like small nucleolar RNAs in Leishmania major indicates conservation among trypanosomatids in the repertoire and in their rRNA targets.
Eukaryot Cell. 2007 Mar;6(3):361-77.
Small nucleolar RNAs (snoRNAs) are a large group of noncoding RNAs that exist in eukaryotes and archaea and guide modifications such as 2'-O-ribose methylations and pseudouridylation on rRNAs and snRNAs. Recently, we described a genome-wide screening approach with Trypanosoma brucei that revealed over 90 guide RNAs. In this study, we extended this approach to analyze the repertoire of the closely related human pathogen Leishmania major. We describe 23 clusters that encode 62 C/Ds that can potentially guide 79 methylations and 37 H/ACA-like RNAs that can potentially guide 30 pseudouridylation reactions. Like T. brucei, Leishmania also contains many modifications and guide RNAs relative to its genome size. This study describes 10 H/ACAs and 14 C/Ds that were not found in T. brucei. Mapping of 2'-O-methylations in rRNA regions rich in modifications suggests the existence of trypanosomatid-specific modifications conserved in T. brucei and Leishmania. Structural features of C/D snoRNAs, such as copy number, conservation of boxes, K turns, and intragenic and extragenic base pairing, were examined to elucidate the great variation in snoRNA abundance. This study highlights the power of comparative genomics for determining conserved features of noncoding RNAs. [Abstract/Link to Full Text]

Xie R, Clark KM, Gorovsky MA
Endoplasmic reticulum retention signal-dependent glycylation of the Hsp70/Grp170-related Pgp1p in Tetrahymena.
Eukaryot Cell. 2007 Mar;6(3):388-97.
Glycylation is an uncommon posttranslational modification. It has been found that tubulin glycylation is essential for cell survival in Tetrahymena. Here we describe PGP1, a Tetrahymena gene encoding an Hsp70 homologue that is a novel glycylated protein. Pgp1p is a conserved glycoprotein that localizes within the lumen of the endoplasmic reticulum (ER). We demonstrate that PGP1 is essential for viability and present evidence that both glycosylation and ER retention are necessary but not sufficient for glycylation. [Abstract/Link to Full Text]

Vargas RC, García-Salcedo R, Tenreiro S, Teixeira MC, Fernandes AR, Ramos J, Sá-Correia I
Saccharomyces cerevisiae multidrug resistance transporter Qdr2 is implicated in potassium uptake, providing a physiological advantage to quinidine-stressed cells.
Eukaryot Cell. 2007 Feb;6(2):134-42.
The QDR2 gene of Saccharomyces cerevisiae encodes a putative plasma membrane drug:H(+) antiporter that confers resistance against quinidine, barban, bleomycin, and cisplatin. This work provides experimental evidence of defective K(+) (Rb(+)) uptake in the absence of QDR2. The direct involvement of Qdr2p in K(+) uptake is reinforced by the fact that increased K(+) (Rb(+)) uptake due to QDR2 expression is independent of the Trk1p/Trk2p system. QDR2 expression confers a physiological advantage for the yeast cell during the onset of K(+) limited growth, due either to a limiting level of K(+) in the growth medium or to the presence of quinidine. This drug decreases the K(+) uptake rate and K(+) accumulation in the yeast cell, especially in the Deltaqdr2 mutant. Qdr2p also helps to sustain the decrease of intracellular pH in quinidine-stressed cells in growth medium at pH 5.5 by indirectly promoting H(+) extrusion affected by the drug. The results are consistent with the hypothesis that Qdr2p may also couple K(+) movement with substrate export, presumably with quinidine. Other clues to the biological role of QDR2 in the yeast cell come from two additional lines of experimental evidence. First, QDR2 transcription is activated under nitrogen (NH(4)(+)) limitation or when the auxotrophic strain examined enters stationary phase due to leucine limitation, this regulation being dependent on general amino acid control by Gcn4p. Second, the amino acid pool is higher in Deltaqdr2 cells than in wild-type cells, indicating that QDR2 expression is, directly or indirectly, involved in amino acid homeostasis. [Abstract/Link to Full Text]

Hicks JK, Heitman J
Divergence of protein kinase A catalytic subunits in Cryptococcus neoformans and Cryptococcus gattii illustrates evolutionary reconfiguration of a signaling cascade.
Eukaryot Cell. 2007 Mar;6(3):413-20.
Gene duplication and divergence via both the loss and gain of gene activities are powerful evolutionary forces underlying the origin of new biological functions. Here a comparative genetics approach was applied to examine the roles of protein kinase A (PKA) catalytic subunits in three closely related varieties or sibling species of the pathogenic fungus genus Cryptococcus. Previous studies revealed that two PKA catalytic subunits, Pka1 and Pka2, control virulence factor production and mating. However, only one of the two plays the predominant physiological role, and this function has been exchanged between Pka1 and Pka2 in strains of the Cryptococcus neoformans var. grubii serotype A lineage compared to divergent C. neoformans var. neoformans serotype D isolates. To understand the basis for this functional plasticity, here the activities of Pka1 and Pka2 were defined in the two varieties and the related sibling species Cryptococcus gattii by gene disruption and characterization, heterologous complementation, and analysis of serotype AD hybrid mutant strains. The findings provide evidence for a shared ancestral role of PKA in governing mating and virulence factor production and indicate that the exchange of catalytic subunit roles is attributable to loss of function. Our studies illustrate the plasticity of signaling networks enabling rapid rewiring during speciation of a clade of common human fungal pathogens. [Abstract/Link to Full Text]

Ferrer-Sevillano F, Fernández-Cańón JM
Novel phacB-encoded cytochrome P450 monooxygenase from Aspergillus nidulans with 3-hydroxyphenylacetate 6-hydroxylase and 3,4-dihydroxyphenylacetate 6-hydroxylase activities.
Eukaryot Cell. 2007 Mar;6(3):514-20.
Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain (delta phacB) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B). [Abstract/Link to Full Text]

Ragni E, Coluccio A, Rolli E, Rodriguez-Peńa JM, Colasante G, Arroyo J, Neiman AM, Popolo L
GAS2 and GAS4, a pair of developmentally regulated genes required for spore wall assembly in Saccharomyces cerevisiae.
Eukaryot Cell. 2007 Feb;6(2):302-16.
The GAS multigene family of Saccharomyces cerevisiae is composed of five paralogs (GAS1 to GAS5). GAS1 is the only one of these genes that has been characterized to date. It encodes a glycosylphosphatidylinositol-anchored protein functioning as a beta(1,3)-glucan elongase and required for proper cell wall assembly during vegetative growth. In this study, we characterize the roles of the GAS2 and GAS4 genes. These genes are expressed exclusively during sporulation. Their mRNA levels showed a peak at 7 h from induction of sporulation and then decreased. Gas2 and Gas4 proteins were detected and reached maximum levels between 8 and 10 h from induction of sporulation, a time roughly coincident with spore wall assembly. The double null gas2 gas4 diploid mutant showed a severe reduction in the efficiency of sporulation, an increased permeability of the spores to exogenous substances, and production of inviable spores, whereas the single gas2 and gas4 null diploids were similar to the parental strain. An analysis of spore ultrastructure indicated that the loss of Gas2 and Gas4 proteins affected the proper attachment of the glucan to the chitosan layer, probably as a consequence of the lack of coherence of the glucan layer. The ectopic expression of GAS2 and GAS4 genes in a gas1 null mutant revealed that these proteins are redundant versions of Gas1p specialized to function in a compartment at a pH value close to neutral. [Abstract/Link to Full Text]

Richard ML, Plaine A
Comprehensive analysis of glycosylphosphatidylinositol-anchored proteins in Candida albicans.
Eukaryot Cell. 2007 Feb;6(2):119-33. [Abstract/Link to Full Text]

Heinrich M, Köhler T, Mösch HU
Role of Cdc42-Cla4 interaction in the pheromone response of Saccharomyces cerevisiae.
Eukaryot Cell. 2007 Feb;6(2):317-27.
In Saccharomyces cerevisiae, the highly conserved Rho-type GTPase Cdc42 is essential for cell division and controls cellular development during mating and invasive growth. The role of Cdc42 in mating has been controversial, but a number of previous studies suggest that the GTPase controls the mitogen-activated protein (MAP) kinase cascade by activating the p21-activated protein kinase (PAK) Ste20. To further explore the role of Cdc42 in pheromone-stimulated signaling, we isolated novel alleles of CDC42 that confer resistance to pheromone. We find that in CDC42(V36A) and CDC42(V36A, I182T) mutant strains, the inability to undergo pheromone-induced cell cycle arrest correlates with reduced phosphorylation of the mating MAP kinases Fus3 and Kss1 and with a decrease in mating efficiency. Furthermore, Cdc42(V36A) and Cdc42(V36A, I182T) proteins show reduced interaction with the PAK Cla4 but not with Ste20. We also show that deletion of CLA4 in a CDC42(V36A, I182T) mutant strain suppresses pheromone resistance and that overexpression of CLA4 interferes with pheromone-induced cell cycle arrest and MAP kinase phosphorylation in CDC42 wild-type strains. Our data indicate that Cla4 has the potential to act as a negative regulator of the mating pathway and that this function of the PAK might be under control of Cdc42. In conclusion, our study suggests that control of pheromone signaling by Cdc42 not only depends on Ste20 but also involves interaction of the GTPase with Cla4. [Abstract/Link to Full Text]

Wang A, Lane S, Tian Z, Sharon A, Hazan I, Liu H
Temporal and spatial control of HGC1 expression results in Hgc1 localization to the apical cells of hyphae in Candida albicans.
Eukaryot Cell. 2007 Feb;6(2):253-61.
The human fungal pathogen Candida albicans can undergo a morphological transition from a unicellular yeast growth form to a multicellular hyphal growth form. During hyphal growth, cell division is asymmetric. Only the apical cell divides, whereas subapical cells remain in G(1), and cell surface growth is highly restricted to the tip of the apical cell. Hgc1, a hypha-specific, G(1) cyclin-like protein, is essential for hyphal development. Here, we report, using indirect immunofluorescence, that Hgc1 is preferentially localized to the dividing apical cells of hyphae. Hgc1 protein is rapidly degraded in a cell cycle-independent manner, and the protein turnover likely occurs in both the apical and the subapical cells of hyphae. In addition to rapid protein turnover, the HGC1 transcript is also dynamically regulated during cell cycle progression in hyphal growth. It is induced upon germ tube formation in early G(1); the transcript level is reduced during the G(1)/S transition and peaks again around the G(2)/M phase in the subsequent cell cycles. Transcription from the HGC1 promoter is essential for its apical cell localization, as Hgc1 no longer exhibits preferential apical localization when expressed under the MAL2 promoter. Using fluorescence in situ hybridization, the HGC1 transcript is detected only in the apical cells of hyphae, suggesting that HGC1 is transcribed in the apical cell. Therefore, the preferential localization of Hgc1 to the apical cells of hyphae results from the dynamic temporal and spatial control of HGC1 expression. [Abstract/Link to Full Text]

Stewart MS, Krause SA, McGhie J, Gray JV
Mpt5p, a stress tolerance- and lifespan-promoting PUF protein in Saccharomyces cerevisiae, acts upstream of the cell wall integrity pathway.
Eukaryot Cell. 2007 Feb;6(2):262-70.
Pumilio family (PUF) proteins affect specific genes by binding to, and inhibiting the translation or stability of, their transcripts. The PUF domain is required and sufficient for this function. One Saccharomyces cerevisiae PUF protein, Mpt5p (also called Puf5p or Uth4p), promotes stress tolerance and replicative life span (the maximum number of doublings a mother cell can undergo before entering into senescence) by an unknown mechanism thought to partly overlap with, but to be independent of, the cell wall integrity (CWI) pathway. Here, we found that mpt5Delta mutants also display a short chronological life span (the time cells stay alive in saturated cultures in synthetic medium), a defect that is suppressed by activation of CWI signaling. We found that Mpt5p is an upstream activator of the CWI pathway: mpt5Delta mutants display the appropriate phenotypes and genetic interactions, display low basal activity of the pathway, and are defective in activation of the pathway upon thermal stress. A set of mRNAs that specifically bind to Mpt5p was recently reported. One such putative target, LRG1, encodes a GTPase-activating protein for Rho1p that directly links Mpt5p to CWI signaling: Lrg1p inhibits CWI signaling, LRG1 mRNA contains a consensus Mpt5p-binding site in its putative 3' untranslated region, loss of Lrg1p suppresses the temperature sensitivity and CWI signaling defects of mpt5Delta mutants, and LRG1 mRNA abundance is inhibited by Mpt5p. We conclude that Mpt5p is required for normal replicative and chronological life spans and that the CWI pathway is a key and direct downstream target of this PUF protein. [Abstract/Link to Full Text]

Thamatrakoln K, Hildebrand M
Analysis of Thalassiosira pseudonana silicon transporters indicates distinct regulatory levels and transport activity through the cell cycle.
Eukaryot Cell. 2007 Feb;6(2):271-9.
An analysis of the expression and activity of silicon transporters (SITs) was done on synchronously growing cultures of the diatom Thalassiosira pseudonana to provide insight into the role these proteins play in cellular silicon metabolism during the cell cycle. The first SIT-specific polyclonal peptide antibody was generated and used in the immunoblot analysis of whole-cell protein lysates to monitor SIT protein levels during synchronized progression through the cell cycle. Peaks in SIT protein levels correlated with active periods of silica incorporation into cell wall substructures. Quantitative real-time PCR on each of the three distinct SIT genes (TpSIT1, TpSIT2, and TpSIT3) showed that mRNA levels for the most highly expressed SIT genes peaked during the S phase of the cell cycle, a period prior to maximal silicon uptake and during which cell wall silicification does not occur. Variations in protein and mRNA levels did not correlate, suggesting that a significant regulatory step of SITs is at the translational or posttranslational level. Surge uptake rates also did not correlate with SIT protein levels, suggesting that SIT activity is internally controlled by the rate of silica incorporation. This is the first study to characterize SIT mRNA and protein expression and cellular uptake kinetics during the course of the cell cycle and cell wall synthesis, and it provides novel insight into SIT regulation. [Abstract/Link to Full Text]

Mukherjee M, Brown MT, McArthur AG, Johnson PJ
Proteins of the glycine decarboxylase complex in the hydrogenosome of Trichomonas vaginalis.
Eukaryot Cell. 2006 Dec;5(12):2062-71.
Trichomonas vaginalis is a unicellular eukaryote that lacks mitochondria and contains a specialized organelle, the hydrogenosome, involved in carbohydrate metabolism and iron-sulfur cluster assembly. We report the identification of two glycine cleavage H proteins and a dihydrolipoamide dehydrogenase (L protein) of the glycine decarboxylase complex in T. vaginalis with predicted N-terminal hydrogenosomal presequences. Immunofluorescence analyses reveal that both H and L proteins are localized in hydrogenosomes, providing the first evidence for amino acid metabolism in this organelle. All three proteins were expressed in Escherichia coli and purified to homogeneity. The experimental Km of L protein for the two H proteins were 2.6 microM and 3.7 microM, consistent with both H proteins serving as substrates of L protein. Analyses using purified hydrogenosomes showed that endogenous H proteins exist as monomers and endogenous L protein as a homodimer in their native states. Phylogenetic analyses of L proteins revealed that the T. vaginalis homologue shares a common ancestry with dihydrolipoamide dehydrogenases from the firmicute bacteria, indicating its acquisition via a horizontal gene transfer event independent of the origins of mitochondria and hydrogenosomes. [Abstract/Link to Full Text]

Nardelli SC, Avila AR, Freund A, Motta MC, Manhăes L, de Jesus TC, Schenkman S, Fragoso SP, Krieger MA, Goldenberg S, Dallagiovanna B
Small-subunit rRNA processome proteins are translationally regulated during differentiation of Trypanosoma cruzi.
Eukaryot Cell. 2007 Feb;6(2):337-45.
We used differential display to select genes differentially expressed during differentiation of epimastigotes into metacyclic trypomastigotes in the protozoan parasite Trypanosoma cruzi. One of the selected clones had a sequence similar to that of the small-subunit (SSU) processome protein Sof1p, which is involved in rRNA processing. The corresponding T. cruzi protein, TcSof1, displayed a nuclear localization and is downregulated during metacyclogenesis. Heterologous RNA interference assays showed that depletion of this protein impaired growth but did not affect progression through the cell cycle, suggesting that ribosome synthesis regulation and the cell cycle are uncoupled in this parasite. Quantitative PCR (qPCR) assays of several SSU processome-specific genes in T. cruzi also showed that most of them were regulated posttranscriptionally. This process involves the accumulation of mRNA in the polysome fraction of metacyclic trypomastigotes, where TcSof1 cannot be detected. Metacyclic trypomastigote polysomes were purified and separated by sucrose gradient sedimentation. Northern blot analysis of the sucrose gradient fractions showed the association of TcSof1 mRNA with polysomes, confirming the qPCR data. The results suggest that the mechanism of regulation involves the blocking of translation elongation and/or termination. [Abstract/Link to Full Text]

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Recent Articles in The Journal of Biological Chemistry

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Desmots F, Russell HR, Michel D, McKinnon PJ
Scythe regulates apoptosis inducing factor stability during endoplasmic reticulum stress induced apoptosis.
J Biol Chem. 2007 Dec 3; .
Scythe (BAT3; HLA-B Associated Transcript 3, Bag 6) is a protein that has been implicated in apoptosis because it can modulate the Drosophila melanogaster apoptotic regulator, Reaper. Mice lacking Scythe show pronounced defects in organogenesis and in the regulation of apoptosis and proliferation during mammalian development. However, the biochemical pathways important for Scythe function are unknown. We report here multiple levels of interaction between Scythe and the apoptogenic mitochondrial intermembrane protein AIF (Apoptosis-Inducing Factor). Scythe physically interacts with AIF and regulates its stability. AIF stability is markedly reduced in Scythe-/- cells which are more resistant to endoplasmic reticulum stress induced by thapsigargin. Reintroduction of Scythe or overexpression of AIF in Scythe-/- cells restores their sensitivity to apoptosis. Together, these data implicate Scythe as a regulator of AIF. [Abstract/Link to Full Text]

Emami N, Diamandis EP
Human kallikrein-related peptidase 14 (KLK14) is a new activator component of the KLK proteolytic cascade: Possible function in seminal plasma and skin.
J Biol Chem. 2007 Dec 3;
Human kallikrein- related peptidases (KLKs) are a family of fifteen serine proteases mainly known for their biomarker utility in various neoplastic and non-neoplastic diseases. Despite significant progress in understanding their clinical application, little is known about activation mechanism(s) of this important family of enzymes. Emerging evidence indicates that KLKs are activated in a step-wise manner, which is a characteristic of proteolytic cascades. Thus far, KLK cascades have been implicated in semen liquefaction and skin desquamation. Many members of the KLK family have been reported to be active in seminal plasma and/or skin, suggesting their involvement in common proteolytic cascades. KLK14 in particular, is highly active and has recently been proposed as one of the key trypsin-like proteases involved in skin desquamation. This study aims to elucidate a probable cascade-mediated role of KLK14 by: 1). examining KLK14- mediated cleavage of a heptapeptide library encompassing activation sites of the fifteen KLKs. 2). verifying activation of certain candidate downstream targets of KLK14, i.e. pro-KLK1, -KLK3, and -KLK11. Heptapeptides encompassing activation motifs of KLK2, 3, 5, and 11 were cleaved with a high (= 85%) cleavage efficiency. Activation of these candidates was confirmed, using full-length recombinant proteins. Pro-KLK11, -KLK3, and -KLK1 were rapidly activated in a concentration-dependent manner. ProKLK3 regulation was bidirectional, as activation was followed by inactivation via internal cleavage of active KLK3. Accumulatively, we are proposing a putative cascade model, operating through multiple KLKs. Identification of novel members of such proteolytic cascades will aid in further defining mechanisms involved in seminal/skin homeostasis. [Abstract/Link to Full Text]

Ochi H, Hans S, Westerfield M
Smarcd3 regulates the timing of zebrafish myogenesis onset.
J Biol Chem. 2007 Dec 3;
A cascade of signaling events triggers myogenesis in vertebrates. Although studies of zebrafish indicate that Fibroblast growth factor (Fgf), Hedgehog (Hh) and the T-box transcription factors, No tail (Ntl) and T-box gene 16 (Tbx16), regulate myogenesis, the hierarchy of these factors has not been determined. Recently, another transcriptional cofactor, Smarcd3, a subunit of the SWI/SNF chromatin-remodeling complex, has been shown to be required for heart muscle formation in mouse. In zebrafish, fgf8 and ntl expression commences during blastula stages, whereas myogenesis, as indicated by myod expression, does not begin until much later during mid-gastrula stages. smarcd3b expression, on the other hand, becomes enriched in the marginal zone just prior to the beginning of myod expression. Over expression of smarcd3 shifts the onset of myod and myf5 expression earlier, and myod and myf5 expression in adaxial cells, the earliest muscle precursors, requires Smarcd3, indicating that Smarcd3 is the limiting factor that regulates the onset of myogenesis. Smarcd3 physically interacts with Ntl, and Smarcd3 over expression fails to rescue myod expression in ntl mutants, demonstrating that function of Smarcd3 depends on Ntl activity. We propose a model in which cooperative activity of Fgf, Ntl and Smarcd3 is required for the onset of myogenesis, with Smarcd3b serving as the primary regulator of the timing of myogenesis onset. [Abstract/Link to Full Text]

Lajoie-Mazenc I, Tovar D, Penary M, Lortal B, Allart S, Favard C, Brihoum M, Pradines A, Favre G
MAP1A light chain-2 interacts with GTP-RHOB to control EGF-dependent EGF-R signaling.
J Biol Chem. 2007 Dec 3;
Rho GTPases have been implicated in the control of several cellular functions, including regulation of the actin cytoskeleton, cell proliferation, and oncogenesis. Unlike RhoA and RhoC, RhoB localizes in part to endosomes and controls endocytic trafficking. Using a yeast two-hybrid screen and a GST pull-down assay, we identified LC2, the light chain of the microtubule-associated protein MAP1A, as a novel binding partner for RhoB. GTP-binding and the 18 amino acid C-terminal hypervariable domain of RhoB are critical for its binding to MAP1A/LC2. Co-immunoprecipitation and immunofluorescence experiments showed that this interaction occurs in U87 cells. Downregulation of MAP1A/LC2 expression decreased EGF-R expression, and modified the signaling response to EGF treatment. We concluded that MAP1A/LC2 is critical for RhoB function in EGF-induced EGF-R regulation. Because MAP1A/LC2 is thought to function as an adaptator between microtubules and other molecules, we postulate that the RhoB and MAP1A/LC2 interaction facilitates endocytic vesicle trafficking and regulates the trafficking of signaling molecules. [Abstract/Link to Full Text]

Li Z, Nardi MA, Wu J, Pan R, Zhang W, Karpatkin S
Platelet fragmentation requires a specific structural conformation of human monoclonal antibody against beta3 integrin.
J Biol Chem. 2007 Dec 3;
We have described an autoantibody against beta3 (GPIIIa49-66), a region of platelet integrin alphaIIbbeta3 which is unique. It induces platelet fragmentation in the absence of complement via Ab activation of platelet NADPH oxidase and 12-lipoxygenase to release reactive oxygen species (ROS) which destroy platelets. In order to study the mechanism of anti-GPIIIa antibody induced platelet fragmentation, we screened a human scFv antibody library with the GPIIIa49-66 peptide. Nine monoclonal antibodies were identified which were capable of binding to GPIIIa49-66. Surprisingly, binding avidity to GPIIIa49-66 did not correlate with activity of induction of platelet fragmentation. We therefore investigated the requirements for platelet fragmentation. Mutations were introduced into the heavy chain CDR3 region of clones 11, 43 and 54 by site directed mutagenesis. The capability of these clones to induce platelet fragmentation or bind to GPIIIa49-66 subsequently changed. Molecular modeling of these clones with their mutants revealed that the ability to induce platelet fragmentation is affected by the side chain orientation of positively charged amino acids in the heavy chain of residues 99 to 102. Thus, a structural change in the conformation of anti-GPIIIa49-66 antibody contributes to its binding to the beta3 integrin and subsequent Ab-induced platelet fragmentation and aggregate dissolution. [Abstract/Link to Full Text]

Bierings R, Beato M, Edel MJ
An endothelial cell genetic screen identifies the GTPase rem2 as a suppressor of p19ARF expression that promotes endothelial cell proliferation and angiogenesis.
J Biol Chem. 2007 Dec 3;
Angiogenesis requires an increase in endothelial cell proliferation to support an increase in mass of blood vessels. We designed an in vitro endothelial cell model to functionally screen for genes that regulate endothelial cell proliferation. A gain of function screen for genes that by-pass p53 endothelial cell-arrest identified Rem2, a Ras-like GTPase. We show that Rem2 suppresses p14ARF (human) or p19ARF (mouse) expression that leads to increased endothelial cell proliferation. Conversely, loss of Rem2 by RNAi restores p19ARF expression in endothelial cells. We further show that Rem2-interacting 14-3-3 proteins are involved in the cell localization of Rem2, regulation of p19ARF expression, and endothelial cell proliferation. Finally, we demonstrate using the RIP1 tag2 mouse model of pancreatic disease that Rem2 is upregulated in endothelial cells of stage IV disease. The data unravel a possible molecular mechanism for Rem2-induced angiogenesis and suggests Rem2 as a potential novel target for treating pathological angiogenesis. [Abstract/Link to Full Text]

Ma J, Yan R, Zu X, Cheng JM, Rao K, Liao DF, Cao D
Aldo-keto reductase family 1 B10 affects fatty acid synthesis by regulating the stability of acetyl-coa carboxylase-alpha in breast cancer cells.
J Biol Chem. 2007 Dec 1;
Recent studies have demonstrated that aldo-keto reductase family 1 B10 (AKR1B10), a novel protein overexpressed in human hepatocellular carcinoma and non-small cell lung carcinoma, may facilitate cancer cell growth by detoxifying intracellular reactive carbonyls. This study presents a novel function of AKR1B10 in tumorigenic mammary epithelial cells (RAO-3), regulating fatty acid synthesis. In RAO-3 cells, Sephacryl-S 300 gel filtration and DEAE-Sepharose ion exchange chromatography demonstrated that AKR1B10 exists in two distinct forms, monomers (~40 kDa) bound to DEAE- Sepharose column and protein complexes (~300 kDa) remaining in flow-through. Co-immunoprecipitation with AKR1B10 antibody and protein mass spectrometry analysis identified that AKR1B10 associates with acetyl-CoA carboxylase-a (ACCA), a rate-limiting enzyme of de novo fatty acid synthesis. This association between AKR1B10 and ACCA proteins was further confirmed by the co-immunoprecipitation with ACCA antibody and pull-down assays with recombinant AKR1B10 protein. Intracellular fluorescent studies showed that AKR1B10 and ACCA proteins co-localize in the cytoplasm of RAO-3 cells. More interestingly, siRNA-mediated AKR1B10 knockdown increased ACCA degradation through ubiquitination- proteasome pathway and resulted in more than 50% decrease of fatty acid synthesis in RAO-3 cells. These data suggest that AKR1B10 is a novel regulator of the biosynthesis of fatty acid, an essential component of the cell membrane, in breast cancer cells. [Abstract/Link to Full Text]

Esteban O, Bernal RA, Donohoe M, Videler H, Sharon M, Robinson CV, Stock D
Stoichiometry and localisation of the stator subunits E and G in T. thermophilus H+ ATPase/synthase.
J Biol Chem. 2007 Nov 30;
Proton translocating ATPases are central to biological energy conversion. While eukaryotes contain specialised F-ATPases for ATP synthesis and V-ATPases for proton pumping, eubacteria and archaea typically contain only one enzyme for both tasks. Although many eubacteria contain ATPases of the F-type, some eubacteria and all known archaea contain ATPases of the A-type. A-ATPases are closely related to V-ATPases, but simpler in design. While the nucleotide binding and transmembrane rotor subunits share sequence homology between A-, V- and F-ATPases, the peripheral stalk is strikingly different in sequence, composition and stoichiometry. We have analysed the peripheral stalk of Thermus thermophilus A-ATPase by using phage display derived single-domain antibody fragments in combination with electron microscopy and tandem mass spectrometry. Our data provide the first direct evidence for the existence of two peripheral stalks in the A-ATPase, each one composed of heterodimers of subunits E and G arranged symmetrically around the soluble A1 domain. To our knowledge, this is the first description of phage display-derived antibody selection against a multi-subunit membrane protein used for purification and single particle analysis by electron microscopy. It is also the first instance of the derivation of subunit stoichiometry by tandem mass spectrometry to an intact membrane protein complex. Both approaches could be applicable to the structural analysis of other membrane protein complexes. [Abstract/Link to Full Text]

Hostetler HA, Huang H, Kier AB, Schroeder F
Glucose directly links to lipid metabolism through high-affinity interaction with PPARalpha.
J Biol Chem. 2007 Nov 30;
The pathophysiology of diabetes is characterized not only by elevated glucose, but also elevated long-chain fatty acid (LCFA) levels. We show for the first time that the peroxisome proliferator-activated receptor-alpha (PPARalpha) binds glucose and glucose metabolites with high affinity, resulting in significantly altered PPARalpha secondary structure. Glucose decreased PPARalpha interaction with fatty acid metabolites and steroid receptor coactivator-1 (SRC-1), while increasing PPARalpha interaction with DNA. Concomitantly, glucose increased PPARalpha interaction with SRC-1, DNA binding, and transactivation of beta-oxidation pathways in the presence of activating ligands. Heterodimerization of PPARalpha to the retinoid X receptor-alpha (RXRalpha) resulted in even larger increases in transactivation with the addition of glucose. These data suggest that PPARalpha is responsible for maintaining energy homeostasis through a concentration dependent regulation of both lipids and sugars and that hyperglycemic injury mediated by PPARalpha occurs not only indirectly through elevated LCFA levels, but also through direct action of glucose on PPARalpha. [Abstract/Link to Full Text]

Bellon SF, Kaplan-Lefko P, Yang Y, Zhang Y, Moriguchi J, Rex K, Johnson CW, Rose PE, Long AM, O'Connor AB, Gu Y, Coxon A, Kim TS, Tasker A, Burgess TL, Dussault I
c-Met inhibitors with novel binding mode show activity against several hereditary papillary renal cell carcinoma related mutations.
J Biol Chem. 2007 Nov 30;
c-Met is a receptor tyrosine kinase often deregulated in human cancers thus making it an attractive drug target. One mechanism by which c-Met deregulation leads to cancer is through gain-of-function mutations. Therefore, small molecules capable of targeting these mutations could offer therapeutic benefits for affected patients. SU11274 was recently described and reported to inhibit the activity of the wild-type and some mutant forms of c-Met while other mutants are resistant to inhibition. We identified a novel series of c-Met small molecule inhibitors that are active against multiple mutants previously identified in hereditary papillary renal cell carcinoma patients. AM7 is active against wild-type c-Met as well as several mutants, inhibits c-Met-mediated signaling in MKN-45 and U-87 MG cells, and inhibits tumor growth in these two models grown as xenografts. The crystal structures of AM7 and SU11274 bound to unphosphorylated c-Met have been determined. The AM7 structure reveals a novel binding mode compared with other published c-Met inhibitors and SU11274. The molecule binds the kinase linker, and then extends into a new hydrophobic binding site. This binding site is created by a significant movement of the C-helix, and so represents an inactive conformation of the c-Met kinase. Thus, our results demonstrate that it is possible to identify and design inhibitors that will likely be active against mutants found in different cancers. [Abstract/Link to Full Text]

Liu C, Mao K, Zhang M, Sun Z, Hong W, Li C, Peng B, Chang Z
The SH3-like domain switches its interaction partners to modulate the repression activity of mycobacterial Iron-dependent transcription regulator (IdeR) in response to metal Ion fluctuations.
J Biol Chem. 2007 Nov 30;
Iron-dependent regulator (IdeR), a metal ion-activated pleiotropic transcription factor, plays a critical role in maintaining the intracellular iron homeostasis in Mycobacteria, which is important for the normal growth of the cells. This study was initially performed in an attempt to elucidate all potential interactions between the various domains of IdeR that occur in living mycobacterial cells. This led to a hitherto unidentified self-association for the SH3-like domain of IdeR. Further studies demonstrate that the SH3-like domain interacts with different partners in the dimeric forms of IdeR depending on the levels of metal ions in the environment: it undergoes inter-subunit self-association in the metal-free DNA-non-binding form, but interacts with the N-terminal domain in the metal-bound DNA-binding form in an intra-subunit manner to finely modulate the transcription repression activity of IdeR. Our more detailed mapping studies reveal that the SH3-like domain uses an overlapping surface to participate in these two interactions, which therefore occur in a mutually exclusive fashion. This novel mechanism would allow an effective and cooperative interconversion between the two functional forms of IdeR. Our data also demonstrate that a disturbance of the interactions involving the SH3-like domain impairs the transcription repression activity of IdeR and delays the growth of mycobacterial cells. [Abstract/Link to Full Text]

Watatani Y, Ichikawa K, Nakanishi N, Fujimoto M, Takeda H, Kimura N, Hirose H, Takahashi S, Takahashi Y
Stress-induced translation of ATF5 mRNA is regulated by the 5' untranslated region.
J Biol Chem. 2007 Nov 30;
Activating transcription factor (ATF) 5 is a transcription factor belonging to the ATF/cAMP-responsive element-binding protein gene family. We previously reported that ATF5 mRNA expression increased in response to amino acid limitation. The ATF5 gene allows transcription of mRNAs with at least two alternative 5' untranslated regions (5'UTRs), 5'UTRa and 5UTRss, derived from exon1a and exon1ss. 5'UTRa contains highly conserved sequences, in which the upstream open reading frames (uORFs) uORF1 and uORF2 are found in many species. The present study was designed to investigate the potential role of 5'UTRs in translational control. These 5'UTRs differentially determine translation efficiency from mRNA. The presence of 5'UTRa or 5'UTRss represses translation from the downstream ATF5 ORF. Moreover, 5'UTRa-regulated translational repression is released by amino acid limitation or NaAsO2 exposure. This release was not seen for 5'UTRss. Mutation of uAUG2 in the uORF2 of 5'UTRa restored the basal expression and abolished the positive regulation by amino acid limitation or As exposure. We demonstrated that phosphorylation of eukaryotic initiation factor (eIF) 2a was required for amino acid limitation-induced translational regulation of ATF5. Furthermore, As exposure activated the exogenously expressed heme-regulated inhibitor kinase and induced the phosphorylation of eIF2a in non-erythroid cells. These results suggest that translation of ATF5 is regulated by the alternative 5'UTR region of its mRNA, and ATF5 may play a role in protecting cells from amino acid limitation or As-induced oxidative stress. [Abstract/Link to Full Text]

Kitagawa N, Mazon H, Heck AJ, Wilkens S
Stoichiometry of the peripheral stalk subunits E and G of Yeast V1-ATPase determined by mass spectrometry.
J Biol Chem. 2007 Nov 30;
The stoichiometry of yeast V1-ATPase peripheral stalk subunits E and G was determined by two independent approaches using mass spectrometry (MS). First, the subunit ratio was inferred from measuring the molecular mass of the intact V1-ATPase complex, and each of the individual protein components, using native electrospray ionization-mass spectrometry (ESI-MS). The major observed intact complex had a mass of 593,600 Da, with minor components displaying masses of 553,550 Da and 428,300 Da, respectively. Second, defined amounts of V1-ATPase purified from yeast grown on 14N containing media were titrated with defined amounts of 15N labeled E and G subunits as internal standards. Following protease digestion of subunit bands, 14N and 15N containing peptide pairs were used for quantification of subunit stoichiometry using MALDI-TOF MS. Results from both approaches are in excellent agreement and reveal that the subunit composition of yeast V1-ATPase is A3B3DE3FG3H. [Abstract/Link to Full Text]

Pietrement C, Da Silva N, Silberstein C, James M, Marsolais M, Van Hoek A, Brown D, Pastor-Soler N, Ameen N, Laprade R, Ramesh V, Breton S
Role of NHERF1, CFTR and cAMP in the regulation of aquaporin 9.
J Biol Chem. 2007 Nov 30;
Water and solute transport across the plasma membrane of cells is a crucial biological function that is mediated mainly by aquaporins and aquaglyceroporins. The regulation of these membrane proteins is still incompletely understood. Using the male reproductive tract as a model system in which water and glycerol transport are critical for the establishment of fertility, we now report a novel pathway for the regulation of aquaporin 9 (AQP9) permeability. AQP9 is the major aquaglyceroporin of the epididymis, liver and peripheral leukocytes, and its c-terminal portion contains a putative PDZ binding motif (SVIM). Here we show that NHERF1, CFTR and AQP9 co-localize in the apical membrane of principal cells of the epididymis and the vas deferens, and that both NHERF1 and CFTR co-immunoprecipitate with AQP9. Overlay assays revealed that AQP9 binds to both the PDZ1 and PDZ2 domains of NHERF1, with an apparently higher affinity for PDZ1 vs PDZ2. Pull-down assays showed that the AQP9 c-terminal SVIM motif is essential for interaction with NHERF1. Functional assays on isolated tubules perfused in vitro showed a high permeability of the apical membrane to glycerol, which is inhibited by the AQP9 inhibitor, phloretin, and is markedly activated by cAMP. The CFTR inhibitors DPC, GlyH-101 and CFTRinh-172 all significantly reduced the cAMP-activated glycerol-induced cell swelling. We propose that CFTR is an important regulator of AQP9 and that the interaction between AQP9, NHERF1 and CFTR may facilitate the activation of AQP9 by cAMP. [Abstract/Link to Full Text]

Bergamaschi C, Rosati M, Jalah R, Valentin A, Kulkarni V, Alicea C, Zhang GM, Patel V, Felber BK, Pavlakis GN
Intracellular interaction of IL-15 with its receptor alpha during production leads to mutual stabilization and increased bioactivity.
J Biol Chem. 2007 Nov 30;
We show that co-expression of interleukin 15 (IL-15) and IL-15 receptor alpha (IL-15Ra) in the same cell allows for the intracellular interaction of the two proteins early after translation resulting in increased stability and secretion of both molecules as a complex. In the absence of co-expressed IL-15Ra, a large portion of the produced IL-15 is rapidly degraded immediately after synthesis. Co-injection into mice of IL-15 and IL-15Ra expression plasmids led to significantly increased levels of the cytokine in serum as well as increased biological activity of IL-15. Examination of natural killer (NK) cells and T lymphocytes in mouse organs showed a great expansion of both cell types in the lung, liver and spleen. The presence of IL-15Ra also increased the number of CD44high memory cells with effector phenotype (CD44highCD62L-). Thus, mutual stabilization of IL-15 and IL-15Ra leads to remarkable increases in production, stability and tissue availability of bioactive IL-15 in vivo. The in vivo data show that the most potent form of IL-15 is as part of a complex with its receptor alpha either on the surface of the producing cells or as a soluble extracellular complex. These results explain the reason for co-ordinate expression of IL-15 and IL-15Ra in the same cell and suggest that the IL-15Ra is a part of the active IL-15 cytokine rather than part of the receptor. [Abstract/Link to Full Text]

Job V, Carapito R, Vernet T, Dessen A, Zapun A
Common alterations in PBP1a from resistant streptococcus pneumoniae decrease its reactivity towards beta -lactams: Structural insights.
J Biol Chem. 2007 Nov 30;
The development of high level beta-lactam resistance in the pneumococcus requires the expression of an altered form of PBP1a, in addition to modified forms of PBP2b and PBP2x, which are necessary for the appearance of low levels of resistance. Here, we present the crystal structure of a soluble form of PBP1a from the highly resistant S. pneumoniae strain 5204 (minimal inhibitory concentration of cefotaxime = 12 mg.L(-1)). Mutations T371A, which is adjacent to the catalytic nuclophile Ser370, and TSQF(574-577)NTGY, which lie in a loop bordering the active site cleft, were investigated by site-directed mutagenesis. The consequences of these substitutions on reaction kinetics with beta-lactams were probed in vitro, and their effect on resistance were measured in vivo. The results are interpreted in the framework of the crystal structure, which displays a narrower, discontinuous active site cavity, compared to that of PBP1a from the beta-lactam susceptible strain R6, as well as a reorientation of the catalytic Ser370. [Abstract/Link to Full Text]

Huang YH, Lin Y, Brown TE, Han MH, Saal DB, Neve RL, Zukin RS, Sorg BA, Nestler EJ, Malenka RC, Dong Y
CREB modulates the functional output of nucleus accumbens neurons: A critical role of NMDA receptors.
J Biol Chem. 2007 Nov 30;
Nucleus accumbens (NAc) medium spiny neurons cycle between two states, a functionally inactive downstate and a functionally active upstate. Here, we show that activation of the transcription factor cAMP-response element binding protein (CREB), a common molecular response to several drugs of abuse, increases both duration of the upstate and action-potential firing during the upstate. This effect of CREB is mediated by enhanced N-methyl-D-aspartate glutamate receptor (NMDAR) function: increased CREB activity increases both NMDAR-mediated synaptic currents and surface level of NMDARs, while inhibition of NMDARs abolishes the effect of CREB on upstate duration. Furthermore, mimicking the effect of CREB by pharmacological enhancement of NMDAR function in the NAc in vivo suppressed novelty- and cocaine-elicited locomotor activity. These findings suggest that by enhancing NMDAR-mediated synaptic transmission, CREB activation promotes the proportion of time NAc neurons spend in the upstate. This effect, along with CREB's enhancement of NAc membrane excitability (1), may counteract drug-induced maladaptations in the NAc, and thus ameliorate the addictive state. [Abstract/Link to Full Text]

Cheng TS, Hsiao YL, Lin CC, Yu RC, Hsu CM, Chang MS, Lee CI, Huang CY, Howng SL, Hong YR
Glycogen synthase kinase 3beta interacts with and phosphorylates the spindle-associated protein astrin.
J Biol Chem. 2007 Nov 30;
Emerging evidence shows that glycogen synthase kinase 3beta (GSK3ss) is involved in mitotic division, and that inhibiting of GSK3ss kinase activity causes defects in spindle microtubule length and chromosome alignment. However, the purpose of GSK3ss involvement in spindle microtubule assembly and accurate chromosome segregation remains obscure. Here, we report that GSK3ss interacts with the spindle-associated protein Astrin both in vitro and in vivo. Additionally, Astrin acts as a substrate for GSK3ss and is phosphorylated at Thr-111, Thr-937 (S/TP motif) and Ser-974/Thr-978 (S/T-XXX-S/T motif). Inhibition of GSK3ss impairs spindle and kinetochore accumulation of Astrin and spindle formation at mitosis, suggesting that Astrin association with the spindle microtubule and kinetochore may be dependent on phosphorylation by GSK3ss. Conversely, depletion of Astrin by siRNA has no detectable influence on the localization of GSK3ss. Interestingly, in vitro assays demonstrated that Astrin enhances GSK3ss-mediated phosphorylation of other substrates. Moreover, we showed that coexpression of Astrin and GSK3ss differentially increases GSK3ss-mediated Tau phosphorylation on an unprimed site. Collectively, these data indicate that GSK3ss interacts with and phosphorylates the spindle-associated protein Astrin, resulting in targeting Astrin to the spindle microtubules and kinetochores. In turn, the GSK3ss-Astrin complex may also facilitate further physiological and pathological phosphorylation. [Abstract/Link to Full Text]

Fredenburgh JC, Stafford AR, Leslie BA, Weitz JI
Bivalent binding to gamma A/gamma -fibrin engages both exosites of thrombin and protects it from inhibition by the antithrombin-heparin complex.
J Biol Chem. 2007 Nov 30;
Thrombin exosite 1 binds the predominant A/A-fibrin form with low affinity. A subpopulation of fibrin molecules, A/-fibrin, has an extended COOH-terminal -chain that binds exosite 2 of thrombin. Bivalent binding to A/-fibrin increases thrombin's affinity 10-fold, as determined by surface plasmon resonance. Because of its higher affinity, thrombin dissociates 7-fold more slowly from A/-fibrin clots than from A/A-fibrin clots. After 24 hours of washing, however, both A/- and A/A-fibrin clots generate fibrinopeptide A when incubated with fibrinogen, indicating the retention of active thrombin. Previous studies demonstrated that heparin heightens thrombin's affinity for fibrin by simultaneously binding to fibrin and exosite 2 on thrombin to generate a ternary heparin-thrombin-fibrin complex that protects thrombin from inhibition by antithrombin and heparin cofactor II. In contrast, dermatan sulfate does not promote ternary complex formation because it does not bind to fibrin. Heparin-catalyzed rates of thrombin inhibition by antithrombin were 5-fold slower in A/-fibrin clots than they were in A/A-fibrin clots. This difference reflects bivalent binding of thrombin to A/-fibrin because (a) it is abolished by addition of a -chain-directed antibody that blocks exosite 2-mediated binding of thrombin to the -chain, and (b) the dermatan sulfate-catalyzed rate of thrombin inhibition by heparin cofactor II also is lower with A/-fibrin than with A/A-fibrin clots. Thus, bivalent binding of thrombin to A/-fibrin protects thrombin from inhibition, raising the possibility that A/-fibrin serves as a reservoir of active thrombin that renders thrombi thrombogenic. [Abstract/Link to Full Text]

Zhao X, Nicholls JM, Chen YG
Sars-cov nucleocapsid protein interacts with smad3 and modulates TGF-beta signaling.
J Biol Chem. 2007 Nov 30;
Severe acute respiratory syndrome (SARS) is an acute infectious disease with significant mortality. A typical clinical feature associated with SARS is pulmonary fibrosis and the associated lung failure. However, the underlying mechanism remains elusive. In this study, we demonstrate that SARS-CoV nucleocapsid (N) protein potentiates transforming growth factor-ss (TGF-ss)-induced expression of plasminogen activator inhibitor-1 but attenuates Smad3/Smad4-mediated apoptosis of human peripheral lung epithelial HPL1 cells. The promoting effect of N protein on the transcriptional responses of TGF-ss is Smad3-specific. N protein associates with Smad3 and promotes Smad3-p300 complex formation while it interferes with the complex formation between Smad3 and Smad4. These findings provide evidence of a novel mechanism whereby N protein modulates TGF-ss signaling to block apoptosis of SARS-CoV-infected host cells and meanwhile promote tissue fibrosis. Our results reveal a novel mode of Smad3 action in a Smad4-independent manner and may lead to successful strategies for SARS treatment by targeting the TGF-ss signaling molecules. [Abstract/Link to Full Text]

Anelli VV, Gault CR, Cheng AB, Obeid LM
Sphingosine kinase 1 is up-regulated during hypoxia in U87MG glioma cells: Role of hypoxia-inducible factors 1 and 2.
J Biol Chem. 2007 Nov 30;
Sphingosine-1-phosphate (S1P), a sphingolipid metabolite that plays an important role in the regulation of cell survival, growth, migration, and angiogenesis acts both inside the cells and as an extracellular mediator through binding to five G protein-coupled receptors (S1P1-5). Sphingosine kinase 1 (SK1), the enzyme responsible for S1P production, is over-expressed in many solid tumors, including gliomas. One common feature of these tumors is the presence of "hypoxic regions", characterized by cells expressing high levels of hypoxia-inducible factors HIF-1a and HIF-2a, two transcription regulators that modulate the levels of proteins with crucial roles in tumor progression. So far nothing is known about the role and the regulation of SK1 during tumor-induced hypoxia or about SK1 regulation and HIFs. Here we investigated the role of HIF-1a and HIF-2a in the regulation of SK1 during hypoxic stress in glioma-derived cells U87MG. We report that hypoxia increases SK1 mRNA levels, protein expression and enzyme activity, followed by intracellular S1P production and S1P release. Interestingly, knock-down of HIF-2a by siRNA abolished the induction of SK1 and the production of extracellular S1P after CoCl2 treatment, whereas HIF-1a siRNA resulted in an increase of HIF-2a and of SK1 protein levels. Moreover, using Chip analysis we demonstrate that HIF-2a binds the SK1 promoter. Functionally, we demonstrate that conditioned media from hypoxia-treated tumor cells results in neo-angiogenesis in human umbilical vein endothelial cells (HUVEC) in a S1P receptor-dependent manner. These studies provide evidence of a link between S1P production as a potent angiogenic agent and the hypoxic phenotype observed in many tumors. [Abstract/Link to Full Text]

Langelier MF, Servent KM, Rogers EE, Pascal JM
A third zinc-binding domain of human PARP-1 coordinates DNA-dependent enzyme activation.
J Biol Chem. 2007 Nov 30;
PARP-1 is a chromatin-associated enzyme with multiple cellular functions, including DNA repair, transcriptional regulation, and cell signaling. PARP-1 has a modular architecture with six independent domains comprising the 113 kDa polypeptide. Two zinc-finger domains at the N-terminus of PARP-1 bind to DNA and thereby activate the catalytic domain situated at the C-terminus of the enzyme. The tight coupling of DNA binding and catalytic activities is critical to the cellular regulation of PARP-1 function, however, the mechanism for coordinating these activities remains an unsolved problem. Here, we demonstrate using spectroscopic and crystallographic analysis that human PARP-1 has a third zinc-binding domain. Biochemical mutagenesis and deletion analysis indicate that this region mediates inter-domain contacts important for DNA-dependent enzyme activation. The crystal structure of the third zinc-binding domain reveals a zinc ribbon fold and suggests conserved residues that could form inter-domain contacts. The new zinc-binding domain self-associates in the crystal lattice to form a homodimer with a head-to-tail arrangement. The structure of the homodimer provides a scaffold for assembling the activated state of PARP-1 and suggests a mechanism for coupling the DNA binding and catalytic functions of PARP-1. [Abstract/Link to Full Text]

Kiss AZ, Ruban AV, Horton P
The PsbS protein controls the organisation of the photosystem II antenna in higher plant thylakoid membranes.
J Biol Chem. 2007 Nov 29;
The PsbS subunit of photosystem II (PSII) plays a key role in nonphotochemical quenching (NPQ), the major photoprotective regulatory mechanism in higher plant thylakoid membranes, but its mechanism of action is unknown. Here we describe direct evidence that PsbS controls the organisation of PSII and its light harvesting system (LHCII). The changes in chlorophyll fluorescence amplitude associated with the Mg2+-dependent restacking of thylakoid membranes were measured in thylakoids prepared from wild-type plants, a PsbS deficient mutant and a PsbS over-expresser. The Mg2+ requirement and sigmoidicity of the titration curves for the fluorescence rise were negatively correlated with the level of PsbS. Using a range of PsbS mutants this effect of PsbS was shown not to depend upon its efficacy in controlling NPQ, but to be related only to protein concentration. Electron microscopy and fluorescence spectroscopy showed that this effect was due to enhancement of the Mg2+-dependent re-association of PSII and LHCII by PsbS, rather than an effect on stacking per se. In the presence of PsbS the LHCII/PSII complex was also more readily removed from thylakoid membranes by detergent and the level of PsbS protein correlated with the amplitude of the psi-type CD signal originating from features of LHCII/PSII organisation. It is proposed that PsbS regulates the interaction between LHCII and PSII in the grana membranes, explaining how it acts as a pH-dependent trigger of the conformational changes within the PSII light harvesting system that result in NPQ. [Abstract/Link to Full Text]

Kleinschmidt AM, Nassiri M, Stitt MS, Wasserloos K, Watkins SC, Pitt BR, Jahroudi N
Sequences in intron 51 of the VWF gene target promoter activation to a subset of lung endothelial cells in transgenic mice.
J Biol Chem. 2007 Nov 28;
In vivo analyses of the VWF promoter previously demonstrated that a fragment spanning sequences -487 to +247 targets promoter activation to brain vascular endothelial cells while a longer fragment, including 2182 bp of the 5' flanking sequences, the first exon and the first intron activated expression in endothelial cells of the heart and muscles as well as brain of transgenic mice. These results suggested that additional VWF gene sequences were required for expression in other vascular endothelial cells in vivo. We have now identified a region within intron 51 of the VWF gene that is DNase I hypersensitive (HSS) specifically in non-endothelial cells and interacts with endothelial and non-endothelial specific complexes that contain YY1. We demonstrate that beta-actin is associated with YY1 specifically in the nucleus of non-endothelial cells and is a component of the nuclear protein complexes that interact with DNase I hypersensitive region. In vitro transfection analyses demonstrated that HSS sequences containing this YY1 binding site do not significantly affect VWF promoter activity. However, in vivo analyses demonstrated that addition of these sequences to the VWF promoter (-487 to +247) results in promoter activation in lung and brain vascular endothelial cells. These results demonstrate that the HSS sequences in intron 51 of the VWF gene contain cis-acting elements that are necessary for the VWF gene transcription in a subset of lung endothelial cells in vivo. [Abstract/Link to Full Text]

Narang SS, Malone CL, Deschenes RJ, Fassler JS
Modulation of yeast Sln1 kinase activity by the Ccw12 cell wall protein.
J Biol Chem. 2007 Nov 28;
The yeast Sln1p sensor-kinase is best known as an osmosensor involved in the regulation of the HOG (hyperosmolarity glycerol) MAP kinase cascade. Down-regulation of Sln1 kinase activity occurs under hypertonic conditions and leads to phosphorylation of the Hog1p MAP kinase and increased osmotic stress response gene expression. Conditions leading to kinase up-regulation include osmotic imbalance caused by glycerol retention in the glycerol channel mutant, fps1 (1). The hypothesis that Sln1 kinase activity is responsive to turgor was first suggested by the increased Sln1p kinase activity in mutants lacking Fps1p in which glycerol accumulation leads to water uptake. Also consistent with the turgor hypothesis is the observation that reduced turgor caused by treatment of cells with nystatin which increases membrane permeability and causes cell shrinkage reduced Sln1p kinase activity (1,2). The turgor hypothesis is revisited here in the context of the identification and characterization of the cell wall gene, CCW12, as a determinant of Sln1p activity. Results of this analysis suggest that the activity of the plasma membrane localized Sln1p is affected by the presence or absence of specific outer cell wall proteins and that this effect is independent of turgor. [Abstract/Link to Full Text]

Yin Q, Wang X, McBride J, Fewell C, Flemington EK
B-cell receptor activation induces BIC/MIR-155 expression through a conserved AP-1 element.
J Biol Chem. 2007 Nov 28;
microRNA-155, is an oncogenic microRNA that has been shown to be critical for B-cell maturation and immunoglobulin production in response to antigen. In line with its function in B-cell activation, miR-155, and its primary transcript, BIC, is induced by B-cell receptor (BCR) crosslinking. Using pharmacological inhibitors in the human B cell line, Ramos, we show that activation of BIC and miR-155 expression by BCR signaling occurs through the ERK (Extracellular signaling Regulated Kinase) and JNK (c-Jun N-terminal Kinase) pathways but not the p38 pathway. BCR activation results in the induction of c-Fos, FosB, and JunB and expression of these are suppressed by ERK and JNK inhibitors. Reporter analysis established a key role for a conserved AP-1 site approximately 40 bp upstream from the site of initiation but not an upstream NF-kB site or a putative c-Ets located at the site of initiation. Lastly, chromatin immunoprecipitation analysis demonstrated the recruitment of FosB and JunB to the miR-155 promoter following BCR activation. These results identify key determinants of BCR mediated signaling that lead to the induction of BIC/miR-155. [Abstract/Link to Full Text]

Phartiyal P, Sale H, Jones EM, Robertson GA
ER retention and rescue by heteromeric assembly regulates hERG 1a/1b surface Channel composition.
J Biol Chem. 2007 Nov 28;
Defects in the trafficking of subunits encoded by the human Ether-ŕ-go-go Related Gene (hERG1) can lead to catastrophic arrhythmias and sudden cardiac death due to a reduction in I(Kr)-mediated repolarization. Native I(Kr) channels are composed of two alpha subunits, hERG 1a and 1b. In heterologous expression systems, hERG 1b subunits efficiently produce current only in heteromeric combination with hERG 1a. We used western blot analysis and electrophysiological recordings in HEK-293 cells and Xenopus oocytes to monitor hERG 1b maturation in the secretory pathway and determine the factors regulating surface expression of hERG 1b subunits. We found that 1b subunits expressed alone were largely retained in the endoplasmic reticulum (ER), thus accounting for the poor functional expression of homomeric 1b currents. Association with hERG 1a facilitated 1b ER export and surface expression. We show that hERG 1b subunits fail to mature because of an "RXR" ER retention signal specific to the 1b N terminus of the human sequence and not conserved in other species. Mutating the RXR facilitated maturation and functional expression of homomeric hERG 1b channels, and did so in a charge-dependent manner. Co-expression of the 1b RXR mutants with hERG 1a did not further enhance 1b maturation, suggesting hERG 1a promotes 1b trafficking by overcoming the RXR-mediated retention. Thus, selective trafficking mechanisms regulate subunit composition of surface hERG channels. [Abstract/Link to Full Text]

Lito P, Mets BD, Kleff S, O'Reilly S, Maher VM, McCormick JJ
Evidence that sprouty 2 is necessary for sarcoma formation by HRAS oncogene transformed human fibroblasts.
J Biol Chem. 2007 Nov 28;
Sprouty-2 (Spry2) acts as an inhibitor of receptor tyrosine kinase signaling. Spry2 also prevents the c-Cbl-induced degradation of epidermal growth factor receptor (EGFR). We compared human fibroblasts malignantly transformed by overexpression of HRasV12 oncogene to their non-transformed parental cells and found that the malignant cells express a high level of Spry2. These cells also exhibited an increase in the level of EGFR compared to their precursor cells. We found that intact EGFR was required if HRas-transformed cells were to grow in the absence of exogenous growth factors or form large colonies in agarose. When we decreased expression of Spry2, using a Spry2-specific shRNA, the HRasV12-transformed fibroblasts could no longer form large colonies in agarose, grow in reduced levels of serum, or form tumors in athymic mice. The level of active HRas in these cells remained unaltered. A similar, but less pronounced, effect in tumor formation was observed when Spry2 was down-regulated in human patient-derived fibrosarcoma cell lines. In HRas-transformed cells Spry2 sustained the level and the downstream signaling activity of EGFR. In the parental, non-HRas-transformed, fibroblasts expression of Spry2 resulted in the inhibition of HRas and ERK activation, suggesting that the positive effect of Spry2 in tumor formation is specific to HRas-transformation. Co-immunoprecipita-tion studies with HRas-transformed cells revealed that Spry2 and HRas interact, and that HRas interacts with Spry2-binding partners, c-Cbl and CIN85, in a Spry2-dependent manner. These data show that Spry2 plays a critical role in the ability of HRas-transformed cells to form sarcomas in athymic mice. [Abstract/Link to Full Text]

Thompson C, Cloutier A, Bossé Y, Poisson C, Larivée P, McDonald PP, Stankova J, Rola-Pleszczynski M
Signaling by the cysteinyl-leukotriene receptor 2 : Involvement in chemokine gene transcription.
J Biol Chem. 2007 Nov 29;
Cysteinyl-leukotrienes are involved in inflammation and act on at least two G-protein-coupled receptors, CysLT1 and CysLT2. However, the role of the CysLT2 receptor as well as its signaling remain poorly understood. Here we show that leukotriene (LT)C4 induced the production of the chemokine IL-8 in endothelial cells. To further study the signaling cascade involved, HEK293 cells were stably transfected with CysLT2 and used to study the transcriptional regulation of the IL-8 promoter. Stimulation of the cells with increasing concentrations of LTC4 resulted in a time- and concentration-dependent induction of IL-8 transcription and protein synthesis. Use of IL-8 promoter mutants with substitutions in their NF-B, AP-1 or NF-IL-6 binding elements revealed an almost total requirement for NF-B and AP-1 elements, and a lesser requirement for the NF-IL-6 element. Overexpression of dominant-negative IBa prevented the IL-8 transactivation induced by LTC4. LTC4 stimulation induced NF-B and AP-1 DNA binding, which involved the formation of a p50/p65 and a c-Jun/c-Fos complex, respectively. Transfection of the cells with a dominant negative (dn) form of PKCe prevented p65 phosphorylation, whereas dnPKCd prevented AP-1 binding. Moreover, dnPKCd, dnPKCe and dnPKC prevented LTC4-induced IL-8 transcription in response to LTC4. Our data show for the first time that LTC4 can act via the CysLT2 receptor to transcriptionally activate chemokine production through induction of NF-B and AP-1 transcription factors. These findings suggest the potential implication of CysLT2 in the inflammatory response through the modulation of chemokine gene transcription. [Abstract/Link to Full Text]

Gauguin L, Klaproth B, Sajid W, Andersen AS, McNeil KA, Forbes BE, De Meyts P
Structural basis for the lower affinity of the insulin-like growth factors for the insulin receptor.
J Biol Chem. 2007 Nov 29;
Insulin and the insulin-like growth factors (IGFs) bind with high affinity to their cognate receptor and with lower affinity to the non-cognate receptor. The major structural difference between insulin and the IGFs is that the IGFs are single chain polypeptides containing A-, B-, C- and D-domains, whereas the insulin molecule contains separate A- and B-chains. The C-domain of IGF-I is critical for high affinity binding to the insulin-like growth factor I receptor and lack of a C-domain largely explains the low affinity of insulin for the insulin-like growth factor I receptor. It is less clear why the IGFs have lower affinity for the insulin receptor. In this study 24 insulin analogues and 4 IGF analogues were expressed and analyzed to explore the role of amino acid differences in the A and B-domains between insulin and the IGFs in binding affinity for the insulin receptor. Using the information obtained from single substituted analogues, four multiple substituted analogues were produced. A 'quadruple insulin' analogue ([A8Phe,A10Ser, B5Thr,B16Gln]Ins) showed similar affinity as IGF-I for the insulin receptor and a 'sextuple insulin' analogue ([A8Phe,A10Ser,A18Thr,B5Thr, B14Thr,B16Gln]Ins) showed an affinity close to that of IGF-II for the insulin receptor, while a 'quadruple IGF-I' analogue ([4His,15Tyr,49Thr, 51Ile]IGF-I) and a 'sextuple IGF-II' analogue ([7His,16Ala,18Tyr,49Thr,51Ile,58Asn]IGF-II) showed affinities similar to that of insulin for the insulin receptor. The mitogenic potency of these analogues correlated well with the binding properties. Thus, a small number of A- and B-domain substitutions that map to the IGF surface equivalent to the classical binding surface of insulin weaken two hotspots that bind to the insulin receptor site 1. [Abstract/Link to Full Text]

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Recent Articles in Bioscience, Biotechnology, and Biochemistry

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Azuma N, Kanamaru K, Matsushika A, Yamashino T, Mizuno T, Kato M, Kobayashi T
In vitro analysis of His-Asp phosphorelays in Aspergillus nidulans: the first direct biochemical evidence for the existence of His-Asp phosphotransfer systems in filamentous fungi.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2493-502.
His-Asp phosphorelays are widespread signal transduction mechanisms in bacteria, fungi, and higher plants. In order to investigate a His-Asp phosphorelay network in filamentous fungi, which has been genetically characterized in part, we attempted to construct an in vitro phosphotransfer network in Aspergillus nidulans comprising all the necessary components. As a first step, we established an in vitro phosphotransfer system with a histidine-containing phosphotransmitter YpdA, a response regulator SrrA, and a bacterial histidine kinase ArcB as a phosphate donor. We demonstrated the phosphotransfer from ArcB to A. nidulans YpdA and the subsequent transfer from YpdA to SrrA. This is the first direct biochemical evidence for the presence of the phosphotransfer system in filamentous fungi. Furthermore, a retrograde phosphorylation from YpdA to FphA, a histidine kinase similar to bacterial phytochrome, was found. The overall picture of the His-Asp phosphorelays in A. nidulans is discussed based on the results of the in vitro study. [Abstract/Link to Full Text]

Watanabe T, Furukawa S, Kawarai T, Wachi M, Ogihara H, Yamasaki M
Cytoplasmic acidification may occur in high-pressure carbon dioxide-treated Escherichia coli K12.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2522-6.
While studying the mechanism by which high-pressure carbon dioxide treatment (HCT) inactivates bacteria, we found that the efficiency of DNA recovery via phenol extraction was extraordinarily low from E. coli K12 cells that had been subjected to HCT. DAPI staining of the treated cells, however, revealed that nuclear DNA was present. Most DNA from the cells subjected to HCT was probably caught in the denatured protein layer during phenol extraction. The efficiency of DNA recovery from proteinase-treated crude extracts from cells subjected to HCT was high. Crude extracts of E. coli K12 cells that had not undergone HCT were intentionally acidified with acetic acid to pH 5.2 to cause acidic coagulation of cytoplasmic proteins. The efficiency of DNA recovery from the acidified extracts was low. These results suggest that in cells subjected to HCT, cytoplasmic pH is reduced to around pH 5.2, and that nuclear DNA becomes entangled in coagulated cytoplasmic proteins. Acidification of the cytoplasm might be the primary mechanism by which HCT inactivates bacteria. [Abstract/Link to Full Text]

Tamaru S, Kurayama T, Sakono M, Fukuda N, Nakamori T, Furuta H, Tanaka K, Sugano M
Effects of dietary soybean peptides on hepatic production of ketone bodies and secretion of triglyceride by perfused rat liver.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2451-7.
Soybean protein isolate (SPI) was digested with protease to produce a peptides containing the low-molecular fraction (LD3) or a mixture of high- and low-molecular fractions (HD1). Rats were fed a diets containing SPI, LD3, or HD1 at a protein level equivalent to the 20% casein diet for 4 weeks. The serum triglyceride concentration was lower in rats fed SPI, LD3, and HD1 diets than in rats fed the casein diet, and the differences were significant for the cholesterol-enriched diet. The value for the LD3 group was the lowest among all groups for both the cholesterol-free and -enriched diets. The level of triglyceride in the post-perfused liver was significantly lower in the LD3 and HD1 groups and the SPI group than in the casein group irrespective of the presence of cholesterol in the diet. In the cholesterol-free diet, LD3 feeding as compared to casein feeding caused a reduction in triglyceride secretion from the liver to perfusate and an increment of hepatic ketone body production. The addition of cholesterol to the diets somewhat attenuated these effects of LD3. These results suggest that the low-molecular fraction in soybean peptides causes triglyceride-lowering activity through a reduction in triglyceride secretion from the liver to the blood circulation and the stimulation of fatty acid oxidation in the liver. There is a possibility that soybean peptides modulate triglyceride metabolism by changes in the hepatic contribution. [Abstract/Link to Full Text]

Nakano C, Motegi A, Sato T, Onodera M, Hoshino T
Sterol biosynthesis by a prokaryote: first in vitro identification of the genes encoding squalene epoxidase and lanosterol synthase from Methylococcus capsulatus.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2543-50.
Sterol biosynthesis by prokaryotic organisms is very rare. Squalene epoxidase and lanosterol synthase are prerequisite to cyclic sterol biosynthesis. These two enzymes, from the methanotrophic bacterium Methylococcus capsulatus, were functionally expressed in Escherichia coli. Structural analyses of the enzymatic products indicated that the reactions proceeded in a complete regio- and stereospecific fashion to afford (3S)-2,3-oxidosqualene from squalene and lanosterol from (3S)-2,3-oxidosqualene, in full accordance with those of eukaryotes. However, our result obtained with the putative lanosterol synthase was inconsistent with a previous report that the prokaryote accepts both (3R)- and (3S)-2,3-oxidosqualenes to afford 3-epi-lanosterol and lanosterol, respectively. This is the first report demonstrating the existence of the genes encoding squalene epoxidase and lanosterol synthase in prokaryotes by establishing the enzyme activities. The evolutionary aspect of prokaryotic squalene epoxidase and lanosterol synthase is discussed. [Abstract/Link to Full Text]

Arii Y, Yamaguchi H, Fukuoka S
Production of a soluble recombinant prion protein fused to blue fluorescent protein without refolding or detergents in Escherichia coli cells.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2511-4.
The physiological function of prion proteins (PrP) remains unclear. To investigate the physiological relevance of PrP, we constructed a fusion protein of PrP with enhanced blue fluorescent protein (PrP-EBFP) to quantify the interaction of PrP with other molecules. Production of soluble PrP-EBFP was achieved by lowering the expression temperature in Escherichia coli (E. coli) cells to 15 degrees C. Soluble PrP-EBFP was purified on cation exchange and heparin-affinity columns to yield high purity protein. This is the first report of the preparation of soluble recombinant PrP without refolding following solubilization using denaturants or disruption using detergents. To confirm the integrity of PrP-EBFP, anisotropy was estimated under physiological conditions in the presence of heparin, which interacts with PrP. The dissociation constant was determined to be 0.88+/-0.07 microM. PrP-EBFP should be useful in the quantification of PrP interactions with other molecules. [Abstract/Link to Full Text]

Nozaki K, Seki T, Matsui K, Mizuno M, Kanda T, Amano Y
Structure and characteristics of an endo-beta-1,4-glucanase, isolated from Trametes hirsuta, with high degradation to crystalline cellulose.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2375-82.
Trametes hirsuta produced cellulose-degrading enzymes when it was grown in a cellulosic medium such as Avicel or wheat bran. An endo-beta-1,4-glucanase (ThEG) was purified from the culture filtrate, and the gene and the cDNA were isolated. The gene consisted of an open reading frame encoding 384 amino acids, interrupted by 11 introns. The whole sequence showed high homology with that of family 5 glycoside hydrolase. The properties of the recombinant enzyme (rEG) in Aspergillus oryzae were compared with those of the En-1 from Irpex lacteus, which showed the highest homology among all the endoglucanases reported. The rEG activity against Avicel was about 8 times higher than that of En-1 when based on CMC degradation. A remarkable structural difference between the two enzymes was the length of the linker connecting the cellulose-binding domain to the catalytic domain. [Abstract/Link to Full Text]

Usui T, Yanagisawa S, Ohguchi M, Yoshino M, Kawabata R, Kishimoto J, Arai Y, Aida K, Watanabe H, Hayase F
Identification and determination of alpha-dicarbonyl compounds formed in the degradation of sugars.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2465-72.
The alpha-dicarbonyl compounds formed in the degradation of glucose and fructose were analyzed by HPLC using 2,3-diaminonaphthalene as derivatizing reagent, and identified as glucosone (GLUCO), 3-deoxyglucosone (3DG), 3-deoxyxylosone (3DX), tetrosone (TSO), triosone (TRIO), 3-deoxytetrosone (3DT), glyoxal (GO), and methylglyoxal (MGO). The results suggest that alpha-dicarbonyl compounds were formed from glucose via non-oxidative 3-deoxyglucosone formation and oxidative glucosone formation in glucose degradation. In addition, TRIO, GO, and MGO were also formed from glyceraldehyde as intermediate. The alpha-dicarbonyl compounds might be formed from glucose via these pathways in diabetes. [Abstract/Link to Full Text]

Yamada K, Shibuya T, Noda M, Uchiyama N, Kashiwada A, Matsuda K, Hirata M
Influence of position of substituent groups on removal of chlorophenols and cresols by horseradish peroxidase and determination of optimum conditions.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2503-10.
Enzymatic treatment of o-, m-, and p-chlorophenols and o-, m-, and p-cresols from artificial wastewater was undertaken through the enzymatic conversion into the corresponding phenoxy radicals with horseradish peroxidase (HRP) and nonenzymatic radical coupling reaction. The concentration of chlorophenols and cresols decreased sharply over the reaction time and water-insoluble oligomer precipitates were generated. The optimum conditions were determined to be the H2O2 concentration of 2.5 mM and poly(ethylene glycol) with molecular mass of 1.0 x 10(4) (10K-PEG) of 0.10 mg/cm3 at 30 degrees C for treatment of p-chlorophenol at 2.5 mM. The optimum pH values depended on the relative position of a chlorine atom for chlorophenols and on a methyl group for cresols. Concentrations of HRP and 10K-PEG were increased to 1.0 U/cm3 and 1.0 mg/cm3 respectively to treat m-chlorophenol highly. For o-chlorophenol, a decrease in the pH value to 3.0 after the enzymatic treatment led to the enhancement of the aggregation of oligomer precipitates. The % residual value for o-cresol effectively decreased by absorbing water-soluble intermediates on the chitosan films. These results indicate that chlorophenols and cresols were removed to a great degree by this technique, although the detailed procedure depended on the position of substituent groups of chlorophenols and cresols. [Abstract/Link to Full Text]

Makabe H
Synthesis of annonaceous acetogenins from muricatacin.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2367-74.
Synthetic studies of annonaceous acetogenins starting from (-)-muricatacin (1a) or (+)-muricatacin are described, involving (-)-muricatacin (1a), mono-THF acetogenin, solamin (2), reticulatacin (3), (15R, 16R, 19S, 20S)-cis-solamin (4a) and (15S, 16S, 19R, 20R)-cis-solamin (4b), non-adjacent bis-THF acetogenin, 4-deoxygigantecin (5), and epoxide-bearing acetogenin, (15S, 16R, 19S, 20R)-diepomuricanin (6a). [Abstract/Link to Full Text]

Yamakawa H, Akiyama H, Endo Y, Miyatake K, Sakata K, Sakai S, Toyoda M, Urisu A
Specific detection of wheat residues in processed foods by polymerase chain reaction.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2561-4.
A sensitive qualitative detection method for wheat in foods using polymerase chain reaction (PCR) was developed. Trace amounts of wheat in commercial food products could be qualitatively detected by this method. The sensitivity of the proposed PCR method appears to be similar to that of ELISA. The present method should be very useful for detecting wheat residues in processed foods. [Abstract/Link to Full Text]

Sakuno E, Tani H, Nakajima H
2-epi-botcinin A and 3-O-acetylbotcineric acid from Botrytis cinerea.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2592-5.
Two metabolites, 2-epi-botcinin A and 3-O-acetylbotcineric acid, were isolated from Botrytis cinerea (AEM211). The former compound was new, and the latter was known but structurally revised by us. In a test for antifungal activity against Magnaporthe grisea, a pathogen of rice blast disease, 2-epi-botcinin A was 8 times less active than botcinin A (MIC 100 microM), and the MIC value for 3-O-acetylbotcineric acid being 100 microM. [Abstract/Link to Full Text]

Hoshino T, Eguchi T
Functional analysis of type 1 isopentenyl diphosphate isomerase from Halobacterium sp. NRC-1.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2588-91.
Here we report the characterization of the type-1 isopentenyl diphosphate isomerase derived from Halobacterium sp. NRC-1. The expressed purified enzyme showed maximum isomerase activity in the presence of 1 M NaCl at 37 degrees C at pH 6.0. This type-1 enzyme appears to be the first for which the Co2+ ion is required for activity. [Abstract/Link to Full Text]

Bando N, Wakamatsu S, Terao J
Effect of an excessive intake of quercetin on the vitamin E level and antioxidative enzyme activities of mouse liver under paraquat-induced oxidative stress.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2569-72.
The liver alpha-tocopherol level of the paraquat fed mice group was lower than that of the control diet-fed group. An excessive intake of quercetin lowered the liver alpha-tocopherol level of the control diet-fed mice group, but did not affect it in the paraquat-fed mice group. The same quercetin intake significantly increased the superoxide dismutase and glutathione peroxidase activities in the liver of both groups, indicating that excessive quercetin intake can either promote or attenuate oxidative stress in the liver. [Abstract/Link to Full Text]

Furumoto T, Ohara T, Kubo T, Kawanami Y, Fukui H
2-Geranyl-1,4-naphthoquinone, a possible intermediate of anthraquinones in a Sesamum indicum hairy root culture.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2600-2.
2-Geranyl-1,4-naphthoquinone was isolated from the hairy root culture of Sesamum indicum. The structure was determined to be 2-[(E)-3,7-dimethylocta-2,6-dienyl]-1,4-naphthoquinone on the basis of spectroscopic evidence and chemical synthesis. The production of anthrasesamones A, B and C by the hairy root culture was also confirmed for the first time. [Abstract/Link to Full Text]

Kumada H, Mitsutake S, Inagaki Y, Mitsunaga S, Tsuchikawa H, Katsumura S, Igarashi Y
Kinetics of the ceramide kinase inhibitor K1, a suppressor of mast-cell activation.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2581-4.
In a previous study, we synthesized a novel inhibitor of ceramide kinase, K1. In this study, we determined that inhibition by K1 is non-competitive and that four intact six-membered rings are important to the inhibitory activity. Furthermore, we identified an effective in vivo concentration for K1, at which it did not influence any cellular lipid synthesis other than that of ceramide 1-phosphate (C1P) using RBL-2H3 cells, and found that K1 suppressed the activation of mast cells. [Abstract/Link to Full Text]

Yoshimatsu M, Toyoda A, Onizawa N, Nakamura Y, Minato H
Biochemical characterization of cellulose-binding proteins (CBPA and CBPB) from the rumen cellulolytic bacterium Eubacterium cellulosolvens 5.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2577-80.
The cellulose-binding proteins, CBPA and CBPB, of rumen cellulolytic bacterium Eubacterium cellulosolvens 5 were biochemically characterized, and their properties were compared. Recombinant CBPA and CBPB were a typical 1,4-beta-endoglucanase. Both proteins bound to insoluble polysaccharides such as Avicel cellulose, acid swollen cellulose, lichenan, chitin, and oat spelt xylan. On the other hand, only recombinant CBPB bound to agarose and starch. [Abstract/Link to Full Text]

Tachibana S, Yasuda M
Purification and characterization of heterogeneous glucoamylases from Monascus purpureus.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2573-6.
Two forms of an extracellular glucoamylase, MpuGA-I and MpuGA-II, were purified to homogeneity from Monascus purpureus RY3410. The molecular weights of these enzymes were estimated to be 60,000 (MpuGA-I) and 89,000 (MpuGA-II). These enzymes were glycoproteins with a carbohydrate content of 15.0% (MpuGA-I) and 16.2% (MpuGA-II) respectively. The pH optima were 5.0 for both enzymes, and the optimal temperatures were 50 degrees C (MpuGA-I) and 65 degrees C (MpuGA-II). The Km values for soluble starch were calculated to be 4.0+/-0.8 mg/ml (MpuGA-I) and 1.1+/-0.2 mg/ml (MpuGA-II) respectively. [Abstract/Link to Full Text]

Takahashi S, Hata K, Kikuchi K, Gotoh T
High-level expression of recombinant active human renin in Sf-9 cells: rapid purification and characterization.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2610-3.
Recombinant human (rh) renin was expressed in Sf-9 insect cells. Baculovirus-infected Sf-9 cells produced active rh-renin in the late stage of cultivation. The rh-renin was purified after 5 d of culture by two steps of column chromatography. Approximately 0.61 mg of pure rh-renin was obtained from 200 ml of culture medium with a yield of 35.3%. [Abstract/Link to Full Text]

Kim HW, Takagi Y, Hagihara Y, Ishikawa K
Analysis of the putative substrate binding region of hyperthermophilic endoglucanase from Pyrococcus horikoshii.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2585-7.
A hyperthermophophilic beta-1,4 endoglucanase (family 5, cellulase) was identified in a hyperthermophilic archaeon Pyrococcus horikoshii and found to be capable of hydrolyzing crystalline cellulose at high temperatures. This hyperthermophilic enzyme has promise for applications in biomass utilization, but we have no information regarding the catalytic mechanism or structure of the enzyme. To determine its catalytic mechanism, we examined the roles of amino acids located in a loop near the speculative active site by the alanine scanning method. Ten mutants of the enzyme were constructed and expressed in Escherichia coli. The purified mutant enzymes were assayed for their hydrolytic activities on p-nitrophenyl cellobiose (pNG2), carboxylmethyl cellulose, and avicel. The results showed that His155, Arg156, and Ile162 play an important role in pNG2 binding capacity, and that H155 and I162 are important for catalysis. [Abstract/Link to Full Text]

Fukada T, Senda-Murata K, Nishida K, Sakaushi S, Minamikawa H, Dotsu M, Oka S, Sugimoto K
A multi-fluorescent MDA435 cell line for mitosis inhibitor studies: simultaneous visualization of chromatin, microtubules, and nuclear envelope in living cells.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2603-5.
As a part of our efforts to create a pre-made multi-fluorescent cell library for various cytological assays, we made a triple-fluorescent cell line in which microtubules, chromosomes, and nuclear envelopes were visualized for simultaneous observation of spindle structure and chromosome distribution in living cells. Pilot experiments with microtubule-disturbing drugs showed the advantages of this cell line in mitosis inhibitor studies. [Abstract/Link to Full Text]

Yoshioka M, Murayama Y, Miwa T, Miura K, Takata M, Yokoyama T, Nishizawa K, Mohri S
Assessment of prion inactivation by combined use of Bacillus-derived protease and SDS.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2565-8.
Prions, infectious agents causing transmissible spongiform encephalopathy, retain infectivity even after undergoing routine sterilization processes. We found that MSK103 protease, identified in our previous study, effectively reduces infectivity and the level of misfolded isoform of the prion protein in scrapie-infected brain homogenates in the presence of SDS. The treatment therefore can be applied to the decontamination of thermolabile instruments. [Abstract/Link to Full Text]

Suzuki R, Kulkarni A, Yomoda Y, Kawagishi H, Terada Y, Maoka T, Etoh H
Reaction of retinol with peroxynitrite.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2596-9.
The reactivity of retinol with peroxynitrite, which is a strong oxidant and has been reported to induce several biological damages, was investigated. 13-cis-14-nitroretinol (1), 13-trans-14-nitroretinol (2), 13-apo-beta-carotenone (3), retinal (4), 11,14-epoxyretinol (5), and 11,15-epoxyretinol (6) were identified as reaction products of retinol with peroxynitrite. From these results, it was observed that retinol can undergo a nitration reaction with peroxynitrite. Furthermore, the formation mechanisms of 1, 2, and 3 from retinol with peroxynitrite are discussed. [Abstract/Link to Full Text]

Watanabe J, Tanaka H, Akagawa T, Mogi Y, Yamazaki T
Characterization of Aspergillus oryzae aspartyl aminopeptidase expressed in Escherichia coli.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2557-60.
To characterize aspartyl aminopeptidase from Aspergillus oryzae, the recombinant enzyme was expressed in Escherichia coli. The enzyme cleaves N-terminal acidic amino acids. About 30% activity was retained in 20% NaCl. Digestion of defatted soybean by the enzyme resulted in an increase in the glutamic acid content, suggesting that the enzyme is potentially responsible for the release of glutamic acid in soy sauce mash. [Abstract/Link to Full Text]

Tsuchiyama M, Sakamoto T, Tanimori S, Murata S, Kawasaki H
Enzymatic synthesis of hydroxycinnamic acid glycerol esters using type A feruloyl esterase from Aspergillus niger.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2606-9.
We found that hydroxycinnamic acid (HA) glycerol esters such as 1-sinapoyl glycerol and 1-p-coumaroyl glycerol can be synthesized through a direct esterification reaction using a type A feruloyl esterase from Aspergillus niger. The water solubilities of HA glycerol esters were higher than those of the original chemicals. HA glycerol esters absorbed ultraviolet light and scavenged 1,1-diphenyl-2-picrylhydrazyl radicals. [Abstract/Link to Full Text]

Zhong B, Sakai S, Saeki T, Kanamoto R
Excess leucine intake induces serine dehydratase in rat liver.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2614-7.
We have hypothesized that rat liver serine dehydratase (SDH) is induced in response to the amount of surplus amino acids from dietary protein. In the present study, we found that excess leucine intake strongly induced SDH activity in the liver but not in the kidney of rats. The increase in activity was accompanied by increases in the levels of SDH mRNA. On the other hand, isoleucine and valine had little effect on SDH induction. These results support our hypothesis and suggest that leucine is a signal for SDH induction. [Abstract/Link to Full Text]

Ohinata K, Agui S, Yoshikawa M
Soymorphins, novel mu opioid peptides derived from soy beta-conglycinin beta-subunit, have anxiolytic activities.
Biosci Biotechnol Biochem. 2007 Oct;71(10):2618-21.
Based on the amino acid sequence YPFV found in the soy beta-conglycinin beta-subunit, which is common to an opioid peptide human beta-casomorphin-4, peptides YPFVV, YPFVVN, and YPFVVNA were synthesized according to their primary structure. On guinea pig ileum (GPI) assay, they showed opioid activity (IC50 = 6.0, 9.2 and 13 microM respectively) more potent than human beta-casomorphins, and were named soymorphins-5, -6, and -7, respectively. Their opioid activities on mouse vas deferens (MVD) assay were less potent than on GPI assay, suggesting that they are selective for the mu opioid receptor. Human beta-casomorphin-4 and soymorphin-5 were released from the soy 7S fraction (beta-conglycinin) by the action of gastrointestinal proteases. Soymorphins-5, -6, and -7 had anxiolytic activities after oral administration at doses of 10-30 mg/kg in the elevated plus-maze test in mice. [Abstract/Link to Full Text]

Mimitsuka T, Sawai H, Hatsu M, Yamada K
Metabolic engineering of Corynebacterium glutamicum for cadaverine fermentation.
Biosci Biotechnol Biochem. 2007 Sep;71(9):2130-5.
Cadaverine, the expected raw material of polyamides, is produced by decarboxylation of L-lysine. If we could produce cadaverine from the cheapest sugar, and as a renewable resource, it would be an effective solution against global warming, but there has been no attempt to produce cadaverine from glucose by fermentation. We focused on Corynebacterium glutamicum, whose L-lysine fermentation ability is superior, and constructed a metabolically engineered C. glutamicum in which the L-homoserine dehydrogenase gene (hom) was replaced by the L-lysine decarboxylase gene (cadA) of Escherichia coli. In this recombinant strain, cadaverine was produced at a concentration of 2.6 g/l, equivalent to up to 9.1% (molecular yield) of the glucose transformed into cadaverine in neutralizing cultivation. This is the first report of cadaverine fermentation by C. glutamicum. [Abstract/Link to Full Text]

Thao NT, Kashiwagi T, Sawamura M
Characterization by GC-MS of Vietnamese citrus species and hybrids based on the isotope ratio of monoterpene hydrocarbons.
Biosci Biotechnol Biochem. 2007 Sep;71(9):2155-61.
The isotope ratio of monoterpene hydrocarbons was used to characterize the citrus essential oils from different species and hybrids. Citrus cold-pressed peel oils from Vietnam were analyzed for the composition and isotope ratio of monoterpene hydrocarbons by using gas chromatography-mass spectrometry. A profile of citrus essential oils on the basis of their isotope ratio values and levels of monoterpene hydrocarbons was developed for Vietnamese citrus. The molecular isotope ratios were lower than those calculated from natural abundance of 13C and 2H. In addition, the isotope ratio of the base peaks (m/z 94/93) was significantly different among the citrus essential oils from different species and hybrids. The results would be applicable for the characterization of citrus essential oils from different origins. [Abstract/Link to Full Text]

Cheruiyot EK, Mumera LM, Ng'etich WK, Hassanali A, Wachira F
Polyphenols as potential indicators for drought tolerance in tea (Camellia sinensis L.).
Biosci Biotechnol Biochem. 2007 Sep;71(9):2190-7.
Plant polyphenols have gained prominence in quality of plant products and in human health. An experiment was conducted to determine the association of tea polyphenols with water stress and their suitability as indicators for drought tolerance. The experiment was conducted in a 'rain-out' shelter, and consisted of six tea clones (BBK 35, TRFK 6/8, TRFK 76/1, TRFK 395/2, TRFK 31/30, and TRFK 311/287) and four levels of soil water contents (38, 30, 22, and 14% v/v), which were maintained for a period of 12 weeks. The treatments were arranged in a completely randomized design and replicated three times. Plant growth was monitored over 6 weeks, and a water stress index was calculated to determine water-stress tolerant clones. Total polyphenols in tea shoots was analyzed and a regression analysis done. The results indicate that declining soil water content (SWC) reduced both growth and content of polyphenols in tea. Tolerant clones maintained a high polyphenol content at low SWC, and also showed less fluctuation in phenolics when subjected to changes in SWC. There was significant (P<0.001) correlation of total polyphenol content with shoot growth and WSI of tea, and a linear relationship (r2=0.97) between SWC for tea and both, water stress index and shoot polyphenol content. We report that there is a potential to use polyphenols as indicators for selection of drought-tolerant tea cultivars. [Abstract/Link to Full Text]

Abe K, Kushibiki T, Matsue H, Furukawa K, Motomura S
Generation of antitumor active neutral medium-sized alpha-glycan in apple vinegar fermentation.
Biosci Biotechnol Biochem. 2007 Sep;71(9):2124-9.
The physiologically active substances in apple vinegar have not yet been chemically characterized. We studied the biological functions of apple vinegar produced from crushed apples, and found that the constituent neutral medium-sized alpha-glycan (NMalphaG) acts as an antitumor agent against experimental mouse tumors. NMalphaG is a homoglycan composed of glucose having a molecular weight of about 10,000 and a branched structure bearing alpha (1-4,6) linkages.In this study, we clarified the origin of NMalphaG in apple vinegar by examination of its content in alcohol and acetic acid fermentation products sequentially. We found that NMalphaG appeared in acetic acid fermentation, but not in alcohol fermentation. Furthermore we investigated NMalphaG origin using acetic acid fermentation from alcohol fortifiied apple without alcohol fermentation and from raw material with varying amounts of pomace. The results indicate that NMalphaG originated in the apple fruit body and that its production requires both fermentation processes. [Abstract/Link to Full Text]

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Recent Articles in Experimental & Molecular Medicine

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Chae SC, Park BL, Park CS, Ryu HJ, Yang YS, Lee SO, Choi YH, Kim EM, Uh ST, Kim YH, Kim KK, Oh B, Chung HT, Kimm K, Shin HD
Putative association of RUNX1 polymorphisms with IgE levels in a Korean population.
Exp Mol Med. 2006 Oct 31;38(5):583-8.
RUNX1, a member of the runt domain gene family of transcription factors, encodes a heterodimeric transcription factor and regulates the expression of various genes related to hematopoiesis and myeloid differentiation. RUNX1 has been one of the target genes for research into various autoimmune diseases due to its properties as a transcription factor and functional distribution for chromosomal translocation. In an effort to identify additional gene polymorphisms in which variants have been implicated in asthma, we investigated the genetic polymorphisms in RUNX1 to evaluate it as a potential candidate gene for a host genetic study of asthma and IgE production. We identified 19 sequence variants by direct DNA sequencing in 24 individuals of which four common variants were selected for genotyping in our asthma cohort (1,055 asthmatic patients, 384 normal controls). Using logistic regression analysis for association with the risk of asthma, while controlling for age, gender, and smoking status as covariates, no significant associations with the risk of asthma were detected. However, two polymorphisms in the promoter region (-2084G>C and -1282G>A) showed a marginal association with total IgE levels (0.03 and 0.03 in recessive models, respectively). Our findings suggest that polymorphisms in RUNX1 might be one of the genetic factors for the regulation of IgE production. [Abstract/Link to Full Text]

Choi YS, Kim YK, Shim JH, Kim EM, Kang HS, Yoon do Y, Muneta Y, Myung PK
Immunosuppression of xenograft rejection in porcine kidney PK15 cells by porcine IL-18.
Exp Mol Med. 2006 Oct 31;38(5):574-82.
Xenotransplantation, the transplantation of cells, tissues or organs between individuals of different species, would resolve the current shortage of organs, but rejection remains the major hurdle to successful xenotransplantation. In the present study, we analyzed mixed lymphocyte reactions (MLRs) and used 51Cr release assays in order to identify the proliferation and expansion of mouse CD8+ cytotoxic T lymphocyte cells against PK15, PK15/pIL-18 or PK15/mIL-18 cells. In addition, we identified T cell populations in mouse splenocytes and lymph node cells using two-color flow cytometry. It was found that the CD8+ T cells of xenograft recipients proliferated extensively and that the survival rates of populations of PK15/mIL-18 or PK15/pIL-18 cells were higher than untransfected controls. Moreover, CD3+ T cells were increased in mice injected with PK15 cells or PK15/pIL-18 cells but PK15/pIL-18 cell numbers were lower in lymph nodes than untransfected controls. CD8+ T cells numbers were reduced in the lymph nodes of PK15/pIL-18 injected mice. These results suggest that porcine IL-18 regulates anti-pig cellular rejection in C57BL/6 mice. [Abstract/Link to Full Text]

Lee JH, Gao CF, Lee CC, Kim MD, Vande Woude GF
An alternatively spliced form of Met receptor is tumorigenic.
Exp Mol Med. 2006 Oct 31;38(5):565-73.
The Met tyrosine kinase receptor is a widely expressed molecule, which mediates pleiotropic cellular responses following activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF). Previously, one of the authors identified an alternatively spliced form of Met (Met-SM) that lacked a single exon of a 47-amino-acid segment in the juxtamembrane domain. Here we report that Met-SM is a potent transforming gene in NIH3T3 mouse fibroblast cells. Met-SM-transfected NIH3T3 cells show stronger foci-forming activity than wild type- Met-transfected ones. In addition, Met-SM-transfected NIH3T3 cells form colonies in soft agar and are tumorigenic in athymic nu/nu mice. Furthermore, HGF/SF significantly increases the focus-forming activity of Met-SM comparing to wild type Met. The amount of protein and of tyrosine kinase activity of Met-SM accumulates to a high level following HGF/SF treatment. The accumulation of Met-SM correlated well with its delayed ubiquitination and increased stability. These results are consistent with the important role of the juxtamembrane domain in protein stability of Met receptor and suggest that the alternatively-spliced form may contribute to the development and progression of human cancer. [Abstract/Link to Full Text]

Nenoi M, Daino K, Ichimura S, Takahash S, Akuta T
Low-dose radiation response of the p21WAF1/CIP1 gene promoter transduced by adeno-associated virus vector.
Exp Mol Med. 2006 Oct 31;38(5):553-64.
In cancer gene therapy, restriction of antitumor transgene expression in a radiation field by use of ionizing radiation-inducible promoters is one of the promising approaches for tumor-specific gene delivery. Although tumor suppressor protein p53 is induced by low doses (< 1 Gy) of radiation, there have been only a few reports indicating potential utilization of a p53-target gene promoter, such as that of the p21 gene. This is mainly because the transiently transfected promoter of p53-target genes is not much sensitive to radiation. We examined the response of the p21 gene promoter to low-dose radiation when transduced into a human breast cancer cell line MCF-7 by use of recombinant adeno-associated virus (rAAV) vectors. It was shown that the p21 gene promoter transduced by rAAV vectors was more highly radiation-responsive than that transiently transfected by electroporation. A significant induction of the p21 gene promoter by radiation of low doses down to 0.2 Gy was observed. When cells were transduced with the p21 gene promoter-driven HSVtk gene by rAAV vector, they were significantly sensitized to repetitive treatment with low dose radiation (1 Gy) in the presence of the prodrug ganciclovir. It was therefore considered that the p21 gene promoter in combination with a rAAV vector is potentially usable for the development of a low-dose radiation-inducible vector for cancer gene therapy. [Abstract/Link to Full Text]

Byun MS, Choi J, Jue DM
Cysteine-179 of IkappaB kinase beta plays a critical role in enzyme activation by promoting phosphorylation of activation loop serines.
Exp Mol Med. 2006 Oct 31;38(5):546-52.
IkappaB kinase beta (IKKbeta) subunit of IKK complex is essential for the activation of NF-kappaB in response to various proinflammatory signals. Cys-179 in the activation loop of IKKbeta is known to be the target site for IKK inhibitors such as cyclopentenone prostaglandins, arsenite, and antirheumatic gold compounds. Here we show that a mutant IKKbeta in which Cys-179 is substituted with alanine had decreased activity when it was expressed in HEK-293 cells, and TNF stimulation did not restore the activity. Phosphorylation of activation loop serines (Ser-177 and Ser-181) which is required for IKKbeta activation was reduced in the IKKbeta (C179A) mutant. The activity of IKKbeta (C179A) was partially recovered when its phosphorylation was enforced by coexpression with mitogen-activated protein kinase kinase kinases (MAPKKK) such as NF-kappaB inducing kinase (NIK) and MAPK/extracellular signal-regulated kinase kinase kinase 1(MEKK1) or when the serine residues were replaced with phospho-mimetic glutamate. The IKKbeta (C179A) mutant was normal in dimer formation, while its activity abnormally responded to the change in the concentration of substrate ATP in reaction mixture. Our results suggest that Cys-179 of IKKbeta plays a critical role in enzyme activation by promoting phosphorylation of activation-loop serines and interaction with ATP. [Abstract/Link to Full Text]

Kim SY, Kim SJ, Kim BJ, Rah SY, Chung SM, Im MJ, Kim UH
Doxorubicin-induced reactive oxygen species generation and intracellular Ca2+ increase are reciprocally modulated in rat cardiomyocytes.
Exp Mol Med. 2006 Oct 31;38(5):535-45.
Doxorubicin (DOX) is one of the most potent anticancer drugs and induces acute cardiac arrhythmias and chronic cumulative cardiomyopathy. Though DOX-induced cardiotoxicity is known to be caused mainly by ROS generation, a disturbance of Ca2+ homeostasis is also implicated one of the cardiotoxic mechanisms. In this study, a molecular basis of DOX-induced modulation of intracellular Ca2+ concentration ([Ca2+]i) was investigated. Treatment of adult rat cardiomyocytes with DOX increased [Ca2+]i irrespectively of extracellular Ca2+, indicating DOX-mediated Ca2+ release from intracellular Ca2+ stores. The DOX-induced Ca2+ increase was slowly processed and sustained. The Ca2+ increase was inhibited by pretreatment with a sarcoplasmic reticulum (SR) Ca2+ channel blocker, ryanodine or dantrolene, and an antioxidant, alpha-lipoic acid or alpha-tocopherol. DOX-induced ROS generation was observed immediately after DOX treatment and increased in a time-dependent manner. The ROS production was significantly reduced by the pretreatment of the SR Ca2+ channel blockers and the antioxidants. Moreover, DOX-mediated activation of caspase-3 was significantly inhibited by the Ca2+ channel blockers and a-lipoic acid but not a-tocopherol. In addition, cotreatment of ryanodine with alpha-lipoic acid resulted in further inhibition of the casapse-3 activity. These results demonstrate that DOX-mediated ROS opens ryanodine receptor, resulting in an increase in [Ca2+]i and that the increased [Ca2+]i induces ROS production. These observations also suggest that DOX/ROS-induced increase of [Ca2+]i plays a critical role in damage of cardiomyocytes. [Abstract/Link to Full Text]

Won SM, Park YH, Kim HJ, Park KM, Lee WJ
Catechins inhibit angiotensin II-induced vascular smooth muscle cell proliferation via mitogen-activated protein kinase pathway.
Exp Mol Med. 2006 Oct 31;38(5):525-34.
Catechins, components of green tea, reduce the incidence of cardiovascular diseases such as atherosclerosis. Angiotensin II (Ang II) is highly implicated in the proliferation of vascular smooth muscle cells (VSMC), resulting in atherosclerosis. The acting mechanisms of the catechins remain to be defined in the proliferation of VSMC induced by Ang II. Here we report that catechin, epicatechin (EC), epicatechingallate (ECG) or epigallocatechingallate (EGCG) significantly inhibits the Ang II-induced [3H]thymidine incorporation into the primary cultured rat aortic VSMC. Ang II increases the phosphorylation of the extracellular signal-regulated protein kinase 1/2 (ERK 1/2), c-jun-N-terminal kinase 1/2 (JNK 1/2), or p38 mitogen-activated protein kinases (MAPKs) and mRNA expression of c-jun and c-fos. The EGCG pretreatment inhibits the Ang II-induced phosphorylation of ERK 1/2, JNK 1/2, or p38 MAPK, and the expression of c-jun or c-fos mRNA. U0126, a MEK inhibitor, SP600125, a JNK inhibitor, or SB203580, a p38 inhibitor, attenuates the Ang II-induced [3H]thymidine incorporation into the VSMC. In conclusion, catechins inhibit the Ang II-stimulated VSMC proliferation via the inhibition of the Ang II-stimulated activation of MAPK and activator protein-1 signaling pathways. The antiproliferative effect of catechins may be associated with the reduced risk of cardiovascular diseases by the intake of green tea. Catechins may be useful in the development of prevention and therapeutics of vascular diseases. [Abstract/Link to Full Text]

Kim DJ, Park BL, Koh JM, Kim GS, Kim LH, Cheong HS, Shin HD, Hong JM, Kim TH, Shin HI, Park EK, Kim SY
Methionine synthase reductase polymorphisms are associated with serum osteocalcin levels in postmenopausal women.
Exp Mol Med. 2006 Oct 31;38(5):519-24.
Homocysteine (Hcy) is thought to play an important role in the development of osteoporosis and fracture. Methionine synthase reductase (MTRR) is an enzyme involved in the conversion of Hcy to methionine. We hypothesized that certain genetic polymorphisms of MTRR leading to reduced enzyme activity may cause hyperhomocysteinemia and affect bone metabolism. We therefore examined the associations of the A66G and C524T polymorphisms of the MTRR gene with bone mineral density (BMD) and serum osteocalcin levels in postmenopausal women. Although we did not detect any significant associations between MTRR polymorphisms and BMD or serum osteocalcin levels, we found that the 66G/524C haplotype, which has reduced enzyme activity, was significantly associated with serum osteocalcin levels in a gene-dose dependent manner (P = 0.002). That is, the highest osteocalcin levels (34.5 +/- 16.8 ng/ml) were observed in subjects bearing two copies, intermediate osteocalcin levels (32.6 +/- 14.4 ng/ml) were observed in subjects bearing one copy, and the lowest levels of osteocalcin (28.8 +/- 10.9 ng/ml) were observed in subjects bearing no copies. These results suggest that the 66G/524C haplotype of the MTRR gene affect bone turn over rate. [Abstract/Link to Full Text]

Zheng Y, Im CN, Seo JS
Inhibitory effect of Hsp70 on angiotensin II-induced vascular smooth muscle cell hypertrophy.
Exp Mol Med. 2006 Oct 31;38(5):509-18.
Angiotensin II (Ang II), which is an important mediator of both vascular responsiveness and growth, has been shown to induce vascular smooth muscle cell (VSMC) hypertrophy via the activation of a complex series of intracellular signaling events. Heat shock protein 70 (Hsp70) has recently been shown to protect against Ang II-induced hypertension. In this study, we tested the hypothesis that Hsp70 can protect VSMC from Ang II-induced hypertrophy. We treated VSMCs with Ang II to induce hypertrophy and to activate MAPK signaling pathway. We observed that the augmentation of Hsp70 expression inhibited Ang II-stimulated VSMC hypertrophy. This inhibitory effect of Hsp70 appears to be partly due to extracellular signal-regulated kinase (ERK1/2) inactivation, which in turn, may possibly result from the accumulation of MAP kinase phosphatase-1 (MKP-1). [Abstract/Link to Full Text]

Yi EY, Park SY, Song HS, Son MJ, Yi KY, Yoo SE, Kim YJ
KR-31831, a new synthetic anti-ischemic agent, inhibits in vivo and in vitro angiogenesis.
Exp Mol Med. 2006 Oct 31;38(5):502-8.
Angiogenesis is considered to be an integral process to the growth and spread of solid tumors. Anti-angiogenesis therapy recently has been found to be one of the most promising anti-cancer therapeutic strategies. In this study, we provide several lines of evidences showing that KR-31831, a new benzopyran derivative, has anti-angiogenic activities. KR-31831 inhibited the proliferation, migration, invasion and tube formation of bovine aortic endothelial cells (BAECs), and suppressed the release of matrix metalloproteinase-2 (MMP-2) of BAECs. KR-31831 also inhibited in vivo angiogenesis in mouse Matrigel plug assay. Furthermore, the mRNA expressions of basic fibroblast growth factor (bFGF), fibroblast growth factor receptor-2 (FGFR-2), and vascular endothelial growth factor receptor-2 (VEGFR-2) were decreased by KR-31831. Taken together, these results suggest that KR-31831 acts as a novel angiogenesis inhibitor and might be useful for treating hypervascularized cancers. [Abstract/Link to Full Text]

Kim J, Kim HJ, Choi WS, Nam SH, Cho HR, Kwon B
Maintenance of CD8+ T-cell anergy by CD4+CD25+ regulatory T cells in chronic graft-versus-host disease.
Exp Mol Med. 2006 Oct 31;38(5):494-501.
In a murine model of systemic lupus erythematosus (SLE)-like chronic graft-versus-host disease (cGVHD), donor CD8+ T cells rapidly fall into anergy to host cells, while donor CD4+ T cells hyperactivate B cells and break B-cell tolerance to self-Ags in the recipient mouse. The functional recovery of donor CD8+ T cells can result in the conversion of cGVHD to acute GVHD (aGVHD), indicating that donor CD8+ T-cell anergy is a restriction factor in the development of cGVHD. In this report, we present evidence that donor CD4+CD25+ regulatory T cells (Treg cells) are critical in maintaining the donor CD8+ T-cell anergy and thus suppressing the development of aGVHD in mice that are naturally prone to cGVHD. Our results provide a novel insight into the role of Treg cells in determining cGVHD versus aGVHD. [Abstract/Link to Full Text]

Jung SM, Lee WK, Kwak JO, Jung SY, Park J, Kim WY, Kim J, Cha SH
Identification of a novel murine organic anion transporter like protein 1 (OATLP1) expressed in the kidney.
Exp Mol Med. 2006 Oct 31;38(5):485-93.
The organic anion transporters (OATs) are expressed in various tissues, primarily in the kidney and liver, but they are also expressed in the placenta, small intestine, and the choroid plexus, which are all epithelial tissues that transport xenobiotics. Six isoforms of OATs are currently known. Considering the variety of organic anionic compounds, other OATs isoforms can be assumed. In this connection, we have searched for a new isoform in the expressed sequence tag (EST) database. We found the new candidate clone AK052752 in the mouse kidney cDNA library and we named it mouse organic anion transporter like protein 1 (mOATLP1). The mOATLP1 cDNA consisted of 2221 base pairs that encoded a 552 amino acid residue protein with 12 putative transmembrane domains. The deduced amino acid sequence of mOATLP1 showed 37 to 63% identity to other members of the OAT family. According to the tissue distribution based on Northern blot analysis, 2.7 kb and 2.9 kb mOATLP1 transcripts (approximate sizes) were observed in the kidney and liver. An 85-kDa band (approximate) was detected using Western blot analysis of mouse kidney performed with a synthesized oligopeptide-induced mOATLP1 antibody. Immunohistochemical results showed mOATLP1 was stained in the blood vessels, glomeruli (the parietal epithelial cells and podocytes), distal convoluted tubules, connecting tubules, and inner medullary collecting tubules. mOATLP1 appears to be a novel candidate for an organic anion transporter isoform identified in the kidney. [Abstract/Link to Full Text]

Kim JY, Son YO, Park SW, Bae JH, Chung JS, Kim HH, Chung BS, Kim SH, Kang CD
Increase of NKG2D ligands and sensitivity to NK cell-mediated cytotoxicity of tumor cells by heat shock and ionizing radiation.
Exp Mol Med. 2006 Oct 31;38(5):474-84.
In this study, we have investigated if current cancer therapeutic modalities including hyperthermia and ionizing radiation can increase the expression of NKG2D ligands in human cancer cell lines. The expressions of NKG2D ligands were induced by both heat shock and ionizing radiation in various cell lines including KM12, NCI-H23, HeLa and A375 cells with peaks at 2 h and 9 h after treatment, respectively, although inducibility of each NKG2D ligand was various depending on cell lines. During the induction of NKG2D ligands, heat shock protein 70 was induced by heat shock but not by ionizing radiation. These results were followed by increased susceptibilities to NK cell-mediated cytolysis after treatment with heat shock and ionizing radiation. These results suggest that heat shock and ionizing radiation induce NKG2D ligands and consequently might lead to increased NK cell-mediated cytotoxicity in various cancer cells. [Abstract/Link to Full Text]

Choi BR, Kwon JH, Gong SJ, Kwon MS, Cho JH, Kim JH, Oh S, Roh HJ, Kim DE
Expression of glucocorticoid receptor mRNAs in glucocorticoid-resistant nasal polyps.
Exp Mol Med. 2006 Oct 31;38(5):466-73.
Glucocorticoids (GCs) are the most effective group of medications available to treat inflammation. Although most patients with inflammation respond to GC, a small group of patients exhibit persistent GC-resistance with prolonged inflammation. Previously, it was proposed that the GC-resistance is caused by low amount of human GC receptor (hGRalpha) and/or excessive presence of a GC receptor isoform, hGRbeta that was generated from alternative splicing of the hGR message. We have tested this hypothesis by investigating correlation between the expression pattern of hGR mRNAs in patients with inflammatory nasal polyps and the effectiveness of GC treatment.? We have performed reverse transcription PCR analysis of mRNAs coding each hGRalpha and hGRbeta in nasal tissues.? hGRalpha mRNA was more expressed in patients with nasal polyps than in normal subjects. However, the elevated hGRalphamRNA expression was decreased after GC treatment. Compared with hGRalpha mRNA expression, level of hGRbeta mRNA expression was very low in all groups. In patients, hGRbetamRNA was expressed at a similar level regardless of GC efficacy, indicating that there is no correlation between the GC sensitivity and the expression level of hGRbeta mRNA. Thus, persistent GC-resistance is not associated with low expression of hGRa or over- expression of hGRbeta. [Abstract/Link to Full Text]

Lim BH, Cho BI, Kim YN, Kim JW, Park ST, Lee CW
Overexpression of nicotinamide N-methyltransferase in gastric cancer tissues and its potential post-translational modification.
Exp Mol Med. 2006 Oct 31;38(5):455-65.
Gastric cancer is one of the most common cancers worldwide. The purpose of this study was to find out potential markers for gastric cancer. Tumor and normal tissues from 152 gastric cancer cases were analyzed by two-dimensional gel electrophoresis (2-DE). The images of silver stained gels were analyzed and statistical analysis of spot intensities revealed that spot 4262 showed higher expression (5.7-fold increase) in cancer tissues than in normal tissues (P < 0.001). It was identified by peptide mass fingerprinting as nicotinamide N-methyltransferase (NNMT). A monoclonal antibody with a detection limit down to 10 ng was produced against NNMT in mouse. Using the prepared monoclonal antibody, western blot analysis of NNMT was performed for gastric tissues from 15 gastric cancer patients and two gastric ulcer patients. The results corroborated those of 2-DE experiments. A single spot was detected in gastric ulcer tissues while four to five spots were detected in gastric cancer tissues. In cancer tissues, two additional spots of acidic and basic form were mainly detected on 2-DE gels. This suggests that NNMT receives a post-translational modification in cancer- specific manner. [Abstract/Link to Full Text]

Boo YC
Shear stress stimulates phosphorylation of protein kinase A substrate proteins including endothelial nitric oxide synthase in endothelial cells.
Exp Mol Med. 2006 Aug 31;38(4):453. [Abstract/Link to Full Text]

Boo YC
Shear stress stimulates phosphorylation of protein kinase A substrate proteins including endothelial nitric oxide synthase in endothelial cells.
Exp Mol Med. 2006 Feb 28;38(1):63-71.
Fluid shear stress plays a critical role in vascular health and disease. While protein kinase A (PKA) has been implicated in shear-stimulated signaling events in endothelial cells, it remains unclear whether and how PKA is stimulated in response to shear stress. This issue was addressed in the present study by monitoring the phosphorylation of endogenous substrates of PKA. Shear stress stimulated the phosphorylation of cAMP responsive element binding protein (CREB) in a PKA-dependent manner. Western blot analysis using the antibody reactive against the consensus motif of PKA substrates detected two proteins, P135 and P50, whose phosphorylation was increased by shear stress. The phosphorylation of P135 was blocked by a PKA inhibitor, H89, but not by a phosphoinositide 3-kinase inhibitor, wortmannin. Expression of a constitutively active PKA subunit stimulated P135 phosphorylation, supporting the potential of P135 as a PKA substrate. P135 was identified as endothelial nitric oxide synthase (eNOS) by immunoprecipitation study. PKA appeared to mediate shear stress-stimulated eNOS activation. Shear stress stimulated intracellular translocation of PKA activity from 'soluble' to 'particulate' fractions without involving cellular cAMP increase. Taken together, this study suggests that shear stress stimulates PKA-dependent phosphorylation of target proteins including eNOS, probably by enhancing intracellular site-specific interactions between protein kinase and substrates. [Abstract/Link to Full Text]

Nam KH, Choi JH, Seo YJ, Lee YM, Won YS, Lee MR, Lee MN, Park JG, Kim YM, Kim HC, Lee CH, Lee HK, Oh SR, Oh GT
Inhibitory effects of tilianin on the expression of inducible nitric oxide synthase in low density lipoprotein receptor deficiency mice.
Exp Mol Med. 2006 Aug 31;38(4):445-52.
We investigated the effect of tilianin upon inducible nitric oxide synthesis in the plasma of low-density lipoprotein receptor knock-out (Ldlr-/-) mice fed with high cholesterol diet and in primary peritoneal macrophages of Ldlr-/- mice. High cholesterol diet induced nitric oxide production in the plasma of Ldlr-/- mice. Tilianin reduced the level of nitric oxide (NO) in plasma from Ldlr-/- mice induced by the high cholesterol diet. Tilianin also inhibited the NO production from the primary culture of peritoneal macrophages treated with lipopolysaccharide. The inhibition of NO production was caused by the suppression of inducible nitric oxide synthase (iNOS) gene expression in peritoneal macrophages isolated from Ldlr-/- mice. Moreover, tilianin inhibited the transcriptional activation of iNOS promoter that has NF-kappaB binding element. Thus, these results provide the first evidence that tilianin inhibit iNOS expression and production of NO and may act as a potential anti-inflammatory agent. [Abstract/Link to Full Text]

Yang SH, Chien CM, Lu MC, Lin YH, Hu XW, Lin SR
Up-regulation of Bax and endonuclease G, and down-modulation of Bcl-XL involved in cardiotoxin III-induced apoptosis in K562 cells.
Exp Mol Med. 2006 Aug 31;38(4):435-44.
Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. CTX III-induced K562 cell apoptosis was confirmed by DNA fragmentation (DNA ladder, sub-G1 formation) and phosphatidylserine (PS) externalization with an IC(50) value of 1.7 microg/ml at 48 h. A mechanistic analysis demonstrated that CTX III-induced apoptotic cell death was accompanied by up-regulation of both Bax and endonuclease G (Endo G), and downregulation of Bcl-X(L). CTX III had no effect on the levels of Bcl-2, Bid, XIAP survivin, and AIF proteins. CTX III treatment caused loss of the mitochondrial membrane potential (DeltaPsim), release of mitochondrial cytochrome c to the cytosol, and activation of both caspase-9 and -3. CTX III-induced apoptosis was significantly blocked by the broad-spectrum caspase inhibitor Z-VAD-FMK. However, CTX III did not generate reactive oxygen species (ROS) and antioxidants, including N-acetylcysteine and catalase, did not block CTX III-induced apoptosis in K562 cells. Modulation of Bax, Bcl-XL, and the Endo G proteins, release of mitochondrial cytochome c, and activation of caspase-3 and -9 all are involved in the CTX III-triggered apoptotic process in human leukemia K562 cells. [Abstract/Link to Full Text]

Gong X, Wang M, Tashiro S, Onodera S, Ikejima T
Involvement of JNK-initiated p53 accumulation and phosphorylation of p53 in pseudolaric acid B induced cell death.
Exp Mol Med. 2006 Aug 31;38(4):428-34.
A terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was used to determine that apoptosis causes HeLa cell death induced by pseudolaric acid B. The c-Jun N-terminal kinase (JNK) inhibitor SP600125 decreased p53 protein expression during exposure to pseudolaric acid B. SP600125 decreased the phosphorylation of p53 during pseudolaric acid B exposure, indicating that JNK mediates phosphorylation of p53 during the response to pseudolaric acid B. SP600125 reversed pseudolaric acid B-induced down-regulation of phosphorylated extracellular signal-regulated protein kinase (ERK), and protein kinase C (PKC) was activated by pseudolaric acid B, whereas staurosporine, calphostin C, and H7 partly blocked this effect. These results indicate that p53 is partially regulated by JNK in pseudolaric acid B-induced HeLa cell death and that PKC participates in pseudolaric acid B-induced HeLa cell death. [Abstract/Link to Full Text]

Kim DH, Cho IH, Kim HS, Jung JE, Kim JE, Lee KH, Park T, Yang YM, Seong SY, Ye SK, Chung MH
Anti-inflammatory effects of 8-hydroxydeoxyguanosine in LPS-induced microglia activation: suppression of STAT3-mediated intercellular adhesion molecule-1 expression.
Exp Mol Med. 2006 Aug 31;38(4):417-27.
To elucidate the roles of 8-hydroxydeoxyguanosine (oh(8)dG), the nucleoside of 8-hydroxyguanine (oh(8)Gua), we examined the effects of oh(8)dG upon LPS-induced intercellular adhesion molecule-1 (ICAM-1) expression and the underlying mechanisms in brain microglial cells. We found that oh(8)dG reduces LPS-induced reactive oxygen species (ROS) production, STAT3 activation, and ICAM-1 expression. oh(8)dG also suppresses pro-inflammatory cytokines, such as TNF-alpha, IL-6 and IFN-gamma. Overexpression of dominant negative STAT3 completely diminshed STAT3-mediated ICAM-1 transcriptional activity. Chromatin immunoprecipitation studies revealed that oh(8)dG inhibited recruitment of STAT3 to the ICAM-1 promoter, followed by a decrease in ICAM-1 expression. Using mice lacking a functional Toll-like receptor 4 (TLR4), we demonstrated that, while TLR4+/+ microglia were activated by LPS, TLR4-/- microglia exhibited inactivated STAT3 in response to LPS. Evidently, LPS modulates STAT3-dependent ICAM-1 induction through TLR4-mdiated cellular responses. Oh(8)dG apparently plays a role in anti-inflammatory actions via suppression of ICAM-1 gene expression by blockade of the TLR4-STAT3 signal cascade in inflammation-enhanced brain microglia. Therefore, oh(8)dG in the cytosol probably functions as an anti-inflammatory molecule and should be considered as a candidate for development of anti-inflammatory agents. [Abstract/Link to Full Text]

Lee KH, Kim SE, Lee YS
SP600125, a selective JNK inhibitor, aggravates hepatic ischemia-reperfusion injury.
Exp Mol Med. 2006 Aug 31;38(4):408-16.
c-Jun N-terminal kinase (JNK) is activated during hepatic reperfusion, and JNK inhibitors are known to protect other major organs from ischemia-reperfusion (I/R) injury. We attempted to determine the effect of SP600125, a JNK inhibitor, on hepatic I/R injury using a partial ischemia model in mice. Compared to a vehicle-treated group, the SP600125- treated group showed a greater increase in serum ALT levels 24 h after reperfusion with more severe parenchymal destruction and leukocyte infiltration. Similarly, tissue myeloperoxidase and malondialdehyde levels were higher in the SP600125-treated group, and chemokine expression was also higher in the SP600125-treated group. These data, which are contradictory to previous results, indicate that JNK inhibition by SP600125 may be harmful in hepatic I/R injury. Therefore, care must be taken when investigating the therapeutic use of JNK inhibitors in hepatic I/R injury, especially in the context of the effects of JNK inhibition on inflammatory infiltration. [Abstract/Link to Full Text]

Kim DJ, Chung JH, Ryu EK, Rhim JH, Ryu YS, Park SH, Kim KT, Kang HS, Chung HK, Park SC
Metabolic loading of guanosine induces chondrocyte apoptosis via the Fas pathway.
Exp Mol Med. 2006 Aug 31;38(4):401-7.
Although the apoptosis of chondrocytes plays an important role in endochondral ossification, its mechanism has not been elucidated. In this study, we show that guanosine induces chondrocyte apoptosis based on the results of acridine orange/ethidium bromide staining, caspase-3 activation, and sub-G1 fraction analysis. The potent inhibitory effect of dipyridamole, a nucleoside transporter blocker, indicates that extracellular guanosine must enter the chondrocytes to induce apoptosis. We found that guanosine promotes Fas-Fas ligand interaction which, in turn, leads to chondrocyte apoptosis. These findings indicate a novel mechanism for endochondral ossification via metabolic regulation. [Abstract/Link to Full Text]

Jeong GS, Oh GS, Pae HO, Jeong SO, Kim YC, Shin MK, Seo BY, Han SY, Lee HS, Jeong JG, Koh JS, Chung HT
Comparative effects of curcuminoids on endothelial heme oxygenase-1 expression: ortho-methoxy groups are essential to enhance heme oxygenase activity and protection.
Exp Mol Med. 2006 Aug 31;38(4):393-400.
Recently, it has been reported that curcumin, which is known as a potent antioxidant, acts as a non- stressful and non-cytotoxic inducer of the cytoprotective heme oxygenase (HO)-1. In this study, naturally occurring curcuminoids, such as pure curcumin, demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), were compared for their potential ability to modulate HO-1 expression and cytoprotective activity in human endothelial cells. All three curcuminoids could induce HO-1 expression and HO activity with differential levels. The rank order of HO activity was curcumin, DMC and BDMC. In comparison with endothelial protection against H2O2-induced cellular injury, cytoprotective capacity was found to be highest with curcumin, followed by DMC and BDMC. Interestingly, cytoprotective effects afforded by curcuminoids were considerably associated with their abilities to enhance HO activity. Considering that the main difference among the three curcuminoids is the number of methoxy groups (none for BDMC, one for DMC, and two for curcumin), the presence of methoxy groups in the ortho position on the aromatic ring was suggested to be essential to enhance HO-1 expression and cytoprotection in human endothelial cells. Our results may be useful in designing more efficacious HO-1 inducers which could be considered as promising pharmacological agents in the development of therapeutic approaches for the prevention or treatment of endothelial diseases caused by oxidative damages. [Abstract/Link to Full Text]

Kim JM, Kim SJ, Lee HC, Kim KS
Development of ligand-dependent regulatory system and its application to gene therapy of insulin-dependent diabetes mellitus.
Exp Mol Med. 2006 Aug 31;38(4):385-92.
To develop an inducible expression system, the enhanced artificial nuclear receptors and target reporters were constructed. Artificial nuclear receptors were generated by fusing three domains, consisting of DNA-binding domain (DBD) of GAL4, ligand binding domain (LBD) of progesterone or estrogen receptor, and activation domain (AD) of VP16, sterol regulatory element binding protein (SREBP)-1a, or SREBP-2. The activation domain of SREBP-1a showed most potent transcriptional activity. The maximal level of target reporter gene expression was extremely elevated by the usage of ATP citrate-lyase (ACL) minimal promoter -60/+67 in place of artificial TATA promoter, while the SV40 enhancer severely increased the basal transcription in the absence of ligand. The induction system, developed in the present study, was applied to cell therapy, resulting in successful induction of single-chain insulin analogue (SIA) gene expression to correct the hyperglycemia in diabetic animals. By means of subcutaneous cell therapy, the SIA gene expression rapidly occurred after the local topical application of ligand. These results suggest that our system represents a powerful tool for transcriptional regulation of target gene that can be used for diverse applications, ranging from basic research to gene therapy. [Abstract/Link to Full Text]

Yun DH, Jeon ES, Sung SM, Ryu SH, Kim JH
Lysophosphatidylcholine suppresses apoptosis and induces neurite outgrowth in PC12 cells through activation of phospholipase D2.
Exp Mol Med. 2006 Aug 31;38(4):375-84.
Lysophosphatidylcholine (LPC) is a bioactive lipid generated by phospholipase A2-mediated hydrolysis of phosphatidylcholine. In the present study, we demonstrate that LPC stimulates phospholipase D2 (PLD2) activity in rat pheochromocytoma PC12 cells. Serum deprivation induced cell death of PC12 cells, as demonstrated by decreased viability, DNA fragmentation, and increased sub-G1 fraction of cell cycle. LPC treatment protected PC12 cells partially from the cell death and induced neurite outgrowth of the cells. Overexpression of PLD2 drastically enhanced the LPC-induced inhibition of apoptosis and neuritogenesis. Pretreatment of the cells with 1-butanol, a PLD inhibitor, completely abrogated the LPC-induced inhibition of apoptosis and neurite outgrowth in PC12 cells overexpressing PLD2. These results indicate that LPC possesses the neurotrophic effects, such as anti-apoptosis and neurite outgrowth, through activation of PLD2. [Abstract/Link to Full Text]

Lee KJ, Kim YM, Kim DY, Jeoung D, Han K, Lee ST, Lee YS, Park KH, Park JH, Kim DJ, Hahn JH
Release of heat shock protein 70 (Hsp70) and the effects of extracellular Hsp70 on matric metalloproteinase-9 expression in human monocytic U937 cells.
Exp Mol Med. 2006 Aug 31;38(4):364-74.
Heat shock protein 70 (Hsp70) release and its effects on pro-inflammatory cytokine production have been controversial. In this study, we investigated whether Hsp70 could be released from monocytes and activates matrix metalloproteinase-9 (MMP-9) gene expression. Hsp70 overexpression in human monocytic cell line U937 was found to increase PMA-induced MMP-9 expression and enhance cell motility. Hsp70 cDNA transfectants released Hsp70 protein into culture supernatants, and a part of released Hsp70 subsequently was bound to the surface of U937 cells. Addition of culture medium containing the extracelluar Hsp70 led to an increase not only in proMMP-9 secretion, but also the invasiveness of U937 cells through Matrigel or human umbilical vascular endothelial cells (HUVEC) in vitro. Immunodepletion of Hsp70 abolished its effect on MMP-9 expression. The released Hsp70 activated nuclear factor kappa B (NF-kappaB) and activating protein-1 (AP-1), which led to the activation of MMP-9 transcription. Taken together, these results suggest that extracellular Hsp70 induces the expression of MMP-9 gene through activation of NF-KappaB and AP-1. [Abstract/Link to Full Text]

Kim HJ, Park HJ, Park WS, Bae Y
CD43 cross-linking increases the Fas-induced apoptosis through induction of Fas aggregation in Jurkat T-cells.
Exp Mol Med. 2006 Aug 31;38(4):357-63.
CD43 (sialophorin, leukosialin) is a heavily sialylated surface protein expressed on most leukocytes and platelets including T cells. Although CD43 antigen is known to have multiple and complex structure, exact function of CD43 in each cell type is not completely understood. Here we evaluated the role of CD43 in Fas (CD95)-induced cell death in human T lymphoblastoid cell line, Jurkat. Crosslinking CD43 antigen by K06 mAb increased the Fas-mediated Jurkat cell apoptosis and the augmentation was inhibited by treatment with caspase inhibitors. Further, CD43 signaling of Jurkat cells induced Fas oligomerization on the cell surfaces implying that CD43 ligation have effects on early stage of Fas-induced T cell death. These also suggest that CD43 might play an important role in contraction of the immune response by promotion of Fas-induced apoptosis in human T cells. [Abstract/Link to Full Text]

Kim EY, Hong YB, Go SH, Lee B, Jung SC
Downregulation of neurotrophic factors in the brain of a mouse model of Gaucher disease; implications for neuronal loss in Gaucher disease.
Exp Mol Med. 2006 Aug 31;38(4):348-56.
Gaucher disease is a glycosphingolipid storage disease caused by deficiency of glucocerebrosidase, resulting in the accumulation of glucosylceramide in lysosomes. The neuronopathic forms of this disease are associated with neuronal loss and neurodegeneration. However, the pathophysiological mechanisms leading to prenatal and neonatal death remain uncharacterized. To investigate brain dysfunction in Gaucher disease, we studied the effects of neurotrophic factors during development in a mouse model of Gaucher disease. The expression of brain-derived neurotrophic factor and nerve growth factor was reduced in the cerebral cortex, brainstem, and cerebellum of Gaucher mice, compared with that in wild-type mice. Extracellular signal-regulated kinase (ERK) 1/2 expression was downregulated in neurons from Gaucher mice and correlated with a decreased number of neurons. These results suggest that a reduction in neurotrophic factors could be involved in neuronal loss in Gaucher disease. [Abstract/Link to Full Text]

Kim YS, Joh TH
Microglia, major player in the brain inflammation: their roles in the pathogenesis of Parkinson's disease.
Exp Mol Med. 2006 Aug 31;38(4):333-47.
Inflammation, a self-defensive reaction against various pathogenic stimuli, may become harmful self-damaging process. Increasing evidence has linked chronic inflammation to a number of neurodegenerative disorders including Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis. In the central nervous system, microglia, the resident innate immune cells play major role in the inflammatory process. Although they form the first line of defense for the neural parenchyma, uncontrolled activation of microglia may directly toxic to neurons by releasing various substances such as inflammatory cytokines (IL-1beta, TNF-alpha, IL-6), NO, PGE(2), and superoxide. Moreover, our recent study demonstrated that activated microglia phagocytose not only damaged cell debris but also neighboring intact cells. It further supports their active participation in self-perpetuating neuronal damaging cycles. In the following review, we discuss microglial responses to damaging neurons, known activators released from injured neurons and how microglia cause neuronal degeneration. In the last part, microglial activation and their role in PD are discussed in depth. [Abstract/Link to Full Text]

Lee E, Choi MK, Han IO, Lim SJ
Role of p21CIP1 as a determinant of SC-560 response in human HCT116 colon carcinoma cells.
Exp Mol Med. 2006 Jun 30;38(3):325-31.
SC-560, a structural analogue of celecoxib, induces growth inhibition in a wide range of human cancer cells in a cyclooxygenase (COX)-independent manner. Since SC-560 suppresses the growth of cancer cells mainly by inducing cell cycle arrest, we sought to examine the role of p21CIP1, a cell cycle regulator protein, in the cellular response against SC-560 by using p21(+/+) and p21(-/-) isogenic HCT116 colon carcinoma cells. In HCT116 (p21(+/+)) cells, SC-560 dose-dependently induced growth inhibition and cell cycle arrest at the G1 phase without significant apoptosis induction. SC-560-induced cell cycle arrest was accompanied by upregulation of p21CIP1. However, the extent of SC-560-induced accumulation at the G1 phase was approximately equal in the p21(+/+) and the p21(-/-) cells. Nonetheless, the growth inhibition by SC-560 was increased in p21(-/-) cells than p21(+/+)cells. SC-560-induced reactive oxygen species (ROS) generation did not differ between p21(+/+) and p21(-/-) cells but the subsequent activation of apoptotic caspase cascade was more pronounced in p21(-/-) cells compared with p21(+/+) cells. These results suggest that p21CIP1 blocks the SC-560-induced apoptotic response of HCT116 cells. SC-560 combined with other therapy that can block p21 CIP1 expression or function may contribute to the effective treatment of colon cancer. [Abstract/Link to Full Text]

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Recent Articles in BMC Biochemistry

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Guettouche T, Boellmann F, Lane WS, Voellmy R
Analysis of phosphorylation of human heat shock factor 1 in cells experiencing a stress.
BMC Biochem. 2005;64.
BACKGROUND: Heat shock factor (HSF/HSF1) not only is the transcription factor primarily responsible for the transcriptional response of cells to physical and chemical stress but also coregulates other important signaling pathways. The factor mediates the stress-induced expression of heat shock or stress proteins (HSPs). HSF/HSF1 is inactive in unstressed cells and is activated during stress. Activation is accompanied by hyperphosphorylation of the factor. The regulatory importance of this phosphorylation has remained incompletely understood. Several previous studies on human HSF1 were concerned with phosphorylation on Ser303, Ser307 and Ser363, which phosphorylation appears to be related to factor deactivation subsequent to stress, and one study reported stress-induced phosphorylation of Ser230 contributing to factor activation. However, no previous study attempted to fully describe the phosphorylation status of an HSF/HSF1 in stressed cells and to systematically identify phosphoresidues involved in factor activation. The present study reports such an analysis for human HSF1 in heat-stressed cells. RESULTS: An alanine scan of all Ser, Thr and Tyr residues of human HSF1 was carried out using a validated transactivation assay, and residues phosphorylated in HSF1 were identified by mass spectrometry and sequencing. HSF1 activated by heat treatment was phosphorylated on Ser121, Ser230, Ser292, Ser303, Ser307, Ser314, Ser319, Ser326, Ser344, Ser363, Ser419, and Ser444. Phosphorylation of Ser326 but none of the other Ser residues was found to contribute significantly to activation of the factor by heat stress. Phosphorylation on Ser326 increased rapidly during heat stress as shown by experiments using a pSer326 phosphopeptide antibody. Heat stress-induced DNA binding and nuclear translocation of a S326A substitution mutant was not impaired in HSF1-negative cells, but the mutant stimulated HSP70 expression several times less well than wild type factor. CONCLUSION: Twelve Ser residues but no Thr or Tyr residues were identified that were phosphorylated in heat-activated HSF1. Mutagenesis experiments and functional studies suggested that phosphorylation of HSF1 residue Ser326 plays a critical role in the induction of the factor's transcriptional competence by heat stress. PhosphoSer326 also contributes to activation of HSF1 by chemical stress. To date, no functional role could be ascribed to any of the other newly identified phosphoSer residues. [Abstract/Link to Full Text]

Letarte M, Voulgaraki D, Hatherley D, Foster-Cuevas M, Saunders NJ, Barclay AN
Analysis of leukocyte membrane protein interactions using protein microarrays.
BMC Biochem. 2005;62.
BACKGROUND: Protein microarrays represent an emerging class of proteomic tools to investigate multiple protein-protein interactions in parallel. A sufficient proportion of immobilized proteins must maintain an active conformation and an orientation that allows for the sensitive and specific detection of antibody and ligand binding. In order to establish protein array technology for the characterization of the weak interactions between leukocyte membrane proteins, we selected the human leukocyte membrane protein CD200 (OX2) and its cell surface receptor (hCD200R) as a model system. As antibody-antigen reactions are generally of higher affinity than receptor-ligand binding, we first analyzed the reactivity of monoclonal antibodies (mAb) to normal and mutant forms of immobilized CD200R. RESULTS: Fluorescently labelled mAb DX147, DX136 and OX108 were specifically reactive with immobilized recombinant hCD200R extracellular region, over a range of 0.1-40 microg ml(-1) corresponding to a limit of sensitivity of 0.01-0.05 femtomol per spot. Orientating hCD200R using capture antibodies, showed that DX147 reacts with an epitope spatially distinct from the more closely related DX136 and OX108 epitopes. A panel of soluble recombinant proteins with mutations in hCD200R domain 1 produced by transiently transfected cells, was arrayed directly without purification and screened for binding to the three mAb. Several showed decreased binding to the blocking mAb DX136 and OX108, suggesting close proximity of these epitopes to the CD200 binding site. Binding of hCD200 to directly immobilized rat, mouse, and hCD200R was achieved with multimeric ligands, in the form of biotinylated-hCD200 coupled to FITC-labelled avidin coated beads. CONCLUSION: We have achieved sensitive, specific and reproducible detection of immobilized CD200R with different antibodies and mapped antigenic epitopes for two mAb in the vicinity of the ligand binding site using protein microarrays. We also detected CD200 binding to its receptor, a low affinity interaction, using beads presenting multivalent ligands. Our results demonstrate the quantitative aspects of protein arrays and their potential use in detecting simultaneously multiple protein-protein interactions and in particular the weak interactions found between leukocyte membrane proteins. [Abstract/Link to Full Text]

Tanaka S, Igarashi S, Ferri S, Sode K
Increasing stability of water-soluble PQQ glucose dehydrogenase by increasing hydrophobic interaction at dimeric interface.
BMC Biochem. 2005;61.
BACKGROUND: Water-soluble quinoprotein glucose dehydrogenase (PQQGDH-B) from Acinetobacter calcoaceticus has a great potential for application as a glucose sensor constituent. Because this enzyme shows no activity in its monomeric form, correct quaternary structure is essential for the formation of active enzyme. We have previously reported on the increasing of the stability of PQQGDH-B by preventing the subunit dissociation. Previous studies were based on decreasing the entropy of quaternary structure dissociation but not on increasing the interaction between the two subunits. We therefore attempted to introduce a hydrophobic interaction in the dimeric interface to increase the stability of PQQGDH-B. RESULTS: Amino acid residues Asn340 and Tyr418 face each other at the dimer interface of PQQGDH-B, however no interaction exists between their side chains. We simultaneously substituted Asn340 to Phe and Tyr418 to Phe or Ile, to create the two mutants Asn340Phe/Tyr418Phe and Asn340Phe/Tyr418Ile. Furthermore, residues Leu280, Val282 and Val342 form a hydrophobic region that faces, on the other subunit, residues Thr416 and Thr417, again without any specific interaction. We simultaneously substituted Thr416 and Thr417 to Val, to create the mutant Thr416Val/Thr417Val. The temperatures resulting in lose of half of the initial activity of the constructed mutants were increased by 3-4 degrees C higher over wild type. All mutants showed 2-fold higher thermal stability at 55 degrees C than the wild-type enzyme, without decreasing their catalytic activities. From the 3D models of all the mutant enzymes, the predicted binding energies were found to be significantly greater that in the wild-type enzyme, consistent with the increases in thermal stabilities. CONCLUSIONS: We have achieved via site-directed mutagenesis the improvement of the thermal stability of PQQGDH-B by increasing the dimer interface interaction. Through rational design based on the quaternary structure of the enzyme, we selected residues located at the dimer interface that do not contribute to the intersubunit interaction. By substituting these residues to hydrophobic ones, the thermal stability of PQQGDH-B was increased without decreasing its catalytic activity. [Abstract/Link to Full Text]

Baron RA, Casey PJ
Analysis of the kinetic mechanism of recombinant human isoprenylcysteine carboxylmethyltransferase (Icmt).
BMC Biochem. 2004 Dec 29;519.
BACKGROUND: Isoprenylcysteine carboxyl methyltransferase (Icmt) is the third of three enzymes that posttranslationally modify proteins that contain C-terminal CaaX motifs. The processing of CaaX proteins through this so-called prenylation pathway via a route initiated by addition of an isoprenoid lipid is required for both membrane targeting and function of the proteins. The involvement of many CaaX proteins such as Ras GTPases in oncogenesis and other aberrant proliferative disorders has led to the targeting of the enzymes involved in their processing for therapeutic development, necessitating a detailed understanding of the mechanisms of the enzymes. RESULTS: In this study, we have investigated the kinetic mechanism of recombinant human Icmt. In the reaction catalyzed by Icmt, S-adenosyl-L-methionine (AdoMet) provides the methyl group that is transferred to the second substrate, the C-terminal isoprenylated cysteine residue of a CaaX protein, thereby generating a C-terminal prenylcysteine methyl ester on the protein. To facilitate the kinetic analysis of Icmt, we synthesized a new small molecule substrate of the enzyme, biotin-S-farnesyl-L-cysteine (BFC). Initial kinetic analysis of Icmt suggested a sequential mechanism for the enzyme that was further analyzed using a dead end competitive inhibitor, S-farnesylthioacetic acid (FTA). Inhibition by FTA was competitive with respect to BFC and uncompetitive with respect to AdoMet, indicating an ordered mechanism with SAM binding first. To investigate the order of product dissociation, product inhibition studies were undertaken with S-adenosyl-L-homocysteine (AdoHcy) and the N-acetyl-S-farnesyl-L-cysteine methylester (AFCME). This analysis indicated that AdoHcy is a competitive inhibitor with respect to AdoMet, while AFCME shows a noncompetitive inhibition with respect to BFC and a mixed-type inhibition with respect to AdoMet. These studies established that AdoHcy is the final product released, and that BFC and AFCME bind to different forms of the enzyme. CONCLUSIONS: These studies establish that catalysis by human Icmt proceeds through an ordered sequential mechanism and provide a kinetic framework for analysis of specific inhibitors of this key enzyme. [Abstract/Link to Full Text]

Wakata Y, Tokumoto M, Horiguchi R, Ishikawa K, Nagahama Y, Tokumoto T
Identification of alpha-type subunits of the Xenopus 20S proteasome and analysis of their changes during the meiotic cell cycle.
BMC Biochem. 2004 Dec 17;5(1):18.
BACKGROUND: The 26S proteasome is the proteolytic machinery of the ubiquitin-dependent proteolytic system responsible for most of the regulated intracellular protein degradation in eukaryotic cells. Previously, we demonstrated meiotic cell cycle dependent phosphorylation of alpha4 subunit of the 26S proteasome. In this study, we analyzed the changes in the spotting pattern separated by 2-D gel electrophoresis of alpha subunits during Xenopus oocyte maturation. RESULTS: We identified cDNA for three alpha-type subunits (alpha1, alpha5 and alpha6) of Xenopus, then prepared antibodies specific for five subunits (alpha1, alpha3, alpha5, alpha6, and alpha7). With these antibodies and previously described monoclonal antibodies for subunits alpha2 and alpha4, modifications to all alpha-type subunits of the 26S proteasome during Xenopus meiotic maturation were examined by 2D-PAGE. More than one spot for all subunits except alpha7 was identified. Immunoblot analysis of 26S proteasomes purified from immature and mature oocytes showed a difference in the blots of alpha2 and alpha4, with an additional spot detected in the 26S proteasome from immature oocytes (in G2-phase). CONCLUSIONS: Six of alpha-type subunits of the Xenopus 26S proteasome are modified in Xenopus immature oocytes and two subunits (alpha2 and alpha4) are modified meiotic cell cycle-dependently. [Abstract/Link to Full Text]

Bollivar DW, Clauson C, Lighthall R, Forbes S, Kokona B, Fairman R, Kundrat L, Jaffe EK
Rhodobacter capsulatus porphobilinogen synthase, a high activity metal ion independent hexamer.
BMC Biochem. 2004 Nov 22;517.
BACKGROUND: The enzyme porphobilinogen synthase (PBGS), which is central to the biosynthesis of heme, chlorophyll and cobalamins, has long been known to use a variety of metal ions and has recently been shown able to exist in two very different quaternary forms that are related to metal ion usage. This paper reports new information on the metal ion independence and quaternary structure of PBGS from the photosynthetic bacterium Rhodobacter capsulatus. RESULTS: The gene for R. capsulatus PBGS was amplified from genomic DNA and sequencing revealed errors in the sequence database. R. capsulatus PBGS was heterologously expressed in E. coli and purified to homogeneity. Analysis of an unusual phylogenetic variation in metal ion usage by PBGS enzymes predicts that R. capsulatus PBGS does not utilize metal ions such as Zn2+, or Mg2+, which have been shown to act in other PBGS at either catalytic or allosteric sites. Studies with these ions and chelators confirm the predictions. A broad pH optimum was determined to be independent of monovalent cations, approximately 8.5, and the Km value shows an acidic pKa of approximately 6. Because the metal ions of other PBGS affect the quaternary structure, gel permeation chromatography and analytical ultracentrifugation experiments were performed to examine the quaternary structure of metal ion independent R. capsulatus PBGS. The enzyme was found to be predominantly hexameric, in contrast with most other PBGS, which are octameric. A protein concentration dependence to the specific activity suggests that the hexameric R. capsulatus PBGS is very active and can dissociate to smaller, less active, species. A homology model of hexameric R. capsulatus PBGS is presented and discussed. CONCLUSION: The evidence presented in this paper supports the unusual position of the R. capsulatus PBGS as not requiring any metal ions for function. Unlike other wild-type PBGS, the R. capsulatus protein is a hexamer with an unusually high specific activity when compared to other octameric PBGS proteins. [Abstract/Link to Full Text]

King SC
The "Transport Specificity Ratio": a structure-function tool to search the protein fold for loci that control transition state stability in membrane transport catalysis.
BMC Biochem. 2004 Nov 17;516.
BACKGROUND: In establishing structure-function relationships for membrane transport proteins, the interpretation of phenotypic changes can be problematic, owing to uncertainties in protein expression levels, sub-cellular localization, and protein-folding fidelity. A dual-label competitive transport assay called "Transport Specificity Ratio" (TSR) analysis has been developed that is simple to perform, and circumvents the "expression problem," providing a reliable TSR phenotype (a constant) for comparison to other transporters. RESULTS: Using the Escherichia coli GABA (4-aminobutyrate) permease (GabP) as a model carrier, it is demonstrated that the TSR phenotype is largely independent of assay conditions, exhibiting: (i) indifference to the particular substrate concentrations used, (ii) indifference to extreme changes (40-fold) in transporter expression level, and within broad limits (iii) indifference to assay duration. The theoretical underpinnings of TSR analysis predict all of the above observations, supporting that TSR has (i) applicability in the analysis of membrane transport, and (ii) particular utility in the face of incomplete information on protein expression levels and initial reaction rate intervals (e.g., in high-throughput screening situations). The TSR was used to identify gab permease (GabP) variants that exhibit relative changes in catalytic specificity (kcat/Km) for [14C]GABA (4-aminobutyrate) versus [3H]NA (nipecotic acid). CONCLUSIONS: The TSR phenotype is an easily measured constant that reflects innate molecular properties of the transition state, and provides a reliable index of the difference in catalytic specificity that a carrier exhibits toward a particular pair of substrates. A change in the TSR phenotype, called a Delta(TSR), represents a specificity shift attributable to underlying changes in the intrinsic substrate binding energy (DeltaGb) that translocation catalysts rely upon to decrease activation energy (Delta G(T)(++). TSR analysis is therefore a structure-function tool that enables parsimonious scanning for positions in the protein fold that couple to the transition state, creating stability and thereby serving as functional determinants of catalytic power (efficiency, or specificity). [Abstract/Link to Full Text]

Nordgĺrd O, Andersen TŘ, Gabrielsen OS
Selective inhibition of c-Myb DNA-binding by RNA polymers.
BMC Biochem. 2004 Nov 4;515.
BACKGROUND: The transcription factor c-Myb is expressed in hematopoietic progenitor cells and other rapidly proliferating tissues, regulating genes important for proliferation, differentiation and survival. The DNA-binding domain (DBD) of c-Myb contains three tandemly arranged imperfect repeats, designated Myb domain R1, R2 and R3. The three-dimensional structure of the DBD shows that only the second and third Myb domains are directly involved in sequence-specific DNA-binding, while the R1 repeat does not contact DNA and only marginally affects DNA-binding properties. No structural information is available on the N-terminal 30 residues. Since deletion of the N-terminal region including R1 plays an important role in oncogenic activation of c-Myb, we asked whether this region confers properties beyond DNA-binding to the neighbouring c-Myb DBD. RESULTS: Analysis of a putative RNA-binding function of c-Myb DBD revealed that poly(G) preferentially inhibited c-Myb DNA-binding. A strong sequence-selectivity was observed when different RNA polymers were compared. Most interesting, the poly(G) sensitivity was significantly larger for a protein containing the N-terminus and the R1-repeat than for the minimal DNA-binding domain. CONCLUSION: Preferential inhibition of c-Myb DNA binding by poly(G) RNA suggests that c-Myb is able to interact with RNA in a sequence-selective manner. While R2 and R3, but not R1, are necessary for DNA-binding, R1 seems to have a distinct role in enhancing the RNA-sensitivity of c-Myb. [Abstract/Link to Full Text]

López de Heredia M, Jansen RP
RNA integrity as a quality indicator during the first steps of RNP purifications : a comparison of yeast lysis methods.
BMC Biochem. 2004 Oct 1;514.
BACKGROUND: The completion of several genome-sequencing projects has increased our need to assign functions to newly identified genes. The presence of a specific protein domain has been used as the determinant for suggesting a function for these new genes. In the case of proteins that are predicted to interact with mRNA, most RNAs bound by these proteins are still unknown. In yeast, several protocols for the identification of protein-protein interactions in high-throughput analyses have been developed during the last years leading to an increased understanding of cellular proteomics. If any of these protocols or similar approaches shall be used for the identification of mRNA-protein complexes, the integrity of mRNA is a critical factor. RESULTS: We compared the effect of different lysis protocols on RNA integrity. We report dramatic differences in RNA stability depending on the method used for yeast cell lysis. Glass bead milling and French Press lead to degraded mRNAs even in the presence of RNase inhibitors. Thus, they are not suitable to purify intact mRNP complexes or to identify specific mRNAs bound to proteins. CONCLUSION: We suggest a novel protocol, grinding deep-frozen cells, for the preparation of protein extracts that contain intact RNAs, as lysis method for the purification of mRNA-protein complexes from yeast cells. [Abstract/Link to Full Text]

Mozzherin DJ, McConnell M, Miller H, Fisher PA
Site-specific mutagenesis of Drosophila proliferating cell nuclear antigen enhances its effects on calf thymus DNA polymerase delta.
BMC Biochem. 2004 Aug 13;513.
BACKGROUND: We and others have shown four distinct and presumably related effects of mammalian proliferating cell nuclear antigen (PCNA) on DNA synthesis catalyzed by mammalian DNA polymerase delta(pol delta). In the presence of homologous PCNA, pol delta exhibits 1) increased absolute activity; 2) increased processivity of DNA synthesis; 3) stable binding of synthetic oligonucleotide template-primers (t1/2 of the pol deltaPCNAtemplate-primer complex >/=2.5 h); and 4) enhanced synthesis of DNA opposite and beyond template base lesions. This last effect is potentially mutagenic in vivo. Biochemical studies performed in parallel with in vivo genetic analyses, would represent an extremely powerful approach to investigate further, both DNA replication and repair in eukaryotes. RESULTS: Drosophila PCNA, although highly similar in structure to mammalian PCNA (e.g., it is >70% identical to human PCNA in amino acid sequence), can only substitute poorly for either calf thymus or human PCNA (approximately 10% as well) in affecting calf thymus pol delta. However, by mutating one or only a few amino acids in the region of Drosophila PCNA thought to interact with pol delta, all four effects can be enhanced dramatically. CONCLUSIONS: Our results therefore suggest that all four above effects depend at least in part on the PCNA-pol delta interaction. Moreover unlike mammals, Drosophila offers the potential for immediate in vivo genetic analyses. Although it has proven difficult to obtain sufficient amounts of homologous pol delta for parallel in vitro biochemical studies, by altering Drosophila PCNA using site-directed mutagenesis as suggested by our results, in vitro biochemical studies may now be performed using human and/or calf thymus pol delta preparations. [Abstract/Link to Full Text]

Holdsworth G, Osborne DA, Pham TT, Fells JI, Hutchinson G, Milligan G, Parrill AL
A single amino acid determines preference between phospholipids and reveals length restriction for activation of the S1P4 receptor.
BMC Biochem. 2004 Aug 6;512.
BACKGROUND: Sphingosine-1-phosphate and lysophosphatidic acid (LPA) are ligands for two related families of G protein-coupled receptors, the S1P and LPA receptors, respectively. The lysophospholipid ligands of these receptors are structurally similar, however recognition of these lipids by these receptors is highly selective. A single residue present within the third transmembrane domain (TM) of S1P receptors is thought to determine ligand selectivity; replacement of the naturally occurring glutamic acid with glutamine (present at this position in the LPA receptors) has previously been shown to be sufficient to change the specificity of S1P1 from S1P to 18:1 LPA. RESULTS: We tested whether mutation of this "ligand selectivity" residue to glutamine could confer LPA-responsiveness to the related S1P receptor, S1P4. This mutation severely affected the response of S1P4 to S1P in a [35S]GTP gamma S binding assay, and imparted sensitivity to LPA species in the order 14:0 LPA > 16:0 LPA > 18:1 LPA. These results indicate a length restriction for activation of this receptor and demonstrate the utility of using LPA-responsive S1P receptor mutants to probe binding pocket length using readily available LPA species. Computational modelling of the interactions between these ligands and both wild type and mutant S1P4 receptors showed excellent agreement with experimental data, therefore confirming the fundamental role of this residue in ligand recognition by S1P receptors. CONCLUSIONS: Glutamic acid in the third transmembrane domain of the S1P receptors is a general selectivity switch regulating response to S1P over the closely related phospholipids, LPA. Mutation of this residue to glutamine confers LPA responsiveness with preference for short-chain species. The preference for short-chain LPA species indicates a length restriction different from the closely related S1P1 receptor. [Abstract/Link to Full Text]

Sklenar AR, Parthun MR
Characterization of yeast histone H3-specific type B histone acetyltransferases identifies an ADA2-independent Gcn5p activity.
BMC Biochem. 2004 Jul 26;511.
BACKGROUND: The acetylation of the core histone NH2-terminal tails is catalyzed by histone acetyltransferases. Histone acetyltransferases can be classified into two distinct groups (type A and B) on the basis of cellular localization and substrate specificity. Type B histone acetyltransferases, originally defined as cytoplasmic enzymes that acetylate free histones, have been proposed to play a role in the assembly of chromatin through the acetylation of newly synthesized histones H3 and H4. To date, the only type B histone acetyltransferase activities identified are specific for histone H4. RESULTS: To better understand the role of histone acetylation in the assembly of chromatin structure, we have identified additional type B histone acetyltransferase activities specific for histone H3. One such activity, termed HatB3.1, acetylated histone H3 with a strong preference for free histones relative to chromatin substrates. Deletion of the GCN5 and ADA3 genes resulted in the loss of HatB3.1 activity while deletion of ADA2 had no effect. In addition, Gcn5p and Ada3p co-fractionated with partially purified HatB3.1 activity while Ada2p did not. CONCLUSIONS: Yeast extracts contain several histone acetyltransferase activities that show a strong preference for free histone H3. One such activity, termed HatB3.1, appears to be a novel Gcn5p-containing complex which does not depend on the presence of Ada2p. [Abstract/Link to Full Text]

Tieleman DP
The molecular basis of electroporation.
BMC Biochem. 2004 Jul 19;510.
BACKGROUND: Electroporation is a common method to introduce foreign molecules into cells, but its molecular basis is poorly understood. Here I investigate the mechanism of pore formation by direct molecular dynamics simulations of phospholipid bilayers of a size of 256 and of more than 2000 lipids as well as simulations of simpler interface systems with applied electric fields of different strengths. RESULTS: In a bilayer of 26 x 29 nm multiple pores form independently with sizes of up to 10 nm on a time scale of nanoseconds with an applied field of 0.5 V/nm. Pore formation is accompanied by curving of the bilayer. In smaller bilayers of ca. 6 x 6 nm, a single pore forms on a nanosecond time scale in lipid bilayers with applied fields of at least 0.4 V/nm, corresponding to transmembrane voltages of ca. 3 V. The presence of 1 M salt does not seem to change the mechanism. In an even simpler system, consisting of a 3 nm thick octane layer, pores also form, despite the fact that there are no charged headgroups and no salt in this system. In all cases pore formation begins with the formation of single-file like water defects penetrating into the bilayer or octane. CONCLUSIONS: The simulations suggest that pore formation is driven by local electric field gradients at the water/lipid interface. Water molecules move in these field gradients, which increases the probability of water defects penetrating into the bilayer interior. Such water defects cause a further increase in the local electric field, accelerating the process of pore formation. The likelihood of pore formation appears to be increased by local membrane defects involving lipid headgroups. Simulations with and without salt show little difference in the observed pore formation process. The resulting pores are hydrophilic, lined by phospholipid headgroups. [Abstract/Link to Full Text]

Strub C, Alies C, Lougarre A, Ladurantie C, Czaplicki J, Fournier D
Mutation of exposed hydrophobic amino acids to arginine to increase protein stability.
BMC Biochem. 2004 Jul 13;59.
BACKGROUND: One strategy to increase the stability of proteins is to reduce the area of water-accessible hydrophobic surface. RESULTS: In order to test it, we replaced 14 solvent-exposed hydrophobic residues of acetylcholinesterase by arginine. The stabilities of the resulting proteins were tested using denaturation by high temperature, organic solvents, urea and by proteolytic digestion. CONCLUSION: Although the mutational effects were rather small, this strategy proved to be successful since half of the mutants showed an increased stability. This stability may originate from the suppression of unfavorable interactions of nonpolar residues with water or from addition of new hydrogen bonds with the solvent. Other mechanisms may also contribute to the increased stability observed with some mutants. For example, introduction of a charge at the surface of the protein may provide a new coulombic interaction on the protein surface. [Abstract/Link to Full Text]

Biederbick A, Rösser R, Storre J, Elsässer HP
The VSFASSQQ motif confers calcium sensitivity to the intracellular apyrase LALP70.
BMC Biochem. 2004 Jun 16;58.
BACKGROUND: Apyrases are divalent ion dependent tri- and dinucleotide phosphatases with different substrate specificity. The intracellular lysosomal apyrase LALP70 is also expressed as a splice variant (LALP70v) lacking a VSFASSQQ motif in the center of the molecule (aminoacids 287-294). However, the functional significance of this motif is unknown. In this report we used a thin layer chromatography approach to study separately the UTPase and UDPase activity of the two LALP-enzymes. RESULTS: We show, that LALP70 and LALP70v cleaved UTP to UDP in a calcium independent manner. In contrast, the cleavage of UDP to UMP was strongly calcium dependent for LALP70, but calcium independent for LALP70v. CONCLUSIONS: The VSFASSQQ motif not only influences the substrate specificity of LALP70, but it confers calcium sensitivity to LALP70 during the UDP cleavage. Whether this is due to direct binding of calcium to this motif or to a conformational change of the enzyme, remains to be elucidated. [Abstract/Link to Full Text]

Fisher DI, Cartwright JL, Harashima H, Kamiya H, McLennan AG
Characterization of a nudix hydrolase from Deinococcus radiodurans with a marked specificity for (deoxy)ribonucleoside 5'-diphosphates.
BMC Biochem. 2004 May 17;57.
BACKGROUND: Nudix hydrolases form a protein family whose function is to hydrolyse intracellular nucleotides and so regulate their levels and eliminate potentially toxic derivatives. The genome of the radioresistant bacterium Deinococcus radiodurans encodes 25 nudix hydrolases, an unexpectedly large number. These may contribute to radioresistance by removing mutagenic oxidised and otherwise damaged nucleotides. Characterisation of these hydrolases is necessary to understand the reason for their presence. Here, we report the cloning and characterisation of the DR0975 gene product, a nudix hydrolase that appears to be unique to this organism. RESULTS: The DR0975 gene was cloned and expressed as a 20 kDa histidine-tagged recombinant product in Escherichia coli. Substrate analysis of the purified enzyme showed it to act primarily as a phosphatase with a marked preference for (deoxy)nucleoside 5'-diphosphates (dGDP > ADP > dADP > GDP > dTDP > UDP > dCDP > CDP). Km for dGDP was 110 microM and kcat was 0.18 s-1 under optimal assay conditions (pH 9.4, 7.5 mM Mg2+). 8-Hydroxy-2'-deoxyguanosine 5'-diphosphate (8-OH-dGDP) was also a substrate with a Km of 170 microM and kcat of 0.13 s-1. Thus, DR0975 showed no preference for 8-OH-dGDP over dGDP. Limited pyrophosphatase activity was also observed with NADH and some (di)adenosine polyphosphates but no other substrates. Expression of the DR0975 gene was undetectable in logarithmic phase cells but was induced at least 30-fold in stationary phase. Superoxide, but not peroxide, stress and slow, but not rapid, dehydration both caused a slight induction of the DR0975 gene. CONCLUSION: Nucleotide substrates for nudix hydrolases conform to the structure NDP-X, where X can be one of several moieties. Thus, a preference for (d)NDPs themselves is most unusual. The lack of preference for 8-OH-dGDP over dGDP as a substrate combined with the induction in stationary phase, but not by peroxide or superoxide, suggests that the function of DR09075 may be to assist in the recycling of nucleotides under the very different metabolic requirements of stationary phase. Thus, if DR0975 does contribute to radiation resistance, this contribution may be indirect. [Abstract/Link to Full Text]

Serre V, Penverne B, Souciet JL, Potier S, Guy H, Evans D, Vicart P, Hervé G
Integrated allosteric regulation in the S. cerevisiae carbamylphosphate synthetase - aspartate transcarbamylase multifunctional protein.
BMC Biochem. 2004 May 5;56.
BACKGROUND: The S. cerevisiae carbamylphosphate synthetase - aspartate transcarbamylase multifunctional protein catalyses the first two reactions of the pyrimidine pathway. In this organism, these two reactions are feedback inhibited by the end product UTP. In the present work, the mechanisms of these integrated inhibitions were studied. RESULTS: The results obtained show that the inhibition is competitive in the case of carbamylphosphate synthetase and non-competitive in the case of aspartate transcarbamylase. They also identify the substrate whose binding is altered by this nucleotide and the step of the carbamylphosphate synthetase reaction which is inhibited. Furthermore, the structure of the domains catalyzing these two reactions were modelled in order to localize the mutations which, specifically, alter the aspartate transcarbamylase sensitivity to the feedback inhibitor UTP. Taken together, the results make it possible to propose a model for the integrated regulation of the two activities of the complex. UTP binds to a regulatory site located in the vicinity of the carbamylphosphate synthetase catalytic subsite which catalyzes the third step of this enzyme reaction. Through a local conformational change, this binding decreases, competitively, the affinity of this site for the substrate ATP. At the same time, through a long distance signal transmission process it allosterically decreases the affinity of the aspartate transcarbamylase catalytic site for the substrate aspartate. CONCLUSION: This investigation provides informations about the mechanisms of allosteric inhibition of the two activities of the CPSase-ATCase complex. Although many allosteric monofunctional enzymes were studied, this is the first report on integrated allosteric regulation in a multifunctional protein. The positions of the point mutations which specifically abolish the sensitivity of aspartate transcarbamylase to UTP define an interface between the carbamylphosphate synthetase and aspartate transcarbamylase domains, through which the allosteric signal for the regulation of aspartate transcarbamylase must be propagated. [Abstract/Link to Full Text]

Scuro LS, Simioni PU, Grabriel DL, Saviani EE, Modolo LV, Tamashiro WM, Salgado I
Suppression of nitric oxide production in mouse macrophages by soybean flavonoids accumulated in response to nitroprusside and fungal elicitation.
BMC Biochem. 2004 Apr 21;55.
BACKGROUND: The anti-inflammatory properties of some flavonoids have been attributed to their ability to inhibit the production of NO by activated macrophages. Soybean cotyledons accumulate certain flavonoids following elicitation with an extract of the fungal pathogen Diaporthe phaseolorum f. sp. meridionalis (Dpm). Sodium nitroprusside (SNP), a nitric oxide donor, can substitute for Dpm in inducing flavonoid production. In this study, we investigated the effect of flavonoid-containing diffusates obtained from Dpm- and SNP-elicited soybean cotyledons on NO production by lipopolysaccharide (LPS)- and LPS plus interferon-gamma (IFNgamma)-activated murine macrophages. RESULTS: Significant inhibition of NO production, measured as nitrite formation, was observed when macrophages were activated in the presence of soybean diffusates from Dpm- or SNP-elicited cotyledons. This inhibition was dependent on the duration of exposure to the elicitor. Daidzein, genistein, luteolin and apigenin, the main flavonoids present in diffusates of elicited cotyledons, suppressed the NO production by LPS + IFNgamma activated macrophages in a concentration-dependent manner, with IC50 values of 81.4 microM, 34.5 microM, 38.6 microM and 10.4 microM respectively. For macrophages activated with LPS alone, the IC50 values were 40.0 microM, 16.6 microM, 10.4 microM and 2.8 microM, respectively. Western blot analysis showed that iNOS expression was not affected by daidzein, was reduced by genistein, and was abolished by apigenin, luteolin and Dpm- and SNP-soybean diffusates at concentrations that significantly inhibited NO production by activated macrophages. CONCLUSIONS: These results suggest that the suppressive effect of flavonoids on iNOS expression could account for the potent inhibitory effect of Dpm- and SNP-diffusates on NO production by activated macrophages. Since the physiological concentration of flavonoids in plants is normally low, the treatment of soybean tissues with SNP may provide a simple method for substantially increasing the concentration of metabolites that are beneficial for the treatment of chronic inflammatory diseases associated with NO production. [Abstract/Link to Full Text]

Gressent F, Cullimore JV, Ranjeva R, Bono JJ
Radiolabeling of lipo-chitooligosaccharides using the NodH sulfotransferase: a two-step enzymatic procedure.
BMC Biochem. 2004 Apr 13;54.
BACKGROUND: The NodH sulfotransferase from Sinorhizobium meliloti has been used to radiolabel lipochitooligosaccharidic (LCO) Nod factor signals with 35S from inorganic sulfate in a two-step enzymatic procedure. The first step involved the production of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), a sulphate donor, using enzymes contained in a yeast extract, and the second step used the NodH enzyme. However with this established procedure, only a low incorporation of the initial inorganic sulfate into the Nod factors was obtained (about 7% after purification of the labeled compounds). The aim of this work was to optimize the radiolabelling of Nod factors with 35S. RESULTS: The limiting step has been shown to be the sulfation of ATP and its subsequent conversion into PAPS (first step), the sulfate donor for the NodH sulfotransferase activity (second step). By the addition of GTP to the reaction mixture and by manipulating the [ATP]/[Mg2+] ratio the yield of PAPS has been increased from 13% to 80%. Using the radiolabeled PAPS we have shown that the efficiency of sulfate transfer to LCOs, by the recombinant S. meliloti NodH sulfotransferase is strongly influenced by the length of the oligosaccharide chain. Variations in the substitutions on the non-reducing sugar, including the structure of the fatty acyl chain, had little effect and Nod factors from the heterologous bacterium Rhizobium tropici could be sulfated by NodH from S. meliloti. CONCLUSIONS: By characterizing the two steps we have optimized the procedure to radiolabel biologically-important, lipo-chitooligosaccharide (LCO) Nod factors to a specific radioactivity of about 800 Ci x mmol(-1) with an incorporation of 60% of the initial inorganic sulfate. The two-step sulfation procedure may be used to radiolabel a variety of related LCO molecules. [Abstract/Link to Full Text]

Chevillard G, Clémencet MC, Etienne P, Martin P, Pineau T, Latruffe N, Nicolas-Francčs V
Molecular cloning, gene structure and expression profile of two mouse peroxisomal 3-ketoacyl-CoA thiolase genes.
BMC Biochem. 2004 Mar 25;53.
BACKGROUND: In rats, two peroxisomal 3-ketoacyl-CoA thiolase genes (A and B) have been cloned, whereas only one thiolase gene is found in humans. The aim of this study was thus to clone the different mouse thiolase genes in order to study both their tissue expression and their associated enzymatic activity. RESULTS: In this study, we cloned and characterized two mouse peroxisomal 3-ketoacyl-CoA thiolase genes (termed thiolase A and B). Both thiolase A and B genes contain 12 exons and 11 introns. Using RNA extracted from mouse liver, we cloned the two corresponding cDNAs. Thiolase A and B cDNAs possess an open reading frame of 1272 nucleotides encoding a protein of 424 amino acids. In the coding sequence, the two thiolase genes exhibited approximately equal to 97% nucleotide sequence identity and approximately equal to 96% identity at the amino acid level. The tissue-specific expression of the two peroxisomal 3-ketoacyl-CoA thiolase genes was studied in mice. Thiolase A mRNA was mainly expressed in liver and intestine, while thiolase B mRNA essentially exhibited hepatic expression and weaker levels in kidney, intestine and white adipose tissue. Thiolase A and B expressions in the other tissues such as brain or muscle were very low though these tissues were chiefly involved in peroxisomal disorders. At the enzymatic level, thiolase activity was detected in liver, kidney, intestine and white adipose tissue but no significant difference was observed between these four tissues. Moreover, thiolase A and B genes were differently induced in liver of mice treated with fenofibrate. CONCLUSION: Two mouse thiolase genes and cDNAs were cloned. Their corresponding transcripts are mostly expressed in the liver of mice and are differently induced by fenofibrate. [Abstract/Link to Full Text]

Burnett C, Makridou P, Hewlett L, Howard K
Lipid phosphate phosphatases dimerise, but this interaction is not required for in vivo activity.
BMC Biochem. 2004 Jan 16;52.
BACKGROUND: Lipid phosphate phosphatases (LPPs) are integral membrane proteins believed to dephosphorylate bioactive lipid messengers, so modifying or attenuating their activities. Wunen, a Drosophila LPP homologue, has been shown to play a pivotal role in primordial germ cell (PGC) migration and survival during embryogenesis. It has been hypothesised that LPPs may form oligomeric complexes, and may even function as hexamers. We were interested in exploring this possibility, to confirm whether LPPs can oligomerise, and if they do, whether oligomerisation is required for either in vitro or in vivo activity. RESULTS: We present evidence that Wunen dimerises, that these associations require the last thirty-five C-terminal amino-acids and depend upon the presence of an intact catalytic site. Expression of a truncated, monomeric form of Wunen in Drosophila embryos results in perturbation of germ cell migration and germ cell loss, as observed for full-length Wunen. We also observed that murine LPP-1 and human LPP-3 can also form associations, but do not form interactions with Wunen or each other. Furthermore, Wunen does not form dimers with its closely related counterpart Wunen-2. Finally we discovered that addition of a trimeric myc tag to the C-terminus of Wunen does not prevent dimerisation or in vitro activity, but does prevent activity in vivo. CONCLUSION: LPPs do form complexes, but these do not seem to be specifically required for activity either in vitro or in vivo. Since neither dimerisation nor the C-terminus seem to be involved in substrate recognition, they may instead confer structural or functional stability through dimerisation. The results indicate that the associations we see are highly specific and occur only between monomers of the same protein. [Abstract/Link to Full Text]

Toman R, Garidel P, Andrä J, Slaba K, Hussein A, Koch MH, Brandenburg K
Physicochemical characterization of the endotoxins from Coxiella burnetii strain Priscilla in relation to their bioactivities.
BMC Biochem. 2004 Jan 12;51.
BACKGROUND: Coxiella burnetii is the etiological agent of Q fever found worldwide. The microorganism has like other Gram-negative bacteria a lipopolysaccharide (LPS, endotoxin) in its outer membrane, which is important for the pathogenicity of the bacteria. In order to understand the biological activity of LPS, a detailed physico-chemical analysis of LPS is of utmost importance. RESULTS: The lipid A moiety of LPS is tetraacylated and has longer (C-16) acyl chains than most other lipid A from enterobacterial strains. The two ester-linked 3-OH fatty acids found in the latter are lacking. The acyl chains of the C. burnetii endotoxins exhibit a broad melting range between 5 and 25 degrees C for LPS and 10 and 40 degrees C for lipid A. The lipid A moiety has a cubic inverted aggregate structure, and the inclination angle of the D-glucosamine disaccharide backbone plane of the lipid A part with respect to the membrane normal is around 40 degrees. Furthermore, the endotoxins readily intercalate into phospholipid liposomes mediated by the lipopolysaccharide-binding protein (LBP). The endotoxin-induced tumor necrosis factor alpha (TNFalpha) production in human mononuclear cells is one order of magnitude lower than that found for endotoxins from enterobacterial strains, whereas the same activity as in the latter compounds is found in the clotting reaction of the Limulus amebocyte lysate assay. CONCLUSIONS: Despite a considerably different chemical primary structure of the C. burnetii lipid A in comparison with enterobacterial lipid A, the data can be well understood by applying the previously presented conformational concept of endotoxicity, a conical shape of the lipid A moiety of LPS and a sufficiently high inclination of the sugar backbone plane with respect to the membrane plane. Importantly, the role of the acyl chain fluidity in modulating endotoxicity now becomes more evident. [Abstract/Link to Full Text]

Fischer M, Schott AK, Kemter K, Feicht R, Richter G, Illarionov B, Eisenreich W, Gerhardt S, Cushman M, Steinbacher S, Huber R, Bacher A
Riboflavin synthase of Schizosaccharomyces pombe. Protein dynamics revealed by 19F NMR protein perturbation experiments.
BMC Biochem. 2003 Dec 23;418.
BACKGROUND: Riboflavin synthase catalyzes the transformation of 6,7-dimethyl-8-ribityllumazine into riboflavin in the last step of the riboflavin biosynthetic pathway. Gram-negative bacteria and certain yeasts are unable to incorporate riboflavin from the environment and are therefore absolutely dependent on endogenous synthesis of the vitamin. Riboflavin synthase is therefore a potential target for the development of antiinfective drugs. RESULTS: A cDNA sequence from Schizosaccharomyces pombe comprising a hypothetical open reading frame with similarity to riboflavin synthase of Escherichia coli was expressed in a recombinant E. coli strain. The recombinant protein is a homotrimer of 23 kDa subunits as shown by sedimentation equilibrium centrifugation. The protein sediments at an apparent velocity of 4.1 S at 20 degrees C. The amino acid sequence is characterized by internal sequence similarity indicating two similar folding domains per subunit. The enzyme catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 158 nmol mg(-1) min(-1) with an apparent KM of 5.7 microM. 19F NMR protein perturbation experiments using fluorine-substituted intermediate analogs show multiple signals indicating that a given ligand can be bound in at least 4 different states. 19F NMR signals of enzyme-bound intermediate analogs were assigned to ligands bound by the N-terminal respectively C-terminal folding domain on basis of NMR studies with mutant proteins. CONCLUSION: Riboflavin synthase of Schizosaccharomyces pombe is a trimer of identical 23-kDa subunits. The primary structure is characterized by considerable similarity of the C-terminal and N-terminal parts. Riboflavin synthase catalyzes a mechanistically complex dismutation of 6,7-dimethyl-8-ribityllumazine affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The 19F NMR data suggest large scale dynamic mobility in the trimeric protein which may play an important role in the reaction mechanism. [Abstract/Link to Full Text]

Hanna SM, Kirk P, Holt OJ, Puklavec MJ, Brown MH, Barclay AN
A novel form of the membrane protein CD147 that contains an extra Ig-like domain and interacts homophilically.
BMC Biochem. 2003 Nov 7;417.
BACKGROUND: CD147 is a broadly distributed integral membrane glycoprotein with two Ig-like domains implicated in a wide range of functions. It is associated at the cell surface with the monocarboxylate transporters MCT1 and 4 but interactions of the extracellular region have not been characterised. RESULTS: We report the characterisation of a form of CD147 with an additional membrane-distal Ig-like domain. In contrast to the two domain form, this three domain form of CD147 interacts homophilically. Surface plasmon resonance analysis using recombinant proteins showed that the interaction was of low affinity (KD approximately 40 microM) and this is typical of many interactions between membrane proteins. cDNA for the 3 domain form are rare but have been identified in human and mouse retina. CONCLUSION: The finding that the three domain form of CD147 has an extracellular ligand, that is it interacts homophilically, suggests this interaction may be important in aligning lactate transporters in the retina where lactate is an important metabolite. [Abstract/Link to Full Text]

Darin N, Kadhom N, Bričre JJ, Chretien D, Bébéar CM, Rötig A, Munnich A, Rustin P
Mitochondrial activities in human cultured skin fibroblasts contaminated by Mycoplasma hyorhinis.
BMC Biochem. 2003 Nov 4;415.
BACKGROUND: Mycoplasma contaminations are a recurrent problem in the use of cultured cells, including human cells, especially as it has been shown to impede cell cycle, triggering cell death under various conditions. More specific consequences on cell metabolism are poorly known. RESULTS: Here we report the lack of significant consequence of a heavy contamination by the frequently encountered mycoplasma strain, M. hyorhinis, on the determination of respiratory chain activities, but the potential interference when assaying citrate synthase. Contamination by M. hyorhinis was detected by fluorescent imaging and further quantified by the determination of the mycoplasma-specific phosphate acetyltransferase activity. Noticeably, this latter activity was not found equally distributed in various mycoplasma types, being exceptionally high in M. hyorhinis. CONCLUSION: While we observed a trend for respiration reduction in heavily contaminated cells, no significant and specific targeting of any respiratory chain components could be identified. This suggested a potential interference with cell metabolism rather than direct interaction with respiratory chain components. [Abstract/Link to Full Text]

Timson DJ, Reece RJ
Sugar recognition by human galactokinase.
BMC Biochem. 2003 Nov 4;416.
BACKGROUND: Galactokinase catalyses the first committed step of galactose catabolism in which the sugar is phosphorylated at the expense of MgATP. Recent structural studies suggest that the enzyme makes several contacts with galactose--five side chain and two main chain hydrogen bonds. Furthermore, it has been suggested that inhibition of galactokinase may help sufferers of the genetic disease classical galactosemia which is caused by defects in another enzyme of the pathway galactose-1-phosphate uridyl transferase. Galactokinases from different sources have a range of substrate specificities and a diversity of kinetic mechanisms. Therefore only studies on the human enzyme are likely to be of value in the design of therapeutically useful inhibitors. RESULTS: Using recombinant human galactokinase expressed in and purified from E. coli we have investigated the sugar specificity of the enzyme and the kinetic consequences of mutating residues in the sugar-binding site in order to improve our understanding of substrate recognition by this enzyme. D-galactose and 2-deoxy-D-galactose are substrates for the enzyme, but N-acetyl-D-galactosamine, L-arabinose, D-fucose and D-glucose are all not phosphorylated. Mutation of glutamate-43 (which forms a hydrogen bond to the hydroxyl group attached to carbon 6 of galactose) to alanine results in only minor changes in the kinetic parameters of the enzyme. Mutation of this residue to glycine causes a ten-fold drop in the turnover number. In contrast, mutation of histidine 44 to either alanine or isoleucine results in insoluble protein following expression in E. coli. Alteration of the residue that makes hydrogen bonds to the hydroxyl attached to carbons 3 and 4 (aspartate 46) results in an enzyme that although soluble is essentially inactive. CONCLUSIONS: The enzyme is tolerant to small changes at position 2 of the sugar ring, but not at positions 4 and 6. The results from site directed mutagenesis could not have been predicted from the crystal structure alone and needed to be determined experimentally. [Abstract/Link to Full Text]

Guidry JJ, Shewmaker F, Maskos K, Landry S, Wittung-Stafshede P
Probing the interface in a human co-chaperonin heptamer: residues disrupting oligomeric unfolded state identified.
BMC Biochem. 2003 Oct 2;414.
BACKGROUND: The co-chaperonin protein 10 (cpn10) assists cpn60 in the folding of nonnative polypeptides in a wide range of organisms. All known cpn10 molecules are heptamers of seven identical subunits that are linked together by beta-strand interactions at a large and flexible interface. Unfolding of human mitochondrial cpn10 in urea results in an unfolded heptameric state whereas GuHCl additions result in unfolded monomers. To address the role of specific interface residues in the assembly of cpn10 we prepared two point-mutated variants, in each case removing a hydrophobic residue positioned at the subunit-subunit interface. RESULTS: Replacing valine-100 with a glycine (Val100Gly cpn10) results in a wild-type-like protein with seven-fold symmetry although the thermodynamic stability is decreased and the unfolding processes in urea and GuHCl both result in unfolded monomers. In sharp contrast, replacing phenylalanine-8 with a glycine (Phe8Gly cpn10) results in a protein that has lost the ability to assemble. Instead, this protein exists mostly as unfolded monomers. CONCLUSIONS: We conclude that valine-100 is a residue important to adopt an oligomeric unfolded state but it does not affect the ability to assemble in the folded state. In contrast, phenylalanine-8 is required for both heptamer assembly and monomer folding and therefore this mutation results in unfolded monomers at physiological conditions. Despite the plasticity and large size of the cpn10 interface, our observations show that isolated interface residues can be crucial for both the retention of a heptameric unfolded structure and for subunit folding. [Abstract/Link to Full Text]

Walsh HA, O'Shea KC, Botting NP
Comparative inhibition by substrate analogues 3-methoxy- and 3-hydroxydesaminokynurenine and an improved 3 step purification of recombinant human kynureninase.
BMC Biochem. 2003 Sep 24;413.
BACKGROUND: Kynureninase is a key enzyme on the kynurenine pathway of tryptophan metabolism. One of the end products of the pathway is the neurotoxin quinolinic acid which appears to be responsible for neuronal cell death in a number of important neurological diseases. This makes kynureninase a possible therapeutic target for diseases such as Huntington's, Alzheimer's and AIDS related dementia, and the development of potent inhibitors an important research aim. RESULTS: Two new kynurenine analogues, 3-hydroxydesaminokynurenine and 3-methoxydesaminokynurenine, were synthesised as inhibitors of kynureninase and tested on the tryptophan-induced bacterial enzyme from Pseudomonas fluorescens, the recombinant human enzyme and the rat hepatic enzyme. They were found to be mixed inhibitors of all three enzymes displaying both competitive and non competitive inhibition. The 3-hydroxy derivative gave low Ki values of 5, 40 and 100 nM respectively. An improved 3-step purification scheme for recombinant human kynureninase was also developed. CONCLUSION: For kynureninase from all three species the 2-amino group was found to be crucial for activity whilst the 3-hydroxyl group played a fundamental role in binding at the active site presumably via hydrogen bonding. The potency of the various inhibitors was found to be species specific. The 3-hydroxylated inhibitor had a greater affinity for the human enzyme, consistent with its specificity for 3-hydroxykynurenine as substrate, whilst the methoxylated version yielded no significant difference between bacterial and human kynureninase. The modified purification described is relatively quick, simple and cost effective. [Abstract/Link to Full Text]

Klinger C, Rossbach M, Howe R, Kaufmann M
Thermophile-specific proteins: the gene product of aq_1292 from Aquifex aeolicus is an NTPase.
BMC Biochem. 2003 Sep 23;412.
BACKGROUND: To identify thermophile-specific proteins, we performed phylogenetic patterns searches of 66 completely sequenced microbial genomes. This analysis revealed a cluster of orthologous groups (COG1618) which contains a protein from every thermophile and no sequence from 52 out of 53 mesophilic genomes. Thus, COG1618 proteins belong to the group of thermophile-specific proteins (THEPs) and therefore we here designate COG1618 proteins as THEP1s. Since no THEP1 had been analyzed biochemically thus far, we characterized the gene product of aq_1292 which is THEP1 from the hyperthermophilic bacterium Aquifex aeolicus (aaTHEP1). RESULTS: aaTHEP1 was cloned in E. coli, expressed and purified to homogeneity. At a temperature optimum between 70 and 80 degrees C, aaTHEP1 shows enzymatic activity in hydrolyzing ATP to ADP + Pi with kcat = 5 x 10(-3) s(-1) and Km = 5.5 x 10(-6) M. In addition, the enzyme exhibits GTPase activity (kcat = 9 x 10(-3) s(-1) and Km= 45 x 10(-6) M). aaTHEP1 is inhibited competitively by CTP, UTP, dATP, dGTP, dCTP, and dTTP. As shown by gel filtration, aaTHEP1 in its purified state appears as a monomer. The enzyme is resistant to limited proteolysis suggesting that it consists of a single domain. Although THEP1s are annotated as "predicted nucleotide kinases" we could not confirm such an activity experimentally. CONCLUSION: Since aaTHEP1 is the first member of COG1618 that is characterized biochemically and functional information about one member of a COG may be transferred to the entire COG, we conclude that COG1618 proteins are a family of thermophilic NTPases. [Abstract/Link to Full Text]

Kabine M, El Kebbaj MS, Hafiani A, Latruffe N, Cherkaoui-Malki M
Hibernation impact on the catalytic activities of the mitochondrial D-3-hydroxybutyrate dehydrogenase in liver and brain tissues of jerboa (Jaculus orientalis).
BMC Biochem. 2003 Sep 10;411.
BACKGROUND: Jerboa (Jaculus orientalis) is a deep hibernating rodent native to subdesert highlands. During hibernation, a high level of ketone bodies i.e. acetoacetate (AcAc) and D-3-hydroxybutyrate (BOH) are produced in liver, which are used in brain as energetic fuel. These compounds are bioconverted by mitochondrial D-3-hydroxybutyrate dehydrogenase (BDH) E.C. 1.1.1.30. Here we report, the function and the expression of BDH in terms of catalytic activities, kinetic parameters, levels of protein and mRNA in both tissues i.e brain and liver, in relation to the hibernating process. RESULTS: We found that: 1/ In euthemic jerboa the specific activity in liver is 2.4- and 6.4- fold higher than in brain, respectively for AcAc reduction and for BOH oxidation. The same differences were found in the hibernation state. 2/ In euthermic jerboa, the Michaelis constants, KM BOH and KM NAD+ are different in liver and in brain while KM AcAc, KM NADH and the dissociation constants, KD NAD+and KD NADH are similar. 3/ During prehibernating state, as compared to euthermic state, the liver BDH activity is reduced by half, while kinetic constants are strongly increased except KD NAD+. 4/ During hibernating state, BDH activity is significantly enhanced, moreover, kinetic constants (KM and KD) are strongly modified as compared to the euthermic state; i.e. KD NAD+ in liver and KM AcAc in brain decrease 5 and 3 times respectively, while KD NADH in brain strongly increases up to 5.6 fold. 5/ Both protein content and mRNA level of BDH remain unchanged during the cold adaptation process. CONCLUSIONS: These results cumulatively explained and are consistent with the existence of two BDH enzymatic forms in the liver and the brain. The apoenzyme would be subjected to differential conformational folding depending on the hibernation state. This regulation could be a result of either post-translational modifications and/or a modification of the mitochondrial membrane state, taking into account that BDH activity is phospholipid-dependent. [Abstract/Link to Full Text]

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Acharya M, Mookherjee S, Bhattacharjee A, Thakur SK, Bandyopadhyay AK, Sen A, Chakrabarti S, Ray K
Evaluation of the OPTC gene in primary open angle glaucoma: functional significance of a silent change.
BMC Mol Biol. 2007;821.
BACKGROUND: We investigated the molecular basis of primary open-angle glaucoma (POAG) using Opticin (OPTC) as a candidate gene on the basis of its expression in the trabecular meshwork cells involved in the disease pathogenesis. Two hundred POAG patients and 100 controls were enrolled in this study. The coding sequence of OPTC was amplified by PCR from genomic DNA of POAG patients, followed by SSCP, DHPLC and DNA sequencing. Subsequent bioinformatic analysis, site-directed mutagenesis, quantitative RT-PCR and western blot experiments were performed to address the functional significance of a 'silent' change in the OPTC coding region while screening for mutations in POAG patients. RESULTS: We detected two missense (p.Glu66Gly & p.Ile89Thr) and one silent change (p.Phe162Phe; c.602 C>T) that was present in 3 different patients but in none of the 100 controls screened. The mutant (c.602T) mRNA was predicted to have remarkably different secondary structure compared to the wild-type transcript by in silico approaches. Subsequent wet-lab experiments showed lower expression of the gene both at the mRNA and protein levels. CONCLUSION: Our study suggests OPTC as a candidate gene for POAG. Further, it highlights the importance of investigating the 'silent' variations for functional implication that might not be apparent from only in silico analysis. [Abstract/Link to Full Text]

Hantusch B, Kalt R, Krieger S, Puri C, Kerjaschki D
Sp1/Sp3 and DNA-methylation contribute to basal transcriptional activation of human podoplanin in MG63 versus Saos-2 osteoblastic cells.
BMC Mol Biol. 2007;820.
BACKGROUND: Podoplanin is a membrane mucin that, among a series of tissues, is expressed on late osteoblasts and osteocytes. Since recent findings have focussed on podoplanin's potential role as a tumour progression factor, we aimed at identifying regulatory elements conferring PDPN promoter activity. Here, we characterized the molecular mechanism controlling basal PDPN transcription in human osteoblast-like MG63 versus Saos-2 cells. RESULTS: We cloned and sequenced 2056 nucleotides from the 5'-flanking region of the PDPN gene and a computational search revealed that the TATA and CAAT box-lacking promoter possesses features of a growth-related gene, such as a GC-rich 5' region and the presence of multiple putative Sp1, AP-4 and NF-1 sites. Reporter gene assays demonstrated a functional promoter in MG63 cells exhibiting 30-fold more activity than in Saos-2 cells. In vitro DNase I footprinting revealed eight protected regions flanked by DNaseI hypersensitive sites within the region bp -728 to -39 present in MG63, but not in Saos-2 cells. Among these regions, mutation and supershift electrophoretic mobility shift assays (EMSA) identified four Sp1/Sp3 binding sites and two binding sites for yet unknown transcription factors. Deletion studies demonstrated the functional importance of two Sp1/Sp3 sites for PDPN promoter activity. Overexpression of Sp1 and Sp3 independently increased the stimulatory effect of the promoter and podoplanin mRNA levels in MG63 and Saos-2 cells. In SL2 cells, Sp3 functioned as a repressor, while Sp1 and Sp3 acted positively synergistic. Weak PDPN promoter activity of Saos-2 cells correlated with low Sp1/Sp3 nuclear levels, which was confirmed by Sp1/Sp3 chromatin immunoprecipitations in vivo. Moreover, methylation-sensitive Southern blot analyses and bisulfite sequencing detected strong methylation of CpG sites upstream of bp -464 in MG63 cells, but hypomethylation of these sites in Saos-2 cells. Concomitantly, treatment with the DNA methyltransferase inhibitor 5-azaCdR in combination with trichostatin A (TSA) downregulated podoplanin mRNA levels in MG63 cells, and region-specific in vitro methylation of the distal promoter suggested that DNA methylation rather enhanced than hindered PDPN transcription in both cell types. CONCLUSION: These data establish that in human osteoblast-like MG63 cells, Sp1 and Sp3 stimulate basal PDPN transcription in a concerted, yet independent manner, whereas Saos-2 cells lack sufficient nuclear Sp protein amounts for transcriptional activation. Moreover, a highly methylated chromatin conformation of the distal promoter region confers cell-type specific podoplanin upregulation versus Saos-2 cells. [Abstract/Link to Full Text]

Wang H, Yang S, Yang E, Zhu Z, Mu Y, Feng S, Li K
NF-kappaB mediates the transcription of mouse calsarcin-1 gene, but not calsarcin-2, in C2C12 cells.
BMC Mol Biol. 2007;819.
BACKGROUND: The calsarcins comprise a novel family of muscle-specific calcineurin-interaction proteins that play an important role in modulating both the function and substrate specificity of calcineurin in muscle cells. The expression of calsarcin-1 (CS-1) is restricted to slow-twitch skeletal muscle fibres, whereas that of both calsarcin-2 (CS-2) and calsarcin-3 (CS-3) is enriched in fast-twitch fibres. However, the transcriptional control of this selective expression has not been previously elucidated. RESULTS: Our real-time RT-PCR analyses suggest that the expression of CS-1 and CS-2 is increased during the myogenic differentiation of mouse C2C12 cells. Promoter deletion analysis further suggests that an NF-kappaB binding site within the CS-1 promoter is responsible for the up-regulation of CS-1 transcription, but no similar mechanism was evident for CS-2. These findings are further supported by the results of EMSA analysis, as well as by overexpression and inhibition experiments in which NF-kappaB function was blocked by treatment with its inhibitor, PDTC. In addition, the overexpression of NFATc4 induces both the CS-1 and CS-2 promoters, whereas MEF2C only activates CS-1. CONCLUSION: Our present data suggest that NF-kappaB is required for the transcription of mouse CS-1 but not CS-2, and that the regulation of the calsarcins is mediated also by the NFAT and MEF2 transcription factors. These results provide new insights into the molecular mechanisms governing transcription in specific muscle fibre cells. The calsarcins may also serve as a valuable mechanistic tool to better understand the regulation of calcineurin signalling during muscle differentiation. [Abstract/Link to Full Text]

Du Z, Zhao D, Zhao Y, Wang S, Gao Y, Li N
Identification and characterization of bovine regulator of telomere length elongation helicase gene (RTEL): molecular cloning, expression distribution, splice variants and DNA methylation profile.
BMC Mol Biol. 2007;818.
BACKGROUND: The genetic basis of telomere length heterogeneity among mammalian species is still not well understood. Recently, a gene named regulator of telomere length elongation helicase (RTEL) was identified and predicted to be an essential participant in species-specific telomere length regulation in two murine species. To obtain broader insights into its structure and biological functions and to ascertain whether RTEL is also a candidate gene in the regulation of telomere length diversity in other mammalian species, data from other mammals may be helpful. RESULTS: Here we report the cDNA cloning, genomic structure, chromosomal location, alternative splicing pattern, expression distribution and DNA methylation profile of the bovine homolog of RTEL. The longest transcript of bovine RTEL is 4440 nt, encompassing 24.8 kb of genomic sequence that was mapped to chromosome 13q2.2. It encodes a conserved helicase-like protein containing seven characterized helicase motifs in the first 750 aa and a PIP box in the C-terminus. Four splice variants were identified within the transcripts in both the coding and 5'-untranslated regions; Western blot revealed that the most abundant splice variant SV-1 was translated to a truncated isoform of RTEL. The different 5'UTRs imply alternative transcription start sites in the promoter; Bovine RTEL was transcribed at the blastocyst stage, and expression levels were highest in adult testis, liver and ovary. DNA methylation analysis of tissues that differed significantly in expression level indicated that relatively low DNA methylation is associated with higher expression. CONCLUSION: In this study, we have identified and characterized a bovine RTEL homolog and obtained basic information about it, including gene structure, expression distribution, splice variants and profile of DNA methylation around two putative transcription start sites. These data may be helpful for further comparative and functional analysis of RTEL in mammals. [Abstract/Link to Full Text]

Guo Y, German TL, Schultz RD
A cryptic promoter in potato virus X vector interrupted plasmid construction.
BMC Mol Biol. 2007;817.
BACKGROUND: Potato virus X has been developed into an expression vector for plants. It is widely used to express foreign genes. In molecular manipulation, the foreign genes need to be sub-cloned into the vector. The constructed plasmid needs to be amplified. Usually, during amplification stage, the foreign genes are not expressed. However, if the foreign gene is expressed, the construction work could be interrupted. Two different viral genes were sub-cloned into the vector, but only one foreign gene was successfully sub-cloned. The other foreign gene, canine parvovirus type 2 (CPV-2) VP1 could not be sub-cloned into the vector and amplified without mutation (frame shift mutation). RESULTS: A cryptic promoter in the PVX vector was discovered with RT-PCR. The promoter activity was studied with Northern blots and Real-time RT-PCR. CONCLUSION: It is important to recognize the homologous promoter sequences in the vector when a virus is developed as an expression vector. During the plasmid amplification stage, an unexpected expression of the CPV-2 VP1 gene (not in the target plants, but in E. coli) can interrupt the downstream work. [Abstract/Link to Full Text]

Terwilliger DP, Buckley KM, Brockton V, Ritter NJ, Smith LC
Distinctive expression patterns of 185/333 genes in the purple sea urchin, Strongylocentrotus purpuratus: an unexpectedly diverse family of transcripts in response to LPS, beta-1,3-glucan, and dsRNA.
BMC Mol Biol. 2007;816.
BACKGROUND: A diverse set of transcripts called 185/333 is strongly expressed in sea urchins responding to immune challenge. Optimal alignments of full-length 185/333 cDNAs requires the insertion of large gaps that define 25 blocks of sequence called elements. The presence or absence of individual elements also defines a specific element pattern for each message. Individual sea urchins were challenged with pathogen associated molecular patterns (PAMPs) (lipopolysaccharide, beta-1,3-glucan, or double stranded RNA), and changes in the 185/333 message repertoire were followed over time. RESULTS: Each animal expressed a diverse set of 185/333 messages prior to challenge and a 0.96 kb message was the predominant size after challenge. Sequence analysis of the cloned messages indicated that the major element pattern expressed in immunoquiescent sea urchins was either C1 or E2.1. In contrast, most animals responding to lipopolysaccharide, beta-1,3-glucan or injury, predominantly expressed messages of the E2 pattern. In addition to the major patterns, extensive element pattern diversity was observed among the different animals before and after challenge. Nucleotide sequence diversity of the transcripts increased in response to beta-1,3-glucan, double stranded RNA and injury, whereas diversity decreased in response to LPS. CONCLUSION: These results illustrate that sea urchins appear to be able to differentiate among different PAMPs by inducing the transcription of different sets of 185/333 genes. Furthermore, animals may share a suite of 185/333 genes that are expressed in response to common pathogens, while also maintaining a large number of unique genes within the population. [Abstract/Link to Full Text]

Riccardo S, Tortoriello G, Giordano E, Turano M, Furia M
The coding/non-coding overlapping architecture of the gene encoding the Drosophila pseudouridine synthase.
BMC Mol Biol. 2007;815.
BACKGROUND: In eukaryotic cells, each molecule of H/ACA small nucleolar RNA (snoRNA) assembles with four evolutionarily conserved core proteins to compose a specific ribonucleoprotein particle. One of the four core components has pseudouridine synthase activity and catalyzes the conversion of a selected uridine to pseudouridine. Members of the pseudouridine synthase family are highly conserved. In addition to catalyzing pseudouridylation of target RNAs, they carry out a variety of essential functions related to ribosome biogenesis and, in mammals, to telomere maintenance. To investigate further the molecular mechanisms underlying the expression of pseudouridine synthase genes, we analyzed the transcriptional activity of the Drosophila member of this family in great detail. RESULTS: The Drosophila gene for pseudouridine synthase, minifly/Nop60b (mfl), encodes two novel mRNAs ending at a downstream poly(A) site. One species is characterized only by an extended 3'-untranslated region (3'UTR), while a minor mRNA encodes a variant protein that represents the first example of an alternative subform described for any member of the family to date. The rare spliced variant is detected mainly in females and is predicted to have distinct functional properties. We also report that a cluster comprising four isoforms of a C/D box snoRNA and two highly related copies of a small ncRNA gene of unknown function is intron-encoded at the gene-variable 3'UTRs. Because this arrangement, the alternative 3' ends allow mfl not only to produce two distinct protein subforms, but also to release different ncRNAs. Intriguingly, accumulation of all these intron-encoded RNAs was found to be sex-biased and quantitatively modulated throughout development and, within the ovaries, the ncRNAs of unknown function were found not ubiquitously expressed. CONCLUSION: Our results expand the repertoire of coding/non-coding transcripts derived from the gene encoding Drosophila pseudouridine synthase. This gene exhibits a complex and interlaced organization, and its genetic information may be expressed as different protein subforms and/or ncRNAs that may potentially contribute to its biological functions. [Abstract/Link to Full Text]

Chakrabarty A, Roberts MR
Ets-2 and C/EBP-beta are important mediators of ovine trophoblast Kunitz domain protein-1 gene expression in trophoblast.
BMC Mol Biol. 2007;814.
BACKGROUND: The trophoblast Kunitz domain proteins (TKDPs) constitute a highly expressed, placenta-specific, multigene family restricted to ruminant ungulates and characterized by a C-terminal "Kunitz" domain, preceded by one or more unique N-terminal domains. TKDP-1 shares an almost identical expression pattern with interferon-tau, the "maternal recognition of pregnancy protein" in ruminants. Our goal here has been to determine whether the ovine (ov) Tkdp-1 and IFNT genes possess a similar transcriptional code. RESULTS: The ovTkdp-1 promoter has been cloned and characterized. As with the IFNT promoter, the Tkdp-1 promoter is responsive to Ets-2, and promoter-driven reporter activity can be increased over 700-fold in response to over-expression of Ets-2 and a constitutively active form of protein Kinase A (PKA). Unexpectedly, the promoter element of Tkdp-1 responsible for this up-regulation, unlike that of the IFNT, does not bind Ets-2. However, mutation of a CCAAT/enhancer binding element within this control region not only reduced basal transcriptional activity, but prevented Ets-2 as well as cyclic adenosine 5'-monophosphate (cAMP)/PKA and Ras/mitogen-activated protein kinase (MAPK) responsiveness. In vitro binding experiments and in vivo protein-protein interaction assays implicated CCAAT/enhancer binding protein-beta (C/EBP-beta) as involved in up-regulating the Tkdp-1 promoter activity. A combination of Ets-2 and C/EBP-beta can up-regulate expression of the minimal Tkdp-1 promoter as much as 930-fold in presence of a cAMP analog. An AP-1-like element adjacent to the CCAAT enhancer, which binds Jun family members, is required for basal and cAMP/ C/EBP-beta-dependent activation of the gene, but not for Ets-2-dependent activity. CONCLUSION: This paper demonstrates how Ets-2, a key transcription factor for trophoblast differentiation and function, can control expression of two genes (Tkdp-1 and IFNT) having similar spatial and temporal expression patterns via very different mechanisms. [Abstract/Link to Full Text]

Toegel S, Huang W, Piana C, Unger FM, Wirth M, Goldring MB, Gabor F, Viernstein H
Selection of reliable reference genes for qPCR studies on chondroprotective action.
BMC Mol Biol. 2007;813.
BACKGROUND: Chondroprotective agents (CPA) such as glucosamine, curcumin and diacerein represent potential remedies for the management of osteoarthritis and several studies have been performed on their effects in-vitro and in-vivo. For the investigation of chondroprotective action on chondrocyte gene expression, quantitative real-time RT-PCR is the method of choice. However, validation of applied normalization strategies represents a crucial and sometimes neglected step in the analysis process. Therefore, the present study aimed to determine the expression stability of common reference genes (ACTB, Beta actin; GAPDH, Glyceraldehyde-3-phosphate; B2M, Beta-2-microglobulin; HPRT1, Hypoxanthine phosphoribosyl-transferase I; SDHA, Succinate dehydrogenase complex, subunit A; YWHAZ, Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) under the influence of glucosamine, curcumin and diacerein in the IL-1beta-stimulated C-28/I2 chondrocyte model, using the geNorm software tool. RESULTS: CPA treatment of C-28/I2 chondrocytes significantly affected the expression level of many reference genes (p < 0.05). According to their expression stability, geNorm analysis revealed rankings of the 3 most stable genes (from most stable to least stable) as follows: GAPDH, B2M and SDHA in glucosamine treated samples and HPRT1, GAPDH and B2M in curcumin or diacerein treated samples. Interestingly, ACTB was one of the most variably expressed genes throughout all experiments. CONCLUSION: Our study points out the problem of relying on commonly used reference genes without an accurate validation process. For normalization purposes in gene profiling studies on glucosamine action, the genes GAPDH, B2M and SDHA are recommended as single reference genes depending on the expression level of the target gene or more favourably in combination. For experiments with curcumin and diacerein the use of HPRT1, GAPDH and B2M should be considered. [Abstract/Link to Full Text]

Stewart D, Javadi M, Chambers M, Gunsolly C, Gorski G, Borghaei RC
Interleukin-4 inhibition of interleukin-1-induced expression of matrix metalloproteinase-3 (MMP-3) is independent of lipoxygenase and PPARgamma activation in human gingival fibroblasts.
BMC Mol Biol. 2007;812.
BACKGROUND: Interleukin 4 (IL-4) has been shown to suppress interleukin-1 (IL-1) induced expression of matrix metalloproteinase-3 (MMP-3) in human synovial and gingival fibroblasts, but the mechanism of suppression has not been determined. Activators of peroxisome proliferator-activated receptor-gamma (PPARgamma) have been shown to inhibit cytokine induced expression of MMPs in other cell types, and IL-4 has been shown to activate PPARgamma by stimulating production of ligands through the lipoxygenase pathway. It has been suggested that PPARgamma may inhibit expression of MMPs by competing with transcription factor AP-1 for binding to a putative composite binding element in the promoters. The objective of this study was to determine whether the suppressive effects of IL-4 on the IL-1 induced expression of MMP-3 involve activation of lipoxygenase and/or PPARgamma. RESULTS: Western blotting revealed the presence of PPARgamma in nuclear extract of HGF. IL-1 induced binding of nuclear extract to the putative composite PPRE/AP-1 site was diminished in the presence of pioglitazone, but there was no evidence of any change in the composition of the retarded complexes, and no evidence of PPARgamma binding to this site. Nordihydroguaiaretic acid (NDGA), a non-selective lipoxygenase inhibitor, and MK886, a specific inhibitor of 5-lipoxygenase, induced MMP-3 expression synergistically with IL-1. However IL-4 was still able to inhibit MMP-3 expression in the presence of NDGA or MK886 and IL-1. Activation of PPARgamma with pioglitazone not only failed to inhibit IL-1 induced expression of MMP-3 mRNA, but rather super-induced MMP-3 in the presence of IL-1. PPARgamma antagonist GW9662 failed to abolish the suppressive effects of IL-4. Another PPARgamma activator, 15-deoxy-Delta12,14prostaglandin J2 (15dPGJ2), also super-induced MMP-3 mRNA, and this was due at least in part to increased transcription. CONCLUSION: IL-4 suppression of IL-1-induced MMP-3 expression in HGF is independent of lipoxygenase activity and activation of PPARgamma. Super-induction of MMP-3 by pioglitazone may have important implications for patients using pioglitazone to treat type II diabetes in the presence of chronic inflammation. [Abstract/Link to Full Text]

Willenbrock H, Ussery DW
Prediction of highly expressed genes in microbes based on chromatin accessibility.
BMC Mol Biol. 2007;811.
BACKGROUND: It is well known that gene expression is dependent on chromatin structure in eukaryotes and it is likely that chromatin can play a role in bacterial gene expression as well. Here, we use a nucleosomal position preference measure of anisotropic DNA flexibility to predict highly expressed genes in microbial genomes. We compare these predictions with those based on codon adaptation index (CAI) values, and also with experimental data for 6 different microbial genomes, with a particular interest in experimental data from Escherichia coli. Moreover, position preference is examined further in 328 sequenced microbial genomes. RESULTS: We find that absolute gene expression levels are correlated with the position preference in many microbial genomes. It is postulated that in these regions, the DNA may be more accessible to the transcriptional machinery. Moreover, ribosomal proteins and ribosomal RNA are encoded by DNA having significantly lower position preference values than other genes in fast-replicating microbes. CONCLUSION: This insight into DNA structure-dependent gene expression in microbes may be exploited for predicting the expression of non-translated genes such as non-coding RNAs that may not be predicted by any of the conventional codon usage bias approaches. [Abstract/Link to Full Text]

Filby AL, Tyler CR
Appropriate 'housekeeping' genes for use in expression profiling the effects of environmental estrogens in fish.
BMC Mol Biol. 2007;810.
BACKGROUND: Attempts to develop a mechanistic understanding of the effects of environmental estrogens on fish are increasingly conducted at the level of gene expression. Appropriate application of real-time PCR in such studies requires the use of a stably expressed 'housekeeping' gene as an internal control to normalize for differences in the amount of starting template between samples. RESULTS: We sought to identify appropriate genes for use as internal controls in experimental treatments with estrogen by analyzing the expression of eight functionally distinct 'housekeeping' genes (18S ribosomal RNA [18S rRNA], ribosomal protein l8 [rpl8], elongation factor 1 alpha [ef1a], glucose-6-phosphate dehydrogenase [g6pd], beta actin [bactin], glyceraldehyde-3-phosphate dehydrogenase [gapdh], hypoxanthine phosphoribosyltransferase 1 [hprt1], and tata box binding protein [tbp]) following exposure to the environmental estrogen, 17alpha-ethinylestradiol (EE2), in the fathead minnow (Pimephales promelas). Exposure to 10 ng/L EE2 for 21 days down-regulated the expression of ef1a, g6pd, bactin and gapdh in the liver, and bactin and gapdh in the gonad. Some of these effects were gender-specific, with bactin in the liver and gapdh in the gonad down-regulated by EE2 in males only. Furthermore, when ef1a, g6pd, bactin or gapdh were used for normalization, the hepatic expression of two genes of interest, vitellogenin (vtg) and cytochrome P450 1A (cyp1a) following exposure to EE2 was overestimated. CONCLUSION: Based on the data presented, we recommend 18S rRNA, rpl8, hprt1 and/or tbp, but not ef1a, g6pd, bactin and/or gapdh, as likely appropriate internal controls in real-time PCR studies of estrogens effects in fish. Our studies show that pre-validation of control genes considering the scope and nature of the experiments to be performed, including both gender and tissue type, is critical for accurate assessments of the effects of environmental estrogens on gene expression in fish. [Abstract/Link to Full Text]

Engstrom JU, Kmiec EB
Manipulation of cell cycle progression can counteract the apparent loss of correction frequency following oligonucleotide-directed gene repair.
BMC Mol Biol. 2007;89.
BACKGROUND: Single-stranded oligonucleotides (ssODN) are used routinely to direct specific base alterations within mammalian genomes that result in the restoration of a functional gene. Despite success with the technique, recent studies have revealed that following repair events, correction frequencies decrease as a function of time, possibly due to a sustained activation of damage response signals in corrected cells that lead to a selective stalling. In this study, we use thymidine to slow down the replication rate to enhance repair frequency and to maintain substantial levels of correction over time. RESULTS: First, we utilized thymidine to arrest cells in G1 and released the cells into S phase, at which point specific ssODNs direct the highest level of correction. Next, we devised a protocol in which cells are maintained in thymidine following the repair reaction, in which the replication is slowed in both corrected and non-corrected cells and the initial correction frequency is retained. We also present evidence that cells enter a senescence state upon prolonged treatment with thymidine but this passage can be avoided by removing thymidine at 48 hours. CONCLUSION: Taken together, we believe that thymidine may be used in a therapeutic fashion to enable the maintenance of high levels of treated cells bearing repaired genes. [Abstract/Link to Full Text]

Lennard ML, Hikima J, Ross DA, Kruiswijk CP, Wilson MR, Miller NW, Warr GW
Characterization of an Oct1 orthologue in the channel catfish, Ictalurus punctatus: a negative regulator of immunoglobulin gene transcription?
BMC Mol Biol. 2007;88.
BACKGROUND: The enhancer (Emu3') of the immunoglobulin heavy chain locus (IGH) of the channel catfish (Ictalurus punctatus) has been well characterized. The functional core region consists of two variant Oct transcription factor binding octamer motifs and one E-protein binding muE5 site. An orthologue to the Oct2 transcription factor has previously been cloned in catfish and is a functionally active transcription factor. This study was undertaken to clone and characterize the Oct1 transcription factor, which has also been shown to be important in driving immunoglobulin gene transcription in mammals. RESULTS: An orthologue of Oct1, a POU family transcription factor, was cloned from a catfish macrophage cDNA library. The inferred amino acid sequence of the catfish Oct1, when aligned with other vertebrate Oct1 sequences, revealed clear conservation of structure, with the POU specific subdomain of catfish Oct1 showing 96% identity to that of mouse Oct1. Expression of Oct1 was observed in clonal T and B cell lines and in all tissues examined. Catfish Oct1, when transfected into both mammalian (mouse) and catfish B cell lines, unexpectedly failed to drive transcription from three different octamer-containing reporter constructs. These contained a trimer of octamer motifs, a fish VH promoter, and the core region of the catfish Emu3' IGH enhancer, respectively. This failure of catfish Oct1 to drive transcription was not rescued by human BOB.1, a co-activator of Oct transcription factors that stimulates transcription driven by catfish Oct2. When co-transfected with catfish Oct2, Oct1 reduced Oct2 driven transcriptional activation. Electrophoretic mobility shift assays showed that catfish Oct1 (native or expressed in vitro) bound both consensus and variant octamer motifs. Putative N- and C-terminal activation domains of Oct1, when fused to a Gal4 DNA binding domain and co-transfected with Gal4-dependent reporter constructs were transcriptionally inactive, which may be due in part to a lack of residues associated with activation domain function. CONCLUSION: An orthologue to mammalian Oct1 has been found in the catfish. It is similar to mammalian Oct1 in structure and expression. However, these results indicate that the physiological functions of catfish Oct1 differ from those of mammalian Oct1 and include negative regulation of transcription. [Abstract/Link to Full Text]

Schwartz TR, Kmiec EB
Reduction of gene repair by selenomethionine with the use of single-stranded oligonucleotides.
BMC Mol Biol. 2007;87.
BACKGROUND: The repair of single base mutations in mammalian genes can be directed by single-stranded oligonucleotides in a process known as targeted gene repair. The mechanism of this reaction is currently being elucidated but likely involves a pairing step in which the oligonucleotide align in homologous register with its target sequence and a correction step in which the mutant base is replaced by endogenous repair pathways. This process is regulated by the activity of various factors and proteins that either elevate or depress the frequency at which gene repair takes place. RESULTS: In this report, we find that addition of selenomethionine reduces gene repair frequency in a dose-dependent fashion. A correlation between gene repair and altered cell cycle progression is observed. We also find that selenium induces expression of Ref-1 which, in turn, modifies the activity of p53 during the cell cycle. CONCLUSION: We can conclude from the results that the suppression of gene repair by introduction of selenomethionine occurs through a p53-associated pathway. This result indicates that the successful application of gene repair for treatment of inherited disorders may be hampered by indirect activation of endogenous suppressor functions. [Abstract/Link to Full Text]

Marshall KS, Zhang Z, Curran J, Derbyshire S, Mymryk JS
An improved genetic system for detection and analysis of protein nuclear import signals.
BMC Mol Biol. 2007;86.
BACKGROUND: Nuclear import of proteins is typically mediated by their physical interaction with soluble cytosolic receptor proteins via a nuclear localization signal (NLS). A simple genetic assay to detect active NLSs based on their function in the yeast Saccharomyces cerevisiae has been previously described. In that system, a chimera consisting of a modified bacterial LexA DNA binding domain and the transcriptional activation domain of the yeast Gal4 protein is fused to a candidate NLS. A functional NLS will redirect the chimeric fusion to the yeast cell nucleus and activate transcription of a reporter gene. RESULTS: We have reengineered this nuclear import system to expand its utility and tested it using known NLS sequences from adenovirus E1A. Firstly, the vector has been reconstructed to reduce the level of chimera expression. Secondly, an irrelevant "stuffer" sequence from the E. coli maltose binding protein was used to increase the size of the chimera above the passive diffusion limit of the nuclear pore complex. The improved vector also contains an expanded multiple cloning site and a hemagglutinin epitope tag to allow confirmation of expression. CONCLUSION: The alterations in expression level and composition of the fusions used in this nuclear import system greatly reduce background activity in beta-galactosidase assays, improving sensitivity and allowing more quantitative analysis of NLS bearing sequences. [Abstract/Link to Full Text]

Sporstřl M, Mousavi SA, Eskild W, Roos N, Berg T
ABCA1, ABCG1 and SR-BI: hormonal regulation in primary rat hepatocytes and human cell lines.
BMC Mol Biol. 2007;85.
BACKGROUND: Scavenger receptor type B class I (SR-BI), ABC transporter A1 (ABCA1) -and G1 (ABCG1) all play important roles in the reverse cholesterol transport. Reverse cholesterol transport is a mechanism whereby the body can eliminate excess cholesterol. Here, the regulation of SR-BI, ABCA1, and ABCG1 by dexamethasone (a synthetic glucocorticoid) and insulin were studied in order to gain more insight into the role of these two hormones in the cholesterol metabolism. RESULTS: By use of real time RT-PCR and Western blotting we examined the expression of our target genes. The results show that SR-BI, ABCA1 and ABCG1 mRNA expression increased in response to dexamethasone while insulin treatment reduced the expression in primary rat hepatocytes. The stimulatory effect of dexamethasone was reduced by the addition of the anti-glucocorticoid mifepristone. In HepG2 cells and THP-1 macrophages, however, the effect of dexamethasone was absent or inhibitory with no significant change in the presence of mifepristone. The latter observation may be a result of the low protein expression of glucocorticoid receptor (GR) in these cell lines. CONCLUSION: Our results illustrates that insulin and glucocorticoids, two hormones crucial in the carbohydrate metabolism, also play an important role in the regulation of genes central in reverse cholesterol transport. We found a marked difference in mRNA expression between the primary cells and the two established cell lines when studying the effect of dexamethasone which may result from the varying expression levels of GR. [Abstract/Link to Full Text]

Kar AK, Bhattacharya B, Roy P
Bluetongue virus RNA binding protein NS2 is a modulator of viral replication and assembly.
BMC Mol Biol. 2007;84.
BACKGROUND: Bluetongue virus (BTV) particles consist of seven structural proteins that are organized into two capsids. In addition, BTV also encodes three non-structural (NS) proteins of which protein 2 (NS2) is the RNA binding protein and is also the major component of virus encoded inclusion bodies (VIBs), which are believed to be virus assembly sites. To investigate the contribution of NS2 in virus replication and assembly we have constructed inducible mammalian cell lines expressing full-length NS2. In addition, truncated NS2 fragments were also generated in an attempt to create dominant negative mutants for NS2 function. RESULTS: Our data revealed that expression of full-length NS2 was sufficient for the formation of inclusion bodies (IBs) that were morphologically similar to the VIBs formed during BTV infection. By using either, individual BTV proteins or infectious virions, we found that while the VP3 of the inner capsid (termed as "core") that surrounds the transcription complex was closely associated with both NS2 IBs and BTV VIBs, the surface core protein VP7 co-localized with NS2 IBs only in the presence of VP3. In contrast to the inner core proteins, the outer capsid protein VP2 was not associated with either IBs or VIBs. Like the core proteins, newly synthesized BTV RNAs also accumulated in VIBs. Unlike full-length NS2, neither the amino-, nor carboxyl-terminal fragments formed complete IB structures and each appeared to interfere in overall virus replication when similarly expressed. CONCLUSION: Together, these data demonstrate that NS2 is sufficient and necessary for IB formation and a key player in virus replication and core assembly. Perturbation of NS2 IB formation resulted in reduced virus synthesis and both the N terminal (NS2-1) and C terminal (NS2-2) fragments act as dominant negative mutants of NS2 function. [Abstract/Link to Full Text]

Khoo B, Roca X, Chew SL, Krainer AR
Antisense oligonucleotide-induced alternative splicing of the APOB mRNA generates a novel isoform of APOB.
BMC Mol Biol. 2007;83.
BACKGROUND: Apolipoprotein B (APOB) is an integral part of the LDL, VLDL, IDL, Lp(a) and chylomicron lipoprotein particles. The APOB pre-mRNA consists of 29 constitutively-spliced exons. APOB exists as two natural isoforms: the full-length APOB100 isoform, assembled into LDL, VLDL, IDL and Lp(a) and secreted by the liver in humans; and the C-terminally truncated APOB48, assembled into chylomicrons and secreted by the intestine in humans. Down-regulation of APOB100 is a potential therapy to lower circulating LDL and cholesterol levels. RESULTS: We investigated the ability of 2'O-methyl RNA antisense oligonucleotides (ASOs) to induce the skipping of exon 27 in endogenous APOB mRNA in HepG2 cells. These ASOs are directed towards the 5' and 3' splice-sites of exon 27, the branch-point sequence (BPS) of intron 26-27 and several predicted exonic splicing enhancers within exon 27. ASOs targeting either the 5' or 3' splice-site, in combination with the BPS, are the most effective. The splicing of other alternatively spliced genes are not influenced by these ASOs, suggesting that the effects seen are not due to non-specific changes in alternative splicing. The skip 27 mRNA is translated into a truncated isoform, APOB87SKIP27. CONCLUSION: The induction of APOB87SKIP27 expression in vivo should lead to decreased LDL and cholesterol levels, by analogy to patients with hypobetalipoproteinemia. As intestinal APOB mRNA editing and APOB48 expression rely on sequences within exon 26, exon 27 skipping should not affect APOB48 expression unlike other methods of down-regulating APOB100 expression which also down-regulate APOB48. [Abstract/Link to Full Text]

Chen PY, Chang WS, Chou RH, Lai YK, Lin SC, Chi CY, Wu CW
Two non-homologous brain diseases-related genes, SERPINI1 and PDCD10, are tightly linked by an asymmetric bidirectional promoter in an evolutionarily conserved manner.
BMC Mol Biol. 2007;82.
BACKGROUND: Despite of the fact that mammalian genomes are far more spacious than prokaryotic genomes, recent nucleotide sequencing data have revealed that many mammalian genes are arranged in a head-to-head orientation and separated by a small intergenic sequence. Extensive studies on some of these neighboring genes, in particular homologous gene pairs, have shown that these genes are often co-expressed in a symmetric manner and regulated by a shared promoter region. Here we report the identification of two non-homologous brain disease-related genes, with one coding for a serine protease inhibitor (SERPINI1) and the other for a programmed cell death-related gene (PDCD10), being tightly linked together by an asymmetric bidirectional promoter in an evolutionarily conserved fashion. This asymmetric bidirectional promoter, in cooperation with some cis-acting elements, is responsible for the co-regulation of the gene expression pattern as well as the tissue specificity of SERPINI1 and PDCD10. RESULTS: While SERPINI1 is predominantly expressed in normal brain and down-regulated in brain tumors, PDCD10 is ubiquitously expressed in all normal tissues but its gene transcription becomes aberrant in different types of cancers. By measuring the luciferase activity in various cell lysates, their 851-bp intergenic sequence was shown to be capable of driving the reporter gene expression in either direction. A 175-bp fragment from nt 1 to 175 in the vicinity of PDCD10 was further determined to function as a minimal bidirectional promoter. A critical regulatory fragment, from nt 176-473 outside the minimal promoter in the intergenic region, was identified to contain a strong repressive element for SERPINI1 and an enhancer for PDCD10. These cis-acting elements may exist to help coordinate the expression and regulation of the two flanking genes. CONCLUSION: For all non-homologous genes that have been described to be closely adjacent in the mammalian genomes, the intergenic region of the head-to-head PDCD10-SERPINI1 gene pair provides an interesting and informative example of a complex regulatory system that governs the expression of both genes not only through an asymmetric bidirectional promoter, but also through fine-tuned regulations with some cis-acting elements. [Abstract/Link to Full Text]

Fromme T, Reichwald K, Platzer M, Li XS, Klingenspor M
Chicken ovalbumin upstream promoter transcription factor II regulates uncoupling protein 3 gene transcription in Phodopus sungorus.
BMC Mol Biol. 2007;81.
BACKGROUND: Ucp3 is an integral protein of the inner mitochondrial membrane with a role in lipid metabolism preventing deleterious effects of fatty acids in states of high lipid oxidation. Ucp3 is expressed in brown adipose tissue and skeletal muscle and controlled by a transcription factor complex including PPARalpha, MyoD and the histone acetyltransferase p300. Several studies have demonstrated interaction of these factors with chicken ovalbumin upstream promoter transcription factor II (Coup-TFII). This nuclear receptor is involved in organogenesis and other developmental processes including skeletal muscle development, but also co-regulates a number of metabolic genes. In this study we in silico analyzed the upstream region of Ucp3 of the Djungarian hamster Phodopus sungorus and identified several putative response elements for Coup-TFII. We therefore investigated whether Coup-TFII is a further player in the transcriptional control of the Ucp3 gene in rodents. RESULTS: By quantitative PCR we demonstrated a positive correlation of Coup-TFII and Ucp3 mRNA expression in skeletal muscle and brown adipose tissue in response to food deprivation and cold exposure, respectively. In reporter gene assays Coup-TFII enhanced transactivation of the Ucp3 promoter conveyed by MyoD, PPARalpha, RXRalpha and/or p300. Using deletions and mutated constructs, we identified a Coup-TFII enhancer element 816-840 bp upstream of the transcriptional start site. Binding of Coup-TFII to this upstream enhancer was confirmed in electrophoretic mobility shift and supershift assays. CONCLUSION: Transcriptional regulation of the Coup-TFII gene in response to starvation and cold exposure seems to be the regulatory mechanism of Ucp3 mRNA expression in brown adipose and skeletal muscle tissue determining the final appropriate rate of transcript synthesis. These findings add a crucial component to the complex transcriptional machinery controlling expression of Ucp3. Given the substantial evidence for a function of Ucp3 in lipid metabolism, Coup-TFII may not only be a negative regulator of glucose responsive genes but also transactivate genes involved in lipid metabolism. [Abstract/Link to Full Text]

Grote J, König S, Ackermann D, Sopalla C, Benedyk M, Los M, Kerkhoff C
Identification of poly(ADP-ribose)polymerase-1 and Ku70/Ku80 as transcriptional regulators of S100A9 gene expression.
BMC Mol Biol. 2006;748.
BACKGROUND: S100 proteins, a multigenic family of non-ubiquitous cytoplasmic Ca2+-binding proteins, have been linked to human pathologies in recent years. Dysregulated expression of S100 proteins, including S100A9, has been reported in the epidermis as a response to stress and in association with neoplastic disorders. Recently, we characterized a regulatory element within the S100A9 promotor, referred to as MRE that drives the S100A9 gene expression in a cell type-specific, activation- and differentiation-dependent manner (Kerkhoff et al. (2002) J. Biol. Chem. 277, 41879-41887). RESULTS: In the present study, we investigated transcription factors that bind to MRE. Using the MRE motif for a pull-down assay, poly(ADP-ribose)polymerase-1 (PARP-1) and the heterodimeric complex Ku70/Ku80 were identified by mass spectrometry and confirmed by chromatin immunoprecipitation. Furthermore, TPA-induced S100A9 gene expression in HaCaT keratinocytes was blocked after the pharmacologic inhibition of PARP-1 with 1,5-isoquinolinediol (DiQ). CONCLUSION: The candidates, poly(ADP-ribose)polymerase-1 (PARP-1) and the heterodimeric complex Ku70/Ku80, are known to participate in inflammatory disorders as well as tumorgenesis. The latter may indicate a possible link between S100 and inflammation-associated cancer. [Abstract/Link to Full Text]

Vultos TD, Méderlé I, Abadie V, Pimentel M, Moniz-Pereira J, Gicquel B, Reyrat JM, Winter N
Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNAala T-loops in slow- and fast-growing mycobacteria.
BMC Mol Biol. 2006;747.
BACKGROUND: Mycobacteriophage Ms6 integrates into Mycobacterium smegmatis and M. bovis BCG chromosome at the 3' end of tRNAala genes. Homologous recombination occurs between the phage attP core and the attB site located in the T-loop. Integration-proficient vectors derived from Ms6 are useful genetic tools, but their insertion sites in the BCG chromosome remain poorly defined. The primary objective of this study was to identify Ms6 target genes in M. smegmatis and BCG. We then aimed to modify the attP site in Ms6-derived vectors, to switch integration to other tRNAala loci. This provided the basis for the development of recombinant M. bovis BCG strains expressing several reporter genes inserted into different tRNAala genes. RESULTS: The three tRNAala genes are highly conserved in M. smegmatis and BCG. However, in the T-loop of tRNAalaU and tRNAalaV containing the attB site, a single base difference was observed between the two species. We observed that the tRNAalaU gene was the only site into which Ms6-derived integration-proficient vectors integrated in M. smegmatis, whereas in BCG, the tRNAalaV gene was used as the target. No integration occurred in the BCG tRNAalaU T-loop, despite a difference of only one base from the 26-base Ms6 attP core. We mutated the attP core to give a perfect match with the other tRNAala T-loops from M. smegmatis and BCG. Modification of the seven-base T-loop decreased integration efficiency, identifying this site as a possible site of strand exchange. Finally, two Ms6 vectors were constructed to integrate two reporter genes into the tRNAalaU and tRNAalaV T-loops of the same BCG chromosome. CONCLUSION: Small changes in the 7 bp T-loop attP site of Ms6 made it possible to use another attB site, albeit with a lower integration efficiency. These molecular studies on BCG tRNAala genes made it possible to create valuable tools for the site-directed insertion of several genes in the same BCG strain. These tools will be useful for the development of novel multivalent vaccines and genetic studies. [Abstract/Link to Full Text]

Magnani E, Bartling L, Hake S
From Gateway to MultiSite Gateway in one recombination event.
BMC Mol Biol. 2006;746.
BACKGROUND: Invitrogen Gateway technology exploits the integrase/att site-specific recombination system for directional cloning of PCR products and the subsequent subcloning into destination vectors. One or three DNA segments can be cloned using Gateway or MultiSite Gateway respectively. A vast number of single-site Gateway destination vectors have been created while MultiSite Gateway is limited to few destination vectors and therefore to few applications. The aim of this work was to make the MultiSite Gateway technology available for multiple biological purposes. RESULTS: We created a construct, pDONR-R4-R3, to easily convert any available Gateway destination vector to a MultiSite Gateway vector in a single recombination reaction. In addition, we designed pDONR-R4-R3 so that DNA fragments already cloned upstream or downstream of the Gateway cassette in the original destination vectors can still be utilized for promoter-gene or translational fusions after the conversion. CONCLUSION: Our tool makes MultiSite Gateway a more widely accessible technology and expands its applications by exploiting all the features of the Gateway vectors already available. [Abstract/Link to Full Text]

Liddiard K, Welch JS, Lozach J, Heinz S, Glass CK, Greaves DR
Interleukin-4 induction of the CC chemokine TARC (CCL17) in murine macrophages is mediated by multiple STAT6 sites in the TARC gene promoter.
BMC Mol Biol. 2006;745.
BACKGROUND: Macrophages (Mtheta) play a central role in the innate immune response and in the pathology of chronic inflammatory diseases. Macrophages treated with Th2-type cytokines such as Interleukin-4 (IL-4) and Interleukin-13 (IL-13) exhibit an altered phenotype and such alternatively activated macrophages are important in the pathology of diseases characterised by allergic inflammation including asthma and atopic dermatitis. The CC chemokine Thymus and Activation-Regulated Chemokine (TARC/CCL17) and its murine homologue (mTARC/ABCD-2) bind to the chemokine receptor CCR4, and direct T-cell and macrophage recruitment into areas of allergic inflammation. Delineating the molecular mechanisms responsible for the IL-4 induction of TARC expression will be important for a better understanding of the role of Th2 cytokines in allergic disease. RESULTS: We demonstrate that mTARC mRNA and protein are potently induced by the Th2 cytokine, Interleukin-4 (IL-4), and inhibited by Interferon-gamma (IFN-gamma) in primary macrophages (Mtheta). IL-4 induction of mTARC occurs in the presence of PI3 kinase pathway and translation inhibitors, but not in the absence of STAT6 transcription factor, suggesting a direct-acting STAT6-mediated pathway of mTARC transcriptional activation. We have functionally characterised eleven putative STAT6 sites identified in the mTARC proximal promoter and determined that five of these contribute to the IL-4 induction of mTARC. By in vitro binding assays and transient transfection of isolated sites into the RAW 264.7 Mtheta cell-line, we demonstrate that these sites have widely different capacities for binding and activation by STAT6. Site-directed mutagenesis of these sites within the context of the mTARC proximal promoter revealed that the two most proximal sites, conserved between the human and mouse genes, are important mediators of the IL-4 response. CONCLUSION: The induction of mTARC by IL-4 results from cooperative interactions between STAT6 sites within the mTARC gene promoter. Significantly, we have shown that transfer of the nine most proximal mTARC STAT6 sites in their endogenous conformation confers potent (up to 130-fold) IL-4 inducibility on heterologous promoters. These promoter elements constitute important and sensitive IL-4-responsive transcriptional units that could be used to drive transgene expression in sites of Th2 inflammation in vivo. [Abstract/Link to Full Text]

Poidevin L, MacNeill SA
Biochemical characterisation of LigN, an NAD+-dependent DNA ligase from the halophilic euryarchaeon Haloferax volcanii that displays maximal in vitro activity at high salt concentrations.
BMC Mol Biol. 2006;744.
BACKGROUND: DNA ligases are required for DNA strand joining in all forms of cellular life. NAD+-dependent DNA ligases are found primarily in eubacteria but also in some eukaryotic viruses, bacteriophage and archaea. Among the archaeal NAD+-dependent DNA ligases is the LigN enzyme of the halophilic euryarchaeon Haloferax volcanii, the gene for which was apparently acquired by Hfx. volcanii through lateral gene transfer (LGT) from a halophilic eubacterium. Genetic studies show that the LGT-acquired LigN enzyme shares an essential function with the native Hfx. volcanii ATP-dependent DNA ligase protein LigA. RESULTS: To characterise the enzymatic properties of the LigN protein, wild-type and three mutant forms of the LigN protein were separately expressed in recombinant form in E.coli and purified to apparent homogeneity by immobilised metal ion affinity chromatography (IMAC). Non-isotopic DNA ligase activity assays using lambda DNA restriction fragments with 12 bp cos cohesive ends were used to show that LigN activity was dependent on addition of divalent cations and salt. No activity was detected in the absence of KCl, whereas maximum activity could be detected at 3.2 M KCl, close to the intracellular KCl concentration of Hfx. volcanii cells. CONCLUSION: LigN is unique amongst characterised DNA ligase enzymes in displaying maximal DNA strand joining activity at high (> 3 M) salt levels. As such the LigN enzyme has potential both as a novel tool for biotechnology and as a model enzyme for studying the adaptation of proteins to high intracellular salt levels. [Abstract/Link to Full Text]

Shin JH, Kelman Z
DNA unwinding assay using streptavidin-bound oligonucleotides.
BMC Mol Biol. 2006;743.
BACKGROUND: Helicases play essential roles in many cellular processes including replication, transcription and translation. Most helicases translocate along one strand of the duplex while displacing the complementary strand (of either DNA or RNA). Thus, helicases have directionality. They move along nucleic acids in either the 3'--> 5' or 5'--> 3' direction. The directionality of helicases with low activity or of those that cannot initiate duplex unwinding from a substrate that contains only one single-stranded overhang region is difficult to determine. RESULTS: An improved assay to determine helicase directionality was developed that uses a substrate containing biotinylated oligonucleotides. As a proof of concept, it was shown that the substrates substantially improve helicase activity and directionality determination for several DNA helicases in comparison to more traditional substrates. In addition, a universal substrate that can be used to determine the directionality of both 3'--> 5' and 5'--> 3' helicases was developed. CONCLUSION: It is shown here that the use of a biotin-streptavidin complex as a helicase substrate improves helicase activity and the determination of helicase directionality. The method described is simpler that the currently available techniques. [Abstract/Link to Full Text]

Denise H, Poot J, Jiménez M, Ambit A, Herrmann DC, Vermeulen AN, Coombs GH, Mottram JC
Studies on the CPA cysteine peptidase in the Leishmania infantum genome strain JPCM5.
BMC Mol Biol. 2006;742.
BACKGROUND: Visceral leishmaniasis caused by members of the Leishmania donovani complex is often fatal in the absence of treatment. Research has been hampered by the lack of good laboratory models and tools for genetic manipulation. In this study, we have characterised a L. infantum line (JPCM5) that was isolated from a naturally infected dog and then cloned. We found that JPCM5 has attributes that make it an excellent laboratory model; different stages of the parasite life cycle can be studied in vitro, it is accessible to genetic manipulation and it has retained its virulence. Furthermore, the L. infantum JPCM5 genome has now been fully sequenced. RESULTS: We have further focused our studies on LiCPA, the L. infantum homologue to L. mexicana cysteine peptidase CPA. LiCPA was found to share a high percentage of amino acid identity with CPA proteins of other Leishmania species. Two independent LiCPA-deficient promastigote clones (DeltaLicpa) were generated and their phenotype characterised. In contrast to L. mexicana CPA-deficient mutants, both clones of DeltaLicpa were found to have significantly reduced virulence in vitro and in vivo. Re-expression of just one LiCPA allele (giving DeltaLicpa::CPA) was sufficient to complement the reduced infectivity of both DeltaLicpa mutants for human macrophages, which confirms the importance of LiCPA for L. infantum virulence. In contrast, in vivo experiments did not show any virulence recovery of the re-expressor clone DeltaLicpaC1::CPA compared with the CPA-deficient mutant DeltaLicpaC1. CONCLUSION: The data suggest that CPA is not essential for replication of L. infantum promastigotes, but is important for the host-parasite interaction. Further studies will be necessary to elucidate the precise roles that LiCPA plays and why the re-expression of LiCPA in the DeltaLicpa mutants complemented the gene deletion phenotype only in in vitro and not in in vivo infection of hamsters. [Abstract/Link to Full Text]

Xu J, Xu M, Pan YX
Characterizing exons 11 and 1 promoters of the mu opioid receptor (Oprm) gene in transgenic mice.
BMC Mol Biol. 2006;741.
BACKGROUND: The complexity of the mouse mu opioid receptor (Oprm) gene was demonstrated by the identification of multiple alternatively spliced variants and promoters. Our previous studies have identified a novel promoter, exon 11 (E11) promoter, in the mouse Oprm gene. The E11 promoter is located approximately 10 kb upstream of the exon 1 (E1) promoter. The E11 promoter controls the expression of nine splice variants in the mouse Oprm gene. Distinguished from the TATA-less E1 promoter, the E11 promoter resembles a typical TATA-containing eukaryote class II promoter. The aim of this study is to further characterize the E11 and E1 promoters in vivo using a transgenic mouse model. RESULTS: We constructed a approximately 20 kb transgenic construct in which a 3.7 kb E11 promoter region and an 8.9 kb E1 promoter region controlled expression of tau/LacZ and tau/GFP reporters, respectively. The construct was used to establish a transgenic mouse line. The expression of the reporter mRNAs, determined by a RT-PCR approach, in the transgenic mice during embryonic development displayed a temporal pattern similar to that of the endogenous promoters. X-gal staining for tau/LacZ reporter and GFP imaging for tau/GFP reporter showed that the transgenic E11 and E1 promoters were widely expressed in various regions of the central nervous system (CNS). The distribution of tau/GFP reporter in the CNS was similar to that of MOR-1-like immunoreactivity using an exon 4-specific antibody. However, differential expression of both promoters was observed in some CNS regions such as the hippocampus and substantia nigra, suggesting that the E11 and E1 promoters were regulated differently in these regions. CONCLUSION: We have generated a transgenic mouse line to study the E11 and E1 promoters in vivo using tau/LacZ and tau/GFP reporters. The reasonable relevance of the transgenic model was demonstrated by the temporal and spatial expression of the transgenes as compared to those of the endogenous transcripts. We believe that these transgenic mice will provide a useful model for further characterizing the E11 and E1 promoter in vivo under different physiological and pathological circumstances such as chronic opioid treatment and chronic pain models. [Abstract/Link to Full Text]

Zappulla DC, Maharaj AS, Connelly JJ, Jockusch RA, Sternglanz R
Rtt107/Esc4 binds silent chromatin and DNA repair proteins using different BRCT motifs.
BMC Mol Biol. 2006;740.
BACKGROUND: By screening a plasmid library for proteins that could cause silencing when targeted to the HMR locus in Saccharomyces cerevisiae, we previously reported the identification of Rtt107/Esc4 based on its ability to establish silent chromatin. In this study we aimed to determine the mechanism of Rtt107/Esc4 targeted silencing and also learn more about its biological functions. RESULTS: Targeted silencing by Rtt107/Esc4 was dependent on the SIR genes, which encode obligatory structural and enzymatic components of yeast silent chromatin. Based on its sequence, Rtt107/Esc4 was predicted to contain six BRCT motifs. This motif, originally identified in the human breast tumor suppressor gene BRCA1, is a protein interaction domain. The targeted silencing activity of Rtt107/Esc4 resided within the C-terminal two BRCT motifs, and this region of the protein bound to Sir3 in two-hybrid tests. Deletion of RTT107/ESC4 caused sensitivity to the DNA damaging agent MMS as well as to hydroxyurea. A two-hybrid screen showed that the N-terminal BRCT motifs of Rtt107/Esc4 bound to Slx4, a protein previously shown to be involved in DNA repair and required for viability in a strain lacking the DNA helicase Sgs1. Like SLX genes, RTT107ESC4 interacted genetically with SGS1; esc4Delta sgs1Delta mutants were viable, but exhibited a slow-growth phenotype and also a synergistic DNA repair defect. CONCLUSION: Rtt107/Esc4 binds to the silencing protein Sir3 and the DNA repair protein Slx4 via different BRCT motifs, thus providing a bridge linking silent chromatin to DNA repair enzymes. [Abstract/Link to Full Text]