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Recent Articles in Journal of Biology

Millington OR, Di Lorenzo C, Phillips RS, Garside P, Brewer JM
Suppression of adaptive immunity to heterologous antigens during Plasmodium infection through hemozoin-induced failure of dendritic cell function.
J Biol. 2006 Apr 12;5(2):5.
ABSTRACT : BACKGROUND : Dendritic cells (DCs) are central to the initiation and regulation of the adaptive immune response during infection. Modulation of DC function may therefore allow evasion of the immune system by pathogens. Significant depression of the host's systemic immune response to both concurrent infections and heterologous vaccines has been observed during malaria infection, but the mechanisms underlying this immune hyporesponsiveness are controversial. RESULTS : Here, we demonstrate that the blood stages of malaria infection induce a failure of DC function in vitro and in vivo, causing suboptimal activation of T cells involved in heterologous immune responses. This effect on T-cell activation can be transferred to uninfected recipients by DCs isolated from infected mice. Significantly, T cells activated by these DCs subsequently lack effector function, as demonstrated by a failure to migrate to lymphoid-organ follicles, resulting in an absence of B-cell responses to heterologous antigens. Fractionation studies show that hemozoin, rather than infected erythrocyte (red blood cell) membranes, reproduces the effect of intact infected red blood cells on DCs. Furthermore, hemozoin-containing DCs could be identified in T-cell areas of the spleen in vivo. CONCLUSION : Plasmodium infection inhibits the induction of adaptive immunity to heterologous antigens by modulating DC function, providing a potential explanation for epidemiological studies linking endemic malaria with secondary infections and reduced vaccine efficacy. [Abstract/Link to Full Text]

Cheng MK, Disteche CM
A balancing act between the X chromosome and the autosomes.
J Biol. 2006;5(1):2.
Dosage compensation equalizes gene dosage between males and females, but its role in balancing expression between the X chromosome and the autosomes may be far more important. Now, DNA microarrays have shown equality between the average expression of X-linked genes and that of autosomal genes, in male and female tissues of flies, worms and mice. [Abstract/Link to Full Text]

Weitzman JB
Getting the right dose of sex (chromosomes).
J Biol. 2006;5(1):1.
: Gene-expression analysis provides evidence for dosage compensation of the X chromosome in flies, mice and worms. [Abstract/Link to Full Text]

Gupta V, Parisi M, Sturgill D, Nuttall R, Doctolero M, Dudko OK, Malley JD, Eastman PS, Oliver B
Global analysis of X-chromosome dosage compensation.
J Biol. 2006;5(1):3.
BACKGROUND: Drosophila melanogaster females have two X chromosomes and two autosome sets (XX;AA), while males have a single X chromosome and two autosome sets (X;AA). Drosophila male somatic cells compensate for a single copy of the X chromosome by deploying male-specific-lethal (MSL) complexes that increase transcription from the X chromosome. Male germ cells lack MSL complexes, indicating that either germline X-chromosome dosage compensation is MSL-independent, or that germ cells do not carry out dosage compensation. RESULTS: To investigate whether dosage compensation occurs in germ cells, we directly assayed X-chromosome transcripts using DNA microarrays and show equivalent expression in XX;AA and X;AA germline tissues. In X;AA germ cells, expression from the single X chromosome is about twice that of a single autosome. This mechanism ensures balanced X-chromosome expression between the sexes and, more importantly, it ensures balanced expression between the single X chromosome and the autosome set. Oddly, the inactivation of an X chromosome in mammalian females reduces the effective X-chromosome dose and means that females face the same X-chromosome transcript deficiency as males. Contrary to most current dosage-compensation models, we also show increased X-chromosome expression in X;AA and XX;AA somatic cells of Caenorhabditis elegans and mice. CONCLUSION: Drosophila germ cells compensate for X-chromosome dose. This occurs by equilibrating X-chromosome and autosome expression in X;AA cells. Increased expression of the X chromosome in X;AA individuals appears to be phylogenetically conserved. [Abstract/Link to Full Text]

Taneyhill LA, Bronner-Fraser M
Recycling signals in the neural crest.
J Biol. 2005;4(3):10.
Vertebrate neural crest cells are multipotent and differentiate into structures that include cartilage and the bones of the face, as well as much of the peripheral nervous system. Understanding how different model vertebrates utilize signaling pathways reiteratively during various stages of neural crest formation and differentiation lends insight into human disorders associated with the neural crest. [Abstract/Link to Full Text]

Ittner LM, Wurdak H, Schwerdtfeger K, Kunz T, Ille F, Leveen P, Hjalt TA, Suter U, Karlsson S, Hafezi F, Born W, Sommer L
Compound developmental eye disorders following inactivation of TGFbeta signaling in neural-crest stem cells.
J Biol. 2005;4(3):11.
BACKGROUND: Development of the eye depends partly on the periocular mesenchyme derived from the neural crest (NC), but the fate of NC cells in mammalian eye development and the signals coordinating the formation of ocular structures are poorly understood. RESULTS: Here we reveal distinct NC contributions to both anterior and posterior mesenchymal eye structures and show that TGFbeta signaling in these cells is crucial for normal eye development. In the anterior eye, TGFbeta2 released from the lens is required for the expression of transcription factors Pitx2 and Foxc1 in the NC-derived cornea and in the chamber-angle structures of the eye that control intraocular pressure. TGFbeta enhances Foxc1 and induces Pitx2 expression in cell cultures. As in patients carrying mutations in PITX2 and FOXC1, TGFbeta signal inactivation in NC cells leads to ocular defects characteristic of the human disorder Axenfeld-Rieger's anomaly. In the posterior eye, NC cell-specific inactivation of TGFbeta signaling results in a condition reminiscent of the human disorder persistent hyperplastic primary vitreous. As a secondary effect, retinal patterning is also disturbed in mutant mice. CONCLUSION: In the developing eye the lens acts as a TGFbeta signaling center that controls the development of eye structures derived from the NC. Defective TGFbeta signal transduction interferes with NC-cell differentiation and survival anterior to the lens and with normal tissue morphogenesis and patterning posterior to the lens. The similarity to developmental eye disorders in humans suggests that defective TGFbeta signal modulation in ocular NC derivatives contributes to the pathophysiology of these diseases. [Abstract/Link to Full Text]

Moore P
The GTPase switch in ribosomal translocation.
J Biol. 2005;4(2):7. [Abstract/Link to Full Text]

Fraser CS, Hershey JW
Movement in ribosome translocation.
J Biol. 2005;4(2):8.
Translocation of peptidyl-tRNA and mRNA within the ribosome during protein synthesis is promoted by the elongation factor EF-G and by the hydrolysis of GTP. A new study reports that EF-G binds to ribosomes as an EF-G.GDP complex and that GTP is exchanged for GDP on the ribosome. Together with cryo-electron microscopy, this unexpected finding helps clarify the role of GTP in translocation. [Abstract/Link to Full Text]

Zavialov AV, Hauryliuk VV, Ehrenberg M
Guanine-nucleotide exchange on ribosome-bound elongation factor G initiates the translocation of tRNAs.
J Biol. 2005;4(2):9.
BACKGROUND: During the translation of mRNA into polypeptide, elongation factor G (EF-G) catalyzes the translocation of peptidyl-tRNA from the A site to the P site of the ribosome. According to the 'classical' model, EF-G in the GTP-bound form promotes translocation, while hydrolysis of the bound GTP promotes dissociation of the factor from the post-translocation ribosome. According to a more recent model, EF-G operates like a 'motor protein' and drives translocation of the peptidyl-tRNA after GTP hydrolysis. In both the classical and motor protein models, GDP-to-GTP exchange is assumed to occur spontaneously on 'free' EF-G even in the absence of a guanine-nucleotide exchange factor (GEF). RESULTS: We have made a number of findings that challenge both models. First, free EF-G in the cell is likely to be in the GDP-bound form. Second, the ribosome acts as the GEF for EF-G. Third, after guanine-nucleotide exchange, EF-G in the GTP-bound form moves the tRNA2-mRNA complex to an intermediate translocation state in which the mRNA is partially translocated. Fourth, subsequent accommodation of the tRNA2-mRNA complex in the post-translocation state requires GTP hydrolysis. CONCLUSION: These results, in conjunction with previously published cryo-electron microscopy reconstructions of the ribosome in various functional states, suggest a novel mechanism for translocation of tRNAs on the ribosome by EF-G. Our observations suggest that the ribosome is a universal guanosine-nucleotide exchange factor for EF-G as previously shown for the class-II peptide-release factor 3. [Abstract/Link to Full Text]

Herrg�rd MJ, Palsson B�
Untangling the web of functional and physical interactions in yeast.
J Biol. 2005;4(2):5.
An analysis of an integrated network of over 150,000 functional and physical interactions in yeast suggests that the network can be hierarchically decomposed into themes and thematic maps. This decomposition can be used to explore the organizational principles of integrated biological networks within cells. [Abstract/Link to Full Text]

Weitzman JB
A road map of yeast interactions.
J Biol. 2005;4(2):4. [Abstract/Link to Full Text]

Zhang LV, King OD, Wong SL, Goldberg DS, Tong AH, Lesage G, Andrews B, Bussey H, Boone C, Roth FP
Motifs, themes and thematic maps of an integrated Saccharomyces cerevisiae interaction network.
J Biol. 2005;4(2):6.
BACKGROUND: Large-scale studies have revealed networks of various biological interaction types, such as protein-protein interaction, genetic interaction, transcriptional regulation, sequence homology, and expression correlation. Recurring patterns of interconnection, or 'network motifs', have revealed biological insights for networks containing either one or two types of interaction. RESULTS: To study more complex relationships involving multiple biological interaction types, we assembled an integrated Saccharomyces cerevisiae network in which nodes represent genes (or their protein products) and differently colored links represent the aforementioned five biological interaction types. We examined three- and four-node interconnection patterns containing multiple interaction types and found many enriched multi-color network motifs. Furthermore, we showed that most of the motifs form 'network themes' -- classes of higher-order recurring interconnection patterns that encompass multiple occurrences of network motifs. Network themes can be tied to specific biological phenomena and may represent more fundamental network design principles. Examples of network themes include a pair of protein complexes with many inter-complex genetic interactions -- the 'compensatory complexes' theme. Thematic maps -- networks rendered in terms of such themes -- can simplify an otherwise confusing tangle of biological relationships. We show this by mapping the S. cerevisiae network in terms of two specific network themes. CONCLUSION: Significantly enriched motifs in an integrated S. cerevisiae interaction network are often signatures of network themes, higher-order network structures that correspond to biological phenomena. Representing networks in terms of network themes provides a useful simplification of complex biological relationships. [Abstract/Link to Full Text]

Itoh K, Brott BK, Bae GU, Ratcliffe MJ, Sokol SY
Nuclear localization is required for Dishevelled function in Wnt/beta-catenin signaling.
J Biol. 2005;4(1):3.
BACKGROUND: Dishevelled (Dsh) is a key component of multiple signaling pathways that are initiated by Wnt secreted ligands and Frizzled receptors during embryonic development. Although Dsh has been detected in a number of cellular compartments, the importance of its subcellular distribution for signaling remains to be determined. RESULTS: We report that Dsh protein accumulates in cell nuclei when Xenopus embryonic explants or mammalian cells are incubated with inhibitors of nuclear export or when a specific nuclear-export signal (NES) in Dsh is disrupted by mutagenesis. Dsh protein with a mutated NES, while predominantly nuclear, remains fully active in its ability to stimulate canonical Wnt signaling. Conversely, point mutations in conserved amino-acid residues that are essential for the nuclear localization of Dsh impair the ability of Dsh to activate downstream targets of Wnt signaling. When these conserved residues of Dsh are replaced with an unrelated SV40 nuclear localization signal, full Dsh activity is restored. Consistent with a signaling function for Dsh in the nucleus, treatment of cultured mammalian cells with medium containing Wnt3a results in nuclear accumulation of endogenous Dsh protein. CONCLUSIONS: These findings suggest that nuclear localization of Dsh is required for its function in the canonical Wnt/beta-catenin signaling pathway. We discuss the relevance of these findings to existing models of Wnt signal transduction to the nucleus. [Abstract/Link to Full Text]

Weitzman JB
Dishevelled nuclear shuttling.
J Biol. 2005;4(1):1.
Structure-function analysis of the Dishevelled (Dsh) protein in frog embryos has defined sequences that regulate Dsh nuclear localization, which proves critical for Wnt signaling. [Abstract/Link to Full Text]

Habas R, Dawid IB
Dishevelled and Wnt signaling: is the nucleus the final frontier?
J Biol. 2005;4(1):2.
The phosphoprotein Dishevelled (Dsh) is an essential component of Wnt signaling pathways and transduces signals into three separate branches, the canonical, non-canonical and Ca2+ pathways. How Dsh focuses signaling into these branches remains mysterious, but a new study reveals the importance of nuclear localization of Dsh for pathway-specific activation. [Abstract/Link to Full Text]

Holmes C, Brown SD
All systems GO for understanding mouse gene function.
J Biol. 2004;3(5):20.
It is widely supposed that the tissue specificity of gene expression indicates gene function. Now, an extensive analysis of gene expression in the mouse reveals that quantitative measurement of expression levels in different tissues can contribute powerfully to the prediction of gene function. [Abstract/Link to Full Text]

Zhang W, Morris QD, Chang R, Shai O, Bakowski MA, Mitsakakis N, Mohammad N, Robinson MD, Zirngibl R, Somogyi E, Laurin N, Eftekharpour E, Sat E, Grigull J, Pan Q, Peng WT, Krogan N, Greenblatt J, Fehlings M, van der Kooy D, Aubin J, Bruneau BG, Rossant J, Blencowe BJ, Frey BJ, Hughes TR
The functional landscape of mouse gene expression.
J Biol. 2004;3(5):21.
BACKGROUND: Large-scale quantitative analysis of transcriptional co-expression has been used to dissect regulatory networks and to predict the functions of new genes discovered by genome sequencing in model organisms such as yeast. Although the idea that tissue-specific expression is indicative of gene function in mammals is widely accepted, it has not been objectively tested nor compared with the related but distinct strategy of correlating gene co-expression as a means to predict gene function. RESULTS: We generated microarray expression data for nearly 40,000 known and predicted mRNAs in 55 mouse tissues, using custom-built oligonucleotide arrays. We show that quantitative transcriptional co-expression is a powerful predictor of gene function. Hundreds of functional categories, as defined by Gene Ontology 'Biological Processes', are associated with characteristic expression patterns across all tissues, including categories that bear no overt relationship to the tissue of origin. In contrast, simple tissue-specific restriction of expression is a poor predictor of which genes are in which functional categories. As an example, the highly conserved mouse gene PWP1 is widely expressed across different tissues but is co-expressed with many RNA-processing genes; we show that the uncharacterized yeast homolog of PWP1 is required for rRNA biogenesis. CONCLUSIONS: We conclude that 'functional genomics' strategies based on quantitative transcriptional co-expression will be as fruitful in mammals as they have been in simpler organisms, and that transcriptional control of mammalian physiology is more modular than is generally appreciated. Our data and analyses provide a public resource for mammalian functional genomics. [Abstract/Link to Full Text]

Weitzman JB
Co-regulation of mouse genes predicts function.
J Biol. 2004;3(5):19. [Abstract/Link to Full Text]

Copenhaver GP
Who's driving the centromere?
J Biol. 2004;3(4):17.
Centromere function is remarkably conserved between species, yet the satellite sequences that make up centromeric DNA are highly divergent. Proteins that bind these sequences appear to be evolving under positive selection, supporting a model wherein the interplay between centromeric repeats and the proteins that bind them creates an opportunity for an intriguing phenomenon known as centromere-based meiotic drive. [Abstract/Link to Full Text]

Williamson P, Schlegel RA
Hide and seek: the secret identity of the phosphatidylserine receptor.
J Biol. 2004;3(4):14.
Phosphatidylserine on the dying cell surface helps identify apoptotic cells to phagocytes, which then engulf them. A candidate phagocyte receptor for phosphatidylserine was identified using phage display, but the phenotypes of knockout mice lacking this presumptive receptor, as well as the location of the protein within cells, cast doubt on the assignment of this protein as the phosphatidylserine receptor. [Abstract/Link to Full Text]

B�se J, Gruber AD, Helming L, Schiebe S, Wegener I, Hafner M, Beales M, K�ntgen F, Lengeling A
The phosphatidylserine receptor has essential functions during embryogenesis but not in apoptotic cell removal.
J Biol. 2004;3(4):15.
BACKGROUND: Phagocytosis of apoptotic cells is fundamental to animal development, immune function and cellular homeostasis. The phosphatidylserine receptor (Ptdsr) on phagocytes has been implicated in the recognition and engulfment of apoptotic cells and in anti-inflammatory signaling. To determine the biological function of the phosphatidylserine receptor in vivo, we inactivated the Ptdsr gene in the mouse. RESULTS: Ablation of Ptdsr function in mice causes perinatal lethality, growth retardation and a delay in terminal differentiation of the kidney, intestine, liver and lungs during embryogenesis. Moreover, eye development can be severely disturbed, ranging from defects in retinal differentiation to complete unilateral or bilateral absence of eyes. Ptdsr -/- mice with anophthalmia develop novel lesions, with induction of ectopic retinal-pigmented epithelium in nasal cavities. A comprehensive investigation of apoptotic cell clearance in vivo and in vitro demonstrated that engulfment of apoptotic cells was normal in Ptdsr knockout mice, but Ptdsr-deficient macrophages were impaired in pro- and anti-inflammatory cytokine signaling after stimulation with apoptotic cells or with lipopolysaccharide. CONCLUSION: Ptdsr is essential for the development and differentiation of multiple organs during embryogenesis but not for apoptotic cell removal. Ptdsr may thus have a novel, unexpected developmental function as an important differentiation-promoting gene. Moreover, Ptdsr is not required for apoptotic cell clearance by macrophages but seems to be necessary for the regulation of macrophage cytokine responses. These results clearly contradict the current view that the phosphatidylserine receptor primarily functions in apoptotic cell clearance. [Abstract/Link to Full Text]

Talbert PB, Bryson TD, Henikoff S
Adaptive evolution of centromere proteins in plants and animals.
J Biol. 2004;3(4):18.
BACKGROUND: Centromeres represent the last frontiers of plant and animal genomics. Although they perform a conserved function in chromosome segregation, centromeres are typically composed of repetitive satellite sequences that are rapidly evolving. The nucleosomes of centromeres are characterized by a special H3-like histone (CenH3), which evolves rapidly and adaptively in Drosophila and Arabidopsis. Most plant, animal and fungal centromeres also bind a large protein, centromere protein C (CENP-C), that is characterized by a single 24 amino-acid motif (CENPC motif). RESULTS: Whereas we find no evidence that mammalian CenH3 (CENP-A) has been evolving adaptively, mammalian CENP-C proteins contain adaptively evolving regions that overlap with regions of DNA-binding activity. In plants we find that CENP-C proteins have complex duplicated regions, with conserved amino and carboxyl termini that are dissimilar in sequence to their counterparts in animals and fungi. Comparisons of Cenpc genes from Arabidopsis species and from grasses revealed multiple regions that are under positive selection, including duplicated exons in some grasses. In contrast to plants and animals, yeast CENP-C (Mif2p) is under negative selection. CONCLUSIONS: CENP-Cs in all plant and animal lineages examined have regions that are rapidly and adaptively evolving. To explain these remarkable evolutionary features for a single-copy gene that is needed at every mitosis, we propose that CENP-Cs, like some CenH3s, suppress meiotic drive of centromeres during female meiosis. This process can account for the rapid evolution and the complexity of centromeric DNA in plants and animals as compared to fungi. [Abstract/Link to Full Text]

Moore P
Making sense of centromeres.
J Biol. 2004;3(4):16.
Comparative analysis of the proteins that bind exclusively at the centromere provides evidence of an evolutionary battle that may make sense of sex. [Abstract/Link to Full Text]

Clayton J
Wnt signaling and the developmental fate of lung cells.
J Biol. 2004;3(3):9. [Abstract/Link to Full Text]

Okubo T, Hogan BL
Hyperactive Wnt signaling changes the developmental potential of embryonic lung endoderm.
J Biol. 2004;3(3):11.
BACKGROUND: Studies in many model systems have shown that canonical signaling through the pathway downstream of ligands of the Wnt family can regulate multiple steps in organogenesis, including cell proliferation, differentiation, and lineage specification. In addition, misexpression of the Wnt-family member Wingless in Drosophila imaginal disc cells can lead to transdetermination of progenitors from one lineage to another. Conditional deletion of the beta-catenin component of the Wnt signaling pathway has indicated a role for Wnt signaling in mouse lung endoderm development. The full range of effects of this pathway, which includes the transcription factor Lef1, has not been explored, however. RESULTS: To explore this issue, we expressed a constitutively active beta-catenin-Lef1 fusion protein in transgenic embryos using a lung-endoderm-specific promoter from the surfactant protein C gene. Transgenic lungs appeared grossly normal, but internally they contained highly proliferative, cuboidal epithelium lacking fully differentiated lung cell types. Unexpectedly, microarray analysis and in situ hybridization revealed a mosaic of cells expressing marker genes characteristic of intestinal Paneth and goblet cells and other non-lung secretory cell types. In addition, there was strong ectopic expression of genes such as Cdx1 and Atoh1 that normally regulate gut development and early allocation of cells to intestinal secretory lineages. CONCLUSIONS: Our results show that hyperactive Wnt signaling in lung progenitors expressing a lung-specific gene can induce a dramatic switch in lineage commitment and the generation of intestinal cell types. We discuss the relevance of our findings to the poorly understood pathological condition of intestinal metaplasia in humans. [Abstract/Link to Full Text]

Slack JM
Intestine in the lung.
J Biol. 2004;3(3):10.
The phenomenon of metaplasia, in which one tissue type is converted into another, is beginning to be explained in molecular terms. The transformation of lung to intestinal tissue has not previously been described, but it is now reported that it can be brought about by prolonged Wnt signaling in late development. [Abstract/Link to Full Text]

Weihs D
The hydrodynamics of dolphin drafting.
J Biol. 2004;3(2):8.
BACKGROUND: Drafting in cetaceans is defined as the transfer of forces between individuals without actual physical contact between them. This behavior has long been surmised to explain how young dolphin calves keep up with their rapidly moving mothers. It has recently been observed that a significant number of calves become permanently separated from their mothers during chases by tuna vessels. A study of the hydrodynamics of drafting, initiated in the hope of understanding the mechanisms causing the separation of mothers and calves during fishing-related activities, is reported here. RESULTS: Quantitative results are shown for the forces and moments around a pair of unequally sized dolphin-like slender bodies. These include two major effects. First, the so-called Bernoulli suction, which stems from the fact that the local pressure drops in areas of high speed, results in an attractive force between mother and calf. Second is the displacement effect, in which the motion of the mother causes the water in front to move forwards and radially outwards, and water behind the body to move forwards to replace the animal's mass. Thus, the calf can gain a 'free ride' in the forward-moving areas. Utilizing these effects, the neonate can gain up to 90% of the thrust needed to move alongside the mother at speeds of up to 2.4 m/sec. A comparison with observations of eastern spinner dolphins (Stenella longirostris) is presented, showing savings of up to 60% in the thrust that calves require if they are to keep up with their mothers. CONCLUSIONS: A theoretical analysis, backed by observations of free-swimming dolphin schools, indicates that hydrodynamic interactions with mothers play an important role in enabling dolphin calves to keep up with rapidly moving adult school members. [Abstract/Link to Full Text]

Moore P
Examining dolphin hydrodynamics provides clues to calf-loss during tuna fishing.
J Biol. 2004;3(2):6. [Abstract/Link to Full Text]

Alexander RM
Hitching a lift hydrodynamically--in swimming, flying and cycling.
J Biol. 2004;3(2):7.
Swimming animals set the water around them moving, and flying animals generate air movements. Other animals traveling with them can save energy by exploiting these movements of the fluid medium; similarly, a cyclist can save energy by riding close behind another. A new study of dolphin mothers and calves exemplifies the advantages of moving in concert. [Abstract/Link to Full Text]

Martindale D
Budding viral hijackers co-opt the endocytic machinery to make a getaway.
J Biol. 2003;3(1):2. [Abstract/Link to Full Text]

Yap MW, Stoye JP
Bending out and breaking away: host-cell accomplices in retroviral escape.
J Biol. 2003;3(1):3.
Budding through the host-cell membrane is a key step in the life cycle of many viruses. Recent studies of retrovirus replication implicate a large number of cellular proteins in this process. [Abstract/Link to Full Text]

Wang MQ, Kim W, Gao G, Torrey TA, Morse HC, De Camilli P, Goff SP
Endophilins interact with Moloney murine leukemia virus Gag and modulate virion production.
J Biol. 2003;3(1):4.
BACKGROUND: The retroviral Gag protein is the central player in the process of virion assembly at the plasma membrane, and is sufficient to induce the formation and release of virus-like particles. Recent evidence suggests that Gag may co-opt the host cell's endocytic machinery to facilitate retroviral assembly and release. RESULTS: A search for novel partners interacting with the Gag protein of the Moloney murine leukemia virus (Mo-MuLV) via the yeast two-hybrid protein-protein interaction assay resulted in the identification of endophilin 2, a component of the machinery involved in clathrin-mediated endocytosis. We demonstrate that endophilin interacts with the matrix or MA domain of the Gag protein of Mo-MuLV, but not of human immunodeficiency virus, HIV. Both exogenously expressed and endogenous endophilin are incorporated into Mo-MuLV viral particles. Titration experiments suggest that the binding sites for inclusion of endophilin into viral particles are limited and saturable. Knock-down of endophilin with small interfering RNA (siRNA) had no effect on virion production, but overexpression of endophilin and, to a lesser extent, of several fragments of the protein, result in inhibition of Mo-MuLV virion production, but not of HIV virion production. CONCLUSIONS: This study shows that endophilins interact with Mo-MuLV Gag and affect virion production. The findings imply that endophilin is another component of the large complex that is hijacked by retroviruses to promote virion production. [Abstract/Link to Full Text]

Weitzman JB
RNAi and the shape of things to come.
J Biol. 2003;2(4):23. [Abstract/Link to Full Text]

Kyriakis JM
At the crossroads: AMP-activated kinase and the LKB1 tumor suppressor link cell proliferation to metabolic regulation.
J Biol. 2003;2(4):26.
The tumor suppressor kinase LKB1 has been identified as a physiologic activator of the key metabolic regulator 5'-AMP-activated protein kinase, establishing a possible molecular link between the regulation of metabolism and cell proliferation. [Abstract/Link to Full Text]

Pollard TD
Functional genomics of cell morphology using RNA interference: pick your style, broad or deep.
J Biol. 2003;2(4):25.
Several new studies have used RNA interference to screen for protein functions affecting cell shape, mitosis and cytokinesis of Drosophila cells in culture. One broad survey of nearly 1,000 proteins and three studies focused on cytoskeletal and motor proteins have identified key proteins essential for these processes in animal cells. [Abstract/Link to Full Text]

Kiger AA, Baum B, Jones S, Jones MR, Coulson A, Echeverri C, Perrimon N
A functional genomic analysis of cell morphology using RNA interference.
J Biol. 2003;2(4):27.
BACKGROUND: The diversity of metazoan cell shapes is influenced by the dynamic cytoskeletal network. With the advent of RNA-interference (RNAi) technology, it is now possible to screen systematically for genes controlling specific cell-biological processes, including those required to generate distinct morphologies. RESULTS: We adapted existing RNAi technology in Drosophila cell culture for use in high-throughput screens to enable a comprehensive genetic dissection of cell morphogenesis. To identify genes responsible for the characteristic shape of two morphologically distinct cell lines, we performed RNAi screens in each line with a set of double-stranded RNAs (dsRNAs) targeting 994 predicted cell shape regulators. Using automated fluorescence microscopy to visualize actin filaments, microtubules and DNA, we detected morphological phenotypes for 160 genes, one-third of which have not been previously characterized in vivo. Genes with similar phenotypes corresponded to known components of pathways controlling cytoskeletal organization and cell shape, leading us to propose similar functions for previously uncharacterized genes. Furthermore, we were able to uncover genes acting within a specific pathway using a co-RNAi screen to identify dsRNA suppressors of a cell shape change induced by Pten dsRNA. CONCLUSIONS: Using RNAi, we identified genes that influence cytoskeletal organization and morphology in two distinct cell types. Some genes exhibited similar RNAi phenotypes in both cell types, while others appeared to have cell-type-specific functions, in part reflecting the different mechanisms used to generate a round or a flat cell morphology. [Abstract/Link to Full Text]

Recent Articles in The Journal of Experimental Biology

Gardiner JM, Atema J
Sharks need the lateral line to locate odor sources: rheotaxis and eddy chemotaxis.
J Exp Biol. 2007 Jun;210(Pt 11):1925-34.
Odor plumes are complex, dynamic, three-dimensional structures used by many animals to locate food, mates, home sites, etc. Yet odor itself has no directional properties. Animals use a variety of different senses to obtain directional information. Since most odor plumes are composed of dispersing odor patches and dissipating vorticity eddies, aquatic animals may localize odor sources by simultaneous analysis of chemical and hydrodynamic dispersal fields, a process referred to as eddy chemotaxis. This study examines the contributions of olfaction, mechanoreception and vision to odor source localization in a shark, the smooth dogfish Mustelus canis. Two parallel, turbulent plumes were created in an 8 m flume: squid rinse odor and seawater control. Minimally turbulent ;oozing' sources of odor and seawater control were physically separated from sources of major turbulence by placing a brick downstream from each oozing source, creating two turbulent wakes, one or the other flavored with food odor. This created four separate targets for the sharks to locate. Animals were tested under two light conditions (fluorescent and infrared) and in two sensory conditions (lateral line intact and lateral line lesioned by streptomycin). Intact animals demonstrated a preference for the odor plume over the seawater plume and for the source of odor/turbulence (the brick on the odor side) over the source of the odor alone (the odor-oozing nozzle). Plume and target preference and search time were not significantly affected by light condition. In the light, lesioning the lateral line increased search time but did not affect success rate or plume preference. However, lesioned animals no longer discriminated between sources of turbulent and oozing odor. In the dark, search time of lesioned animals further increased, and the few animals that located any of the targets did not discriminate between odor and seawater plumes, let alone targets. These results demonstrate for the first time that sharks require both olfactory and lateral line input for efficient and precise tracking of odor-flavored wakes and that visual input can improve food-finding performance when lateral line information is not available. We distinguish between rheotaxis: orientation to the large-scale flow field (olfaction, vision and superficial lateral line), eddy chemotaxis: tracking the trail of small-scale, odor-flavored turbulence (olfaction and lateral line canals), and pinpointing the source of the plume (lateral line canals and olfaction). [Abstract/Link to Full Text]

Hedrick TL, Usherwood JR, Biewener AA
Low speed maneuvering flight of the rose-breasted cockatoo (Eolophus roseicapillus). II. Inertial and aerodynamic reorientation.
J Exp Biol. 2007 Jun;210(Pt 11):1912-24.
The reconfigurable, flapping wings of birds allow for both inertial and aerodynamic modes of reorientation. We found evidence that both these modes play important roles in the low speed turning flight of the rose-breasted cockatoo Eolophus roseicapillus. Using three-dimensional kinematics recorded from six cockatoos making a 90 degrees turn in a flight corridor, we developed predictions of inertial and aerodynamic reorientation from estimates of wing moments of inertia and flapping arcs, and a blade-element aerodynamic model. The blade-element model successfully predicted weight support (predicted was 88+/-17% of observed, N=6) and centripetal force (predicted was 79+/-29% of observed, N=6) for the maneuvering cockatoos and provided a reasonable estimate of mechanical power. The estimated torque from the model was a significant predictor of roll acceleration (r(2)=0.55, P<0.00001), but greatly overestimated roll magnitude when applied with no roll damping. Non-dimensional roll damping coefficients of approximately -1.5, 2-6 times greater than those typical of airplane flight dynamics (approximately -0.45), were required to bring our estimates of reorientation due to aerodynamic torque back into conjunction with the measured changes in orientation. Our estimates of inertial reorientation were statistically significant predictors of the measured reorientation within wingbeats (r(2) from 0.2 to 0.37, P<0.0005). Components of both our inertial reorientation and aerodynamic torque estimates correlated, significantly, with asymmetries in the activation profile of four flight muscles: the pectoralis, supracoracoideus, biceps brachii and extensor metacarpi radialis (r(2) from 0.27 to 0.45, P<0.005). Thus, avian flight maneuvers rely on production of asymmetries throughout the flight apparatus rather than in a specific set of control or turning muscles. [Abstract/Link to Full Text]

Hedrick TL, Biewener AA
Low speed maneuvering flight of the rose-breasted cockatoo (Eolophus roseicapillus). I. Kinematic and neuromuscular control of turning.
J Exp Biol. 2007 Jun;210(Pt 11):1897-911.
Maneuvering flight has long been recognized as an important component of the natural behavior of many bird species, but has been the subject of little experimental work. Here we examine the kinematics and neuromuscular control of turning flight in the rose-breasted cockatoo Eolophus roseicapillus (N=6), testing predictions of maneuvering flight and control based on aerodynamic theory and prior kinematic and neuromuscular studies. Six cockatoos were trained to navigate between two perches placed in an L-shaped flight corridor, making a 90 degrees turn midway through each flight. Flights were recorded with three synchronized high-speed video cameras placed outside the corridor, allowing a three-dimensional reconstruction of wing and body kinematics through the turn. We simultaneously collected electromyography recordings from bilateral implants in the pectoralis, supracoracoideus, biceps brachii and extensor metacarpi radialis muscles. The cockatoos maneuvered using flapping, banked turns with an average turn radius of 0.92 m. The mean rate of change in heading during a complete wingbeat varied through the turn and was significantly correlated to roll angle at mid-downstroke. Changes in roll angle were found to include both within-wingbeat and among-wingbeat components that bear no direct relationship to one another. Within-wingbeat changes in roll were dominated by the inertial effects while among-wingbeat changes in roll were likely the result of both inertial and aerodynamic effects. [Abstract/Link to Full Text]

Bobbert MF, Alvarez CB, van Weeren PR, Roepstorff L, Weishaupt MA
Validation of vertical ground reaction forces on individual limbs calculated from kinematics of horse locomotion.
J Exp Biol. 2007 Jun;210(Pt 11):1885-96.
The purpose of this study was to determine whether individual limb forces could be calculated accurately from kinematics of trotting and walking horses. We collected kinematic data and measured vertical ground reaction forces on the individual limbs of seven Warmblood dressage horses, trotting at 3.4 m s(-1) and walking at 1.6 m s(-1) on a treadmill. First, using a segmental model, we calculated from kinematics the total ground reaction force vector and its moment arm relative to each of the hoofs. Second, for phases in which the body was supported by only two limbs, we calculated the individual reaction forces on these limbs. Third, we assumed that the distal limbs operated as linear springs, and determined their force-length relationships using calculated individual limb forces at trot. Finally, we calculated individual limb force-time histories from distal limb lengths. A good correspondence was obtained between calculated and measured individual limb forces. At trot, the average peak vertical reaction force on the forelimb was calculated to be 11.5+/-0.9 N kg(-1) and measured to be 11.7+/-0.9 N kg(-1), and for the hindlimb these values were 9.8+/-0.7 N kg(-1) and 10.0+/-0.6 N kg(-1), respectively. At walk, the average peak vertical reaction force on the forelimb was calculated to be 6.9+/-0.5 N kg(-1) and measured to be 7.1+/-0.3 N kg(-1), and for the hindlimb these values were 4.8+/-0.5 N kg(-1) and 4.7+/-0.3 N kg(-1), respectively. It was concluded that the proposed method of calculating individual limb reaction forces is sufficiently accurate to detect changes in loading reported in the literature for mild to moderate lameness at trot. [Abstract/Link to Full Text]

Boller ML, Carrington E
Interspecific comparison of hydrodynamic performance and structural properties among intertidal macroalgae.
J Exp Biol. 2007 Jun;210(Pt 11):1874-84.
Macroalgae use flexibility and reconfiguration, i.e. the alteration of shape, size and orientation as water velocity increases, to reduce the hydrodynamic forces imposed in the wave-swept rocky intertidal zone. Quantifying the effects of flexibility on hydrodynamic performance is difficult, however, because the mechanisms of reconfiguration vary with water velocity and the relationship between algal solid mechanics and hydrodynamic performance is poorly understood. In this study, the hydrodynamic performance, morphology and solid mechanics of 10 rocky shore macroalgal species were quantified to evaluate the influences of flexibility and morphology on reconfiguration. Hydrodynamic performance was measured in a flume by direct measurement of changes in size and shape during reconfiguration across a wide range of velocities, material stiffness was quantified with standard materials testing, and structural properties were calculated from material and morphological data. Hydrodynamic parameters varied significantly among species, indicating variation in the magnitude of reconfiguration and the velocities required for full reconfiguration. Structural properties also varied among species, and were correlated with hydrodynamic performance in some instances. The relationship between hydrodynamic and structural properties is velocity dependent, such that flexibility influences different aspects of reconfiguration at low and high velocities. Groups are identifiable among species based on hydrodynamic and structural properties, suggesting that these properties are useful for addressing functional-form hypotheses and the effects of hydrodynamic disturbance on macroalgal communities. [Abstract/Link to Full Text]

Dabiri JO, Colin SP, Costello JH
Morphological diversity of medusan lineages constrained by animal-fluid interactions.
J Exp Biol. 2007 Jun;210(Pt 11):1868-73.
Cnidarian medusae, commonly known as jellyfish, represent the earliest known animal taxa to achieve locomotion using muscle power. Propulsion by medusae requires the force of bell contraction to generate forward thrust. However, thrust production is limited in medusae by the primitive structure of their epitheliomuscular cells. This paper demonstrates that constraints in available locomotor muscular force result in a trade-off between high-thrust swimming via jet propulsion and high-efficiency swimming via a combined jet-paddling propulsion. This trade-off is reflected in the morphological diversity of medusae, which exhibit a range of fineness ratios (i.e. the ratio between bell height and diameter) and small body size in the high-thrust regime, and low fineness ratios and large body size in the high-efficiency regime. A quantitative model of the animal-fluid interactions that dictate this trade-off is developed and validated by comparison with morphological data collected from 660 extant medusan species ranging in size from 300 microm to over 2 m. These results demonstrate a biomechanical basis linking fluid dynamics and the evolution of medusan bell morphology. We believe these to be the organising principles for muscle-driven motility in Cnidaria. [Abstract/Link to Full Text]

Gilman CA, Wolf BO
Use of portable ultrasonography as a nondestructive method for estimating reproductive effort in lizards.
J Exp Biol. 2007 Jun;210(Pt 11):1859-67.
Obtaining population-level life history data such as egg and clutch size in reptiles has most often required that individuals be sacrificed. This prevents a reexamination of individuals over intra-annual and inter-annual time scales, limiting insight into the effects of varying environmental conditions on reproductive output. Here, we test the use of a laptop-sized portable ultrasound imaging system as a nondestructive means for quantifying reproductive investment in five species of lizards with a range of body sizes, forms and life histories. Ultrasound scans produced egg counts that were accurate for clutch sizes of two to seven eggs, and provided good estimates (within 5.5+/-1.69 eggs, mean +/- s.e.m., relative error 21%) for clutch sizes of between 18 and 41 eggs. Egg measurements using virtual calipers produced average egg volumes that deviated from actual volumes by 0.09+/-0.01 cm(3) (relative error 25.9%), and estimated clutch volumes that differed from actual volumes by 1.03+/-0.26 cm(3) (relative error 29.5%). We also monitored development in five lizard species and found that changes in follicle and egg size and degree of embryonic development can be measured over periods of just a few days. [Abstract/Link to Full Text]

Nikinmaa M, Waser W
Molecular and cellular studies in evolutionary physiology of natural vertebrate populations: influences of individual variation and genetic components on sampling and measurements.
J Exp Biol. 2007 Jun;210(Pt 11):1847-57.
Studies combining ecological, genetic and physiological approaches are needed in evolutionary biology. Although the combination of approaches has been emphasized, such studies have been rare with regard to molecular and cellular studies on natural vertebrate populations. The major reasons for this are that the generation time of vertebrates is long and it is difficult to find a molecular or cell physiological measurement that is both relevant for the fitness of the population and can be repeated an adequate number of times to enable estimations of individual variability. The paucity of suitable physiological parameters is partly due to the fact that most physiological studies have not been directed towards understanding the behaviour of populations but towards understanding the basic mechanisms of the function of individuals. Also, physiological measurements that appear most relevant from the point of view of evolutionary studies are often integrative functions, composed of the function of many genes. When dissecting the integrative functions into components, it is often observed that the same integrative response can be achieved via different routes, i.e. changes in the responses of different genes. To enable cellular and molecular physiological studies to be increasingly combined with ecological and genetic studies, it is important that such studies include and report individual variability and that the sample size is increased. In addition, more sophisticated statistical methods should be used than is traditionally done, and when the function of most genes in the integrative response are not known, techniques such as QTL mapping should be used. Hitherto in vertebrates, the methodology has mainly been used in production biology (e.g. meat or milk production). With regard to combining genomic and physiological studies, one must bear in mind that the massive datasets associated with genomic studies need to be further enlarged to enable estimates of individual variation. It is also important to remember that microarray and proteomic data give the levels of mRNA and proteins, respectively. Since the function of the protein can be regulated independently of its transcription or its level in the cell, direct physiological measurements are also needed if estimations of protein activity in the individuals of a population are wanted. [Abstract/Link to Full Text]

Fonseca PJ, Correia T
Effects of temperature on tuning of the auditory pathway in the cicada Tettigetta josei (Hemiptera, Tibicinidae).
J Exp Biol. 2007 May;210(Pt 10):1834-45.
The effects of temperature on hearing in the cicada Tettigetta josei were studied. The activity of the auditory nerve and the responses of auditory interneurons to stimuli of different frequencies and intensities were recorded at different temperatures ranging from 16 degrees C to 29 degrees C. Firstly, in order to investigate the temperature dependence of hearing processes, we analyzed its effects on auditory tuning, sensitivity, latency and Q(10dB). Increasing temperature led to an upward shift of the characteristic hearing frequency, to an increase in sensitivity and to a decrease in the latency of the auditory response both in the auditory nerve recordings (periphery) and in some interneurons at the metathoracic-abdominal ganglionic complex (MAC). Characteristic frequency shifts were only observed at low frequency (3-8 kHz). No changes were seen in Q(10dB). Different tuning mechanisms underlying frequency selectivity may explain the results observed. Secondly, we investigated the role of the mechanical sensory structures that participate in the transduction process. Laser vibrometry measurements revealed that the vibrations of the tympanum and tympanal apodeme are temperature independent in the biologically relevant range (18-35 degrees C). Since the above mentioned effects of temperature are present in the auditory nerve recordings, the observed shifts in frequency tuning must be performed by mechanisms intrinsic to the receptor cells. Finally, the role of potassium channels in the response of the auditory system was investigated using a specific inhibitor of these channels, tetraethylammonium (TEA). TEA caused shifts on tuning and sensitivity of the summed response of the receptors similar to the effects of temperature. Thus, potassium channels are implicated in the tuning of the receptor cells. [Abstract/Link to Full Text]

Formenti F, Minetti AE
Human locomotion on ice: the evolution of ice-skating energetics through history.
J Exp Biol. 2007 May;210(Pt 10):1825-33.
More than 3000 years ago, peoples living in the cold North European regions started developing tools such as ice skates that allowed them to travel on frozen lakes. We show here which technical and technological changes determined the main steps in the evolution of ice-skating performance over its long history. An in-depth historical research helped identify the skates displaying significantly different features from previous models and that could consequently determine a better performance in terms of speed and energy demand. Five pairs of ice skates were tested, from the bone-skates, dated about 1800 BC, to modern ones. This paper provides evidence for the fact that the metabolic cost of locomotion on ice decreased dramatically through history, the metabolic cost of modern ice-skating being only 25% of that associated with the use of bone-skates. Moreover, for the same metabolic power, nowadays skaters can achieve speeds four times higher than their ancestors could. In the range of speeds considered, the cost of travelling on ice was speed independent for each skate model, as for running. This latter finding, combined with the accepted relationship between time of exhaustion and the sustainable fraction of metabolic power, gives the opportunity to estimate the maximum skating speed according to the distance travelled. Ice skates were probably the first human powered locomotion tools to take the maximum advantage from the biomechanical properties of the muscular system: even when travelling at relatively high speeds, the skating movement pattern required muscles to shorten slowly so that they could also develop a considerable amount of force. [Abstract/Link to Full Text]

Gao Y, Wheatly MG
Molecular characterization of an epithelial Ca2+ channel-like gene from crayfish Procambarus clarkii.
J Exp Biol. 2007 May;210(Pt 10):1813-24.
This study describes the cloning, sequencing and functional characterization of an epithelial Ca(2+) channel (ECaC)-like gene isolated from antennal gland (kidney) of the freshwater crayfish Procambarus clarkii. The full-length cDNA consisted of 2687 bp with an open reading frame of 2169 bp encoding a protein of 722 amino acids with a predicted molecular mass of 81.7 kDa. Crayfish ECaC had 76-78% identity at the mRNA level (80-82% amino acid identity) with published fish sequences and 56-62% identity at the mRNA level (52-60% amino acid identity) with mammalian ECaCs. Secondary structure of the crayfish ECaC closely resembled that of cloned ECaCs. Postmolt ECaC expression was exclusively restricted to epithelia associated with Ca(2+) influx and was virtually undetectable in non-epithelial tissues (eggs, muscle). Compared with expression levels in hepatopancreas, expression in gill was 10-fold greater and expression was highest in antennal gland (15-fold greater than in hepatopancreas). Compared with baseline expression levels in intermolt stage, expression of ECaC in antennal gland increased 7.4- and 23.8-fold, respectively, in pre- and postmolt stages of the molting cycle. This increase was localized primarily in the labyrinth and nephridial canal, regions of the antennal gland associated with renal Ca(2+) reabsorption. The ECaC in crayfish appears to be expressed in epithelia associated with unidirectional Ca(2+) influx and relative expression is correlated with rate of Ca(2+) influx. [Abstract/Link to Full Text]

Narendra A
Homing strategies of the Australian desert ant Melophorus bagoti. II. Interaction of the path integrator with visual cue information.
J Exp Biol. 2007 May;210(Pt 10):1804-12.
Individually foraging ants are known to return to their nest by using path-integration and recording visual information present in the environment. The interaction between the path integrator and the information provided by the visual cues in an Australian desert ant are reported here. Ants were trained to travel in a 1-m wide and 20-m long corridor of cylinders. Homeward paths of trained ants were recorded in the presence and absence of vector information and route cues in both the familiar training field and in an unfamiliar test field. Homing ants used route cue information only in a familiar context. The route cues were not essential but served to reduce the deviation of the homing trajectory from the nest-feeder line. When displaced locally, homebound ants initially oriented towards the nest using distant cues and then headed in a direction intermediate between that dictated by the path integrator and the distant cues. If in the course of travel ants encountered the familiar path they adhered to it. If not, they travelled on average half the distance of the outbound journey and initiated a search directed towards the nest. Following the search, ants headed in a direction intermediate between that dictated by the route cues and the distant cues. In an unfamiliar context neither vector nor route cue information could steer a homing ant towards the nest. The dominance of distant cues, the importance of familiar context and the interaction between different navigation strategies are discussed here. [Abstract/Link to Full Text]

Narendra A
Homing strategies of the Australian desert ant Melophorus bagoti. I. Proportional path-integration takes the ant half-way home.
J Exp Biol. 2007 May;210(Pt 10):1798-803.
Highly evolved eusocial insects such as ants return from a food source to their nest by the shortest possible distance. This form of navigation, called path-integration, involves keeping track of the distance travelled and the angles steered on the outbound journey, which then aids in the computation of the shortest return distance. In featureless terrain, ants rely on the path integrator to travel the entire distance to return to the nest, whereas in landmark-rich habitats ants are guided by visual cues and in the absence of the visual cues homing ants rely on the path integrator to travel only an initial 10-60 cm of the homebound distance. The functioning of the path integrator in a habitat of intermediate landmark density is unknown. The findings reported here show that when the outward journey is on a familiar foraging area, and the inward journey is on an unfamiliar area, the Australian route-following desert ant Melophorus bagoti relies on the path integrator and consistently travels half the distance of the outward trip. However, when both the outward and inward trips are performed in plain and featureless channels, which blocks the distinct terrestrial visual cues, ants travel the entire distance accurately. A similar half-way abbreviation of the home vector occurs when the ant's outward trip is in an L-shaped channel and the homeward trip is over an open and unfamiliar region. The ecological significance of these new findings is discussed. [Abstract/Link to Full Text]

Petersen AM, Gleeson TT
Characterization of circannual patterns of metabolic recovery from activity in Rana catesbeiana at 15 degrees C.
J Exp Biol. 2007 May;210(Pt 10):1786-97.
We characterized carbohydrate metabolism following activity in the American bullfrog, Rana catesbeiana, and compared whole body metabolic profiles between two seasons. Forty-eight adult male Rana catesbeiana were chronically cannulated and injected with [U-(14)C]L-lactic acid sodium salt in either summer (June) or winter (January) after acclimation for 2 weeks at 15 degrees C with a 12 h:12 h L:D photoperiod. Following injection with [(14)C]lactate, frogs were either allowed to rest for 240 min (REST), hopped for 2 min on a treadmill and immediately sacrificed (PE), or hopped for 2 min on a treadmill and allowed to recover for 240 min (REC 4). Exercise caused a significant increase in blood lactate level from 2.7+/-0.1 mmol l(-1) at rest to 17.0+/-2.1 mmol l(-1) immediately following exercise. This increase persisted throughout the recovery period, with average blood lactate level only reduced to 13.7+/-1.1 mmol l(-1) after 240 min of recovery, despite complete recovery of intramuscular lactate levels. Lactate levels were not significantly different between seasons in any treatment (REST, PE, REC4), in either gastrocnemius muscle or blood. The vast majority of [(14)C]lactate was recovered in the muscle, in both winter (86.3%) and summer (87.5%). Season had no effect on total amount of (14)C label recovered. [(14)C]Lactate was measured in the forms of lactate, glucose and glycogen, in the liver and the muscle sampled. The most robust difference found in seasonal metabolism was that both the liver and the gastrocnemius contained significantly higher levels of intracellular free glucose under all treatments in winter. These data suggest that, overall, bullfrogs accumulate and slowly clear lactate in a manner quite similar to findings in fish, other amphibians and lizards. Additionally, our findings indicate that lactate metabolism is not highly influenced by season alone, but that intracellular glucose levels may be sensitive to annual patterns. [Abstract/Link to Full Text]

Ferrer RP, Zimmer RK
Chemosensory reception, behavioral expression, and ecological interactions at multiple trophic levels.
J Exp Biol. 2007 May;210(Pt 10):1776-85.
Chemoreception may function throughout an entire animal lifetime, with independent, stage-specific selection pressures leading to changes in physiological properties, behavioral expression, and hence, trophic interactions. When the California newt (Taricha torosa) metamorphoses from an entirely aquatic larva to a semi-terrestrial juvenile/adult form, its chemosensory organs undergo dramatic reorganization. The relationship between newt life-history stage and chemosensory-mediated behavior was established by comparing responses of adults (as determined here) to those of conspecific larvae (as studied previously). Bioassays were performed in mountain streams, testing responses of free-ranging adults to 13 individual l-amino acids. Relative to stream water (controls), adults turned immediately upcurrent and moved to the source of arginine, glycine or alanine release. These responses were indicative of predatory search. Arginine was the strongest attractant tested, with a response threshold (median effective dose) of 8.3x10(-7) mol l(-1) (uncorrected for dilution associated with chemical release and delivery). In contrast to adult behavior, arginine suppressed cannibal-avoidance and failed to evoke search reactions in larvae. For a common set of arginine analogs, the magnitudes of adult attraction and larval suppression were not positively correlated. Suppression of cannibal-avoidance behavior in larvae was unaffected by most structural modifications of the arginine molecule. Adult behavior, on the other hand, was strongly influenced by even subtle alterations in the parent compound. Reactions to arginine in both adults and larvae were eliminated by blocking the external openings of the nasal cavity. Stimulating adult predatory search in one case and inhibiting larval cannibal avoidance in the other, arginine is a chemical signal with opposing behavioral effects and varying ecological consequences. Significant differences between responses of adults and larvae to changes in arginine structure suggest alternative, chemosensory receptor targets. Although arginine reception functions throughout an entire newt lifetime, an ontogenetic shift in larval and adult chemoreceptive ability changes behavioral expression, and thus, reflects the unique selection pressures that act at each life-history stage. [Abstract/Link to Full Text]

Ferrer RP, Zimmer RK
The scent of danger: arginine as an olfactory cue of reduced predation risk.
J Exp Biol. 2007 May;210(Pt 10):1768-75.
Animal perception of chemosensory cues is a function of ecological context. Larvae of the California newt (Taricha torosa), for example, exhibit predator-avoidance behavior in response to a chemical from cannibalistic adults. The poison tetrodotoxin (TTX), well known as an adult chemical defense, stimulates larval escape to refuges. Although they are cannibals, adult newts feed preferentially on worms (Eisenia rosea) over conspecific young. Hence, larval avoidance reactions to TTX are suppressed in the presence of odor from these alternative prey. The free amino acid, arginine, is abundant in fluids emitted by injured worms. Here, we demonstrate that arginine is a natural suppressant of TTX-stimulated larval escape behavior. Compared to a tapwater control, larvae initiated vigorous swimming in response to 10(-7) mol l(-1) TTX. This excitatory response was eliminated when larval nasal cavities were blocked with an inert gel, but not when gel was placed on the forehead (control). In additional trials, a binary mixture of arginine and 10(-7) mol l(-1) TTX failed to induce larval swimming. The inhibitory effect of arginine was, however, dose dependent. An arginine concentration as low as 0.3-times that of TTX was significantly suppressant. Further analysis showed that suppression by arginine of TTX-stimulated behavior was eliminated by altering the positively-charged guanidinium moiety, but not by modifying the carbon chain, carboxyl group, or amine group. These results are best explained by a mechanism of competitive inhibition between arginine and TTX for common, olfactory receptor binding sites. Although arginine alone has no impact on larval behavior, it nevertheless signals active adult predation on alternative prey, and hence, reduced cannibalism risk. [Abstract/Link to Full Text]

Herrel A, James RS, Van Damme R
Fight versus flight: physiological basis for temperature-dependent behavioral shifts in lizards.
J Exp Biol. 2007 May;210(Pt 10):1762-7.
Previous studies have demonstrated that a behavioral shift from flight to aggressive behavior occurs at low temperatures in some lizards. Our data for the agamid lizard Trapelus pallida demonstrate how the effect of temperature on whole organism performance traits such as sprint speed (much lower performance at lower temperature) and bite force (largely independent of temperature) may explain the shift from flight to fight behavior with decreasing temperature. Moreover, our data hint at the physiological basis for this effect as isolated muscle power output, twitch and tetanus time traits, relevant to sprinting, appear to be strongly temperature-dependent muscle properties. Maximal muscle force production, on the other hand, appears largely independent of temperature. Unexpectedly, differences in the physiological properties of jaw versus limb muscle were observed that enhance the ability of the jaw muscle to generate maximal force at all temperatures tested. Thus our data show how behavioral responses may be determined by the limitations set by temperature on physiological processes. [Abstract/Link to Full Text]

Pontzer H
Effective limb length and the scaling of locomotor cost in terrestrial animals.
J Exp Biol. 2007 May;210(Pt 10):1752-61.
Relative to body size, smaller animals use more energy to travel a given distance than larger animals, but the anatomical variable driving this negative allometry remains the subject of debate. Here, I report a simple inverse relationship between effective limb length (i.e. hip height) and the energy cost of transport (COT; J kg(-1) m(-1)) for terrestrial animals. Using published data for a diverse set of terrestrial species including birds, mammals, reptiles and arthropods, I show that between-species differences in locomotor cost are driven by differences in limb length. Notably, there is no independent effect of body mass on cost. Remarkably, effective limb length explains 98% of the observed variance in locomotor cost across a wide range of terrestrial species including mammals, birds, reptiles and arthropods. Variation about the limb-length/COT scaling relationship is attributable to taxonomic differences in limb design, with birds and arthropods exhibiting greater residuals than mammals. Differences in COT between semi-aquatic, generalist and cursorial species also corresponds to differences in leg length between these groups. These results are discussed in light of previous investigations of the limb length and locomotor cost. [Abstract/Link to Full Text]

Tobalske BW, Dial KP
Aerodynamics of wing-assisted incline running in birds.
J Exp Biol. 2007 May;210(Pt 10):1742-51.
Wing-assisted incline running (WAIR) is a form of locomotion in which a bird flaps its wings to aid its hindlimbs in climbing a slope. WAIR is used for escape in ground birds, and the ontogeny of this behavior in precocial birds has been suggested to represent a model analogous to transitional adaptive states during the evolution of powered avian flight. To begin to reveal the aerodynamics of flap-running, we used digital particle image velocimetry (DPIV) and measured air velocity, vorticity, circulation and added mass in the wake of chukar partridge Alectoris chukar as they engaged in WAIR (incline 65-85 degrees; N=7 birds) and ascending flight (85 degrees, N=2). To estimate lift and impulse, we coupled our DPIV data with three-dimensional wing kinematics from a companion study. The ontogeny of lift production was evaluated using three age classes: baby birds incapable of flight [6-8 days post hatching (d.p.h.)] and volant juveniles (25-28 days) and adults (45+ days). All three age classes of birds, including baby birds with partially emerged, symmetrical wing feathers, generated circulation with their wings and exhibited a wake structure that consisted of discrete vortex rings shed once per downstroke. Impulse of the vortex rings during WAIR was directed 45+/-5 degrees relative to horizontal and 21+/-4 degrees relative to the substrate. Absolute values of circulation in vortex cores and induced velocity increased with increasing age. Normalized circulation was similar among all ages in WAIR but 67% greater in adults during flight compared with flap-running. Estimated lift during WAIR was 6.6% of body weight in babies and between 63 and 86% of body weight in juveniles and adults. During flight, average lift was 110% of body weight. Our results reveal for the first time that lift from the wings, rather than wing inertia or profile drag, is primarily responsible for accelerating the body toward the substrate during WAIR, and that partially developed wings, not yet capable of flight, can produce useful lift during WAIR. We predict that neuromuscular control or power output, rather than external wing morphology, constrain the onset of flight ability during development in birds. [Abstract/Link to Full Text]

Albokhadaim I, Hammond CL, Ashton C, Simbi BH, Bayol S, Farrington S, Stickland N
Larval programming of post-hatch muscle growth and activity in Atlantic salmon (Salmo salar).
J Exp Biol. 2007 May;210(Pt 10):1735-41.
Larval muscle development in Atlantic salmon is known to be affected by temperature; however, the long term effects and possible mechanisms involved are less well understood. Therefore, the aim of this study was to evaluate the influence of egg incubation temperature on post-hatch muscle growth and fish activity. Salmon eggs were incubated at either 10 degrees C or 5 degrees C from fertilization until hatching, then subsequently both groups were reared at 5 degrees C. Fish from both groups were sampled at the eyed stage, 6 and 21 weeks after first feeding, for muscle cellularity analysis and immunocytochemistry. In addition, to try to establish a mechanism for altered growth, the activity of the fish was measured at 3, 6 and 21 weeks after first feeding. Our results demonstrate that whereas fish incubated at 10 degrees C grow faster, the fish incubated at 5 degrees C show a more sustained period of muscle growth and by 21 weeks are significantly longer, heavier and have more muscle fibres than those fish incubated at a higher temperature. We also demonstrate that fish raised at 5 degrees C show increased food seeking activity throughout development and that this may explain their sustained growth and muscle development. These results taken together, demonstrate that egg incubation temperature up to hatching in salmon is critical for longer term muscle growth, twinned with increased activity. This is of interest to the aquaculture industry in term of the production of good quality fish protein. [Abstract/Link to Full Text]

Tracy CR, McWhorter TJ, Korine C, Wojciechowski MS, Pinshow B, Karasov WH
Absorption of sugars in the Egyptian fruit bat (Rousettus aegyptiacus): a paradox explained.
J Exp Biol. 2007 May;210(Pt 10):1726-34.
Two decades ago D. J. Keegan reported results on Egyptian fruit bats (Rousettus aegyptiacus, Megachiroptera) that were strangely at odds with the prevailing understanding of how glucose is absorbed in the mammalian intestine. Keegan's in vitro tests for glucose transport against a concentration gradient and with phloridzin inhibition in fruit bat intestine were all negative, although he used several different tissue preparations and had positive control results with laboratory rats. Because glucose absorption by fruit bats is nonetheless efficient, Keegan postulated that the rapid glucose absorption from the fruit bat intestine is not through the enterocytes, but must occur via spaces between the cells. Thus, we hypothesized that absorption of water-soluble compounds that are not actively transported would be extensive in these bats, and would decline with increasing molecular mass in accord with sieve-like paracellular absorption. We did not presume from Keegan's studies that there is no Na(+)-coupled, mediated sugar transport in these bats, and our study was not designed to rule it out, but rather to quantify the level of possible non-mediated absorption. Using a standard pharmacokinetic technique, we fed, or injected intraperitonealy, the metabolically inert carbohydrates L-rhamnose (molecular mass=164 Da) and cellobiose (molecular mass=342 Da), which are absorbed by paracellular uptake, and 3-O-methyl-D-glucose (3OMD-glucose), a D-glucose analog that is absorbed via both mediated (active) and paracellular uptake. As predicted, the bioavailability of paracellular probes declined with increasing molecular mass (rhamnose, 62+/-4%; cellobiose, 22+/-4%) and was significantly higher in bats than has been reported for rats and other mammals. In addition, fractional absorption of 3OMd-glucose was high (91+/-2%). We estimated that Egyptian fruit bats rely on passive, paracellular absorption for the majority of their glucose absorption (at least 55% of 3OMD-glucose absorption), much more than in non-flying mammals. [Abstract/Link to Full Text]

Farrell AP
Tribute to P. L. Lutz: a message from the heart--why hypoxic bradycardia in fishes?
J Exp Biol. 2007 May;210(Pt 10):1715-25.
The sensing and processing of hypoxic signals, the responses to these signals and the modulation of these responses by other physical and physiological factors are an immense topic filled with numerous novel and exciting discoveries. Nestled among these discoveries, and in contrast to mammals, is the unusual cardiac response of many fish to environmental hypoxia - a reflex slowing of heart rate. The afferent and efferent arms of this reflex have been characterised, but the benefits of the hypoxic bradycardia remain enigmatic since equivocal results have emerged from experiments examining the benefit to oxygen transfer across the gills. The main thesis developed here is that hypoxic bradycardia could afford a number of direct benefits to the fish heart, largely because the oxygen supply to the spongy myocardium is precarious (i.e. it is determined primarily by the partial pressure of oxygen in venous blood, Pv(O(2))) and, secondarily, because the fish heart has an unusual ability to produce large increases in cardiac stroke volume (V(SH)) that allow cardiac output to be maintained during hypoxic bradycardia. Among the putative benefits of hypoxic bradycardia is an increase in the diastolic residence time of blood in the lumen of the heart, which offers an advantage of increased time for diffusion, and improved cardiac contractility through the negative force-frequency effect. The increase in V(SH) will stretch the cardiac chambers, potentially reducing the diffusion distance for oxygen. Hypoxic bradycardia could also reduce cardiac oxygen demand by reducing cardiac dP/dt and cardiac power output, something that could be masked at cold temperature because of a reduced myocardial work load. While the presence of a coronary circulation in certain fishes decreases the reliance of the heart on Pv(O(2)), hypoxic bradycardia could still benefit oxygen delivery via an extended diastolic period during which peak coronary blood flow occurs. The notable absence of hypoxic bradycardia among fishes that breathe air during aquatic hypoxia and thereby raise their Pv(O(2)), opens the possibility that that the evolutionary loss of hypoxic bradycardia may have coincided with some forms of air breathing in fishes. Experiments are needed to test some of these possibilities. Ultimately, any potential benefit of hypoxic bradycardia must be placed in the proper context of myocardial oxygen supply and demand, and must consider the ability of the fish heart to support its routine cardiac power output through glycolysis. [Abstract/Link to Full Text]

Storey KB, Storey JM
Tribute to P. L. Lutz: putting life on 'pause'--molecular regulation of hypometabolism.
J Exp Biol. 2007 May;210(Pt 10):1700-14.
Entry into a hypometabolic state is an important survival strategy for many organisms when challenged by environmental stress, including low oxygen, cold temperatures and lack of food or water. The molecular mechanisms that regulate transitions to and from hypometabolic states, and stabilize long-term viability during dormancy, are proving to be highly conserved across phylogenic lines. A number of these mechanisms were identified and explored using anoxia-tolerant turtles as the model system, particularly from the research contributions made by Dr Peter L. Lutz in his explorations of the mechanisms of neuronal suppression in anoxic brain. Here we review some recent advances in understanding the biochemical mechanisms of metabolic arrest with a focus on ideas such as the strategies used to reorganize metabolic priorities for ATP expenditure, molecular controls that suppress cell functions (e.g. ion pumping, transcription, translation, cell cycle arrest), changes in gene expression that support hypometabolism, and enhancement of defense mechanisms (e.g. antioxidants, chaperone proteins, protease inhibitors) that stabilize macromolecules and promote long-term viability in the hypometabolic state. [Abstract/Link to Full Text]

Overgaard J, Gesser H, Wang T
Tribute to P. L. Lutz: cardiac performance and cardiovascular regulation during anoxia/hypoxia in freshwater turtles.
J Exp Biol. 2007 May;210(Pt 10):1687-99.
Freshwater turtles overwintering in ice-covered ponds in North America may be exposed to prolonged anoxia, and survive this hostile environment by metabolic depression. Here, we review their cardiovascular function and regulation, with particular emphasis on the factors limiting cardiac performance. The pronounced anoxia tolerance of the turtle heart is based on the ability to match energy consumption with the low anaerobic ATP production during anoxia. Together with a well-developed temporal and spatial energy buffering by creatine kinase, this allows for cellular energy charge to remain high during anoxia. Furthermore, the turtle heart is well adapted to handle the adverse effects of free phosphate arising when phosphocreatine stores are used. Anoxia causes tenfold reductions in heart rate and blood flows that match the metabolic depression, and blood pressure is largely maintained through increased systemic vascular resistance. Depression of the heart rate is not driven by the autonomic nervous system and seems to arise from direct effects of oxygen lack and the associated hyperkalaemia and acidosis on the cardiac pacemaker. These intra- and extracellular changes also affect cardiac contractility, and both acidosis and hyperkalaemia severely depress cardiac contractility. However, increased levels of adrenaline and calcium may, at least partially, salvage cardiac function under prolonged periods of anoxia. [Abstract/Link to Full Text]

Nilsson GE, Hobbs JP, Ostlund-Nilsson S
Tribute to P. L. Lutz: respiratory ecophysiology of coral-reef teleosts.
J Exp Biol. 2007 May;210(Pt 10):1673-86.
One of the most diverse vertebrate communities is found on tropical coral reefs. Coral-reef fishes are not only remarkable in color and shape, but also in several aspects of physiological performance. Early in life, at the end of the pelagic larval stage, coral-reef fishes are the fastest swimmers of all fishes in relation to body size, and show the highest specific rates of maximum oxygen uptake. Upon settling on the reef, coral-reef fishes have to adopt a demersal lifestyle, which involves coping with a habitat that can become severely hypoxic, and some fishes may even have to rely on air breathing when their coral homes become air exposed. Oxygen availability appears to be a major ambient selection pressure, making respiratory function a key factor for survival on coral reefs. Consequently, hypoxia tolerance is widespread among coral-reef fishes. Hypoxia can even be a factor to gamble with for those fishes that are mouthbrooders, or a factor that the coral inhabitants may actively seek to reduce by sleep-swimming at night. Here, we summarize the present knowledge of the respiratory ecophysiology of coral-reef teleosts. From an ecophysiological perspective, the coral reef is an exciting and largely unexplored system for testing existing hypotheses and making new discoveries. [Abstract/Link to Full Text]

Wilkinson MT
Sailing the skies: the improbable aeronautical success of the pterosaurs.
J Exp Biol. 2007 May;210(Pt 10):1663-71.
Pterosaur wings bore a striking resemblance to sails, having a bony spar at the leading edge, formed by the forelimb and one enormously elongated digit, and an elastic wing membrane. Such simple wings would be expected to have performed badly due to excessive deformation, membrane flutter and poor control characteristics. Here I discuss how certain anatomical features, specifically a forewing membrane in the inner part of the wing and a system of fibres embedded in the distal part, may have countered these shortcomings. The forewing, supported by the unique pteroid bone, would have reduced the wings' geometric twist, and has been shown in wind tunnel tests to improve membrane stability at low angles of attack and dramatically increase the maximum lift coefficient at high angles of attack. The function of the fibres is poorly understood, but it is suggested that they improved membrane stability and optimised twist nearer the wingtips. [Abstract/Link to Full Text]

Taylor A, Katz B
Motor innervation of the muscle spindle: the contribution of bernhard katz.
J Exp Biol. 2007 May;210(Pt 10):1661-2. [Abstract/Link to Full Text]

Feder ME
Evolvability of physiological and biochemical traits: evolutionary mechanisms including and beyond single-nucleotide mutation.
J Exp Biol. 2007 May;210(Pt 9):1653-60.
A longstanding challenge for biologists has been to explain not just how organisms are adapted to diverse environments, but how these adaptations arise. Although natural selection is clearly sufficient to act on heritable variation, is this heritable variation sufficient to yield complex adaptations and how does this variation itself arise? Much prior focus has been on mutation of single nucleotides in genes. This process is common and can have dramatic phenotypes, but could be limited in its ability to culminate in complex adaptations for two kinds of reasons: (i) because natural selection is powerful, it can purge genetic variation, and (ii) evolutionary transition from the absence to the presence of a complex adaptation seemingly requires multiple mutations at the right place and time and in the right sequence, with each intermediate stage having increased overall fitness; this seems highly improbable. Because the networks that organisms comprise are hierarchical and redundant and have modular structure, however, single-nucleotide mutations can have large and tolerable impacts. Diverse mechanisms, collectively evolutionary capacitors, can shield genetic variation from the purgative of selection. These features can enable evolution to proceed via single-nucleotide mutation. Importantly, single-nucleotide mutation usually only modifies existing genes rather than creating new ones, and numerous other mechanisms eclipse single-nucleotide mutation in creating genetic variation. These include gene duplication (both segmental and whole-genome), lateral gene transfer, hybridization, mobile genetic elements and symbiosis. Other processes can scramble and reassemble nucleotide sequence. The mechanisms beyond single-gene mutation offer considerable promise in detailing the evolution of complex physiological and biochemical traits, and have already done so for several morphological traits. [Abstract/Link to Full Text]

Berenbrink M
Historical reconstructions of evolving physiological complexity: O2 secretion in the eye and swimbladder of fishes.
J Exp Biol. 2007 May;210(Pt 9):1641-52.
The ability of some fishes to inflate their compressible swimbladder with almost pure oxygen to maintain neutral buoyancy, even against the high hydrostatic pressure several thousand metres below the water surface, has fascinated physiologists for more than 200 years. This review shows how evolutionary reconstruction of the components of such a complex physiological system on a phylogenetic tree can generate new and important insights into the origin of complex phenotypes that are difficult to obtain with a purely mechanistic approach alone. Thus, it is shown that oxygen secretion first evolved in the eyes of fishes, presumably for improved oxygen supply to an avascular, metabolically active retina. Evolution of this system was facilitated by prior changes in the pH dependence of oxygen-binding characteristics of haemoglobin (the Root effect) and in the specific buffer value of haemoglobin. These changes predisposed teleost fishes for the later evolution of swimbladder oxygen secretion, which occurred at least four times independently and can be associated with increased auditory sensitivity and invasion of the deep sea in some groups. It is proposed that the increasing availability of molecular phylogenetic trees for evolutionary reconstructions may be as important for understanding physiological diversity in the postgenomic era as the increase of genomic sequence information in single model species. [Abstract/Link to Full Text]

Dow JA
Integrative physiology, functional genomics and the phenotype gap: a guide for comparative physiologists.
J Exp Biol. 2007 May;210(Pt 9):1632-40.
Classical, curiosity-led comparative physiology finds itself at a crossroads. Major funding for classical physiology is becoming harder to find, as grant agencies focus on more molecular approaches or on science with more immediate strategic value to their respective countries. In turn, this shift in funding places Zoology and Animal Science departments under enormous stress: student numbers are buoyant, but how can research funding be maintained at high levels? Our research group has argued for the redefinition of integrative physiology as the investigation of gene function in an organotypic context in the intact animal. Implicit in this definition is the use of transgenics and reverse genetics to manipulate gene function in a cell-specific manner; this in turn implies the use of a genetically tractable 'model organism'. The significance of this definition is that it aligns integrative physiology with functional genomics. Again, functional genomics draws heavily on reverse genetics to elucidate the function of novel genes. The phenotype gap (the mismatch between what a genetic model organism's genome encodes and the reasons that it has historically been studied) emphasises the need to attract and empower functional biologists: can all 13,500 genes in Drosophila really be explained in terms of developmental biology? So, by embracing the integrative physiology manifesto, comparative physiologists can not only accelerate their own research, but their functional skills can make them indispensable in the post-genomic endeavour. [Abstract/Link to Full Text]

Recent Articles in Biological Procedures Online

Pan B, Shi K, Sundaralingam M
Synthesis, Purification and Crystallization of Guanine-rich RNA Oligonucleotides.
Biol Proced Online. 2004;6257-262.
Guanine-rich RNA oligonucleotides display many novel structural motifs in recent crystal structures. Here we describe the procedures of the chemical synthesis and the purification of such RNA molecules that are suitable for X-ray crystallographic studies. Modifications of the previous purification methods allow us to obtain better yields in shorter time. We also provide 24 screening conditions that are very effective in crystallization of the guanine-rich RNA oligonucleotides. Optimal crystallization conditions are usually achieved by adjustment of the concentration of the metal ions and pH of the buffer. Crystals obtained by this method usually diffract to high resolution. [Abstract/Link to Full Text]

Shinohara ET, Hallahan DE, Lu B
The Use of Antisense Oligonucleotides in Evaluating Survivin as a Therapeutic Target for Radiation Sensitization in Lung Cancer.
Biol Proced Online. 2004;6250-256.
Elucidating the mechanism of over and under expression of proteins is critical in developing a better understanding of cancer. Multiple techniques are used to examine differential expression of proteins in cells and assess changes in protein expression in response to therapies such as radiation. Reduced expression can be caused by protein inactivation, mRNA instability, or reduced transcription. The following protocol was used to determine the mechanism for the reduced expression of an antiapoptotic factor, survivin, in normal tissues in response to radiation and the defect in cancer cells that prevents this reduction. We also examined ways to overcome survivin over expression in cancer cells in order to sensitize them to radiation. We will focus on the use of antisense oligonucleotides, cell cycle analysis, and luciferase reporter genes. [Abstract/Link to Full Text]

Rommelaere H, Waterschoot D, Neirynck K, Vandekerckhove J, Ampe C
A method for rapidly screening functionality of actin mutants and tagged actins.
Biol Proced Online. 2004;6235-249.
Recombinant production and biochemical analysis of actin mutants has been hampered by the fact that actin has an absolute requirement for the eukaryotic chaperone CCT to reach its native state. We therefore have developed a method to rapidly screen the folding capacity and functionality of actin variants, by combining in vitro expression of labelled actin with analysis on native gels, band shift assays or copolymerization tests. Additionally, we monitor, using immuno-fluorescence, incorporation of actin variants in cytoskeletal structures in transfected cells. We illustrate the method by two examples. In one we show that tagged versions of actin do not always behave native-like and in the other we study some of the molecular defects of three beta-actin mutants that have been associated with diseases. [Abstract/Link to Full Text]

Pick N, Cameron S, Arad D, Av-Gay Y
Screening of Compounds Toxicity against Human Monocytic cell line-THP-1 by Flow Cytometry.
Biol Proced Online. 2004;6220-225.
The worldwide rapid increase in bacterial resistance to numerous antibiotics requires on-going development of new drugs to enter the market. As the development of new antibiotics is lengthy and costly, early monitoring of compound's toxicity is essential in the development of novel agents. Our interest is in a rapid, simple, high throughput screening method to assess cytotoxicity induced by potential agents. Some intracellular pathogens, such as Mycobacterium tuberculosis primary site of infection is human alveolar macrophages. Thus, evaluation of candidate drugs for macrophage toxicity is crucial. Protocols for high throughput drug toxicity screening of macrophages using flow cytometry are lacking in the literature. For this application we modified a preexisting technique, propidium iodide (PI) exclusion staining and utilized it for rapid toxicity tests. Samples were prepared in 96 well plates and analyzed by flow cytometry, which allowed for rapid, inexpensive and precise assessment of compound's toxicity associated with cell death. [Abstract/Link to Full Text]

Shirley RL, Richards MR, Culbertson MR
Using the cre-lox recombination system to assess functional impairment caused by amino acid substitutions in yeast proteins.
Biol Proced Online. 2004;6209-219.
A method was developed to assess the functional significance of a sequence motif in yeast Upf3p, a protein required for nonsense-mediated mRNA decay (NMD). The motif lies at the edge of the Upf3p-Upf2p interaction domain, but at the same time resembles the canonical leucine-rich nuclear export sequence (NES) found in proteins that bind Crm1p exportin. To test the function of the putative NES, site-directed mutations that cause substitutions of conserved NES-A residues were first selected to identify hypermorphic alleles. Next, a portable Crm1p-binding NES from HIV-1 Rev protein that functions in yeast was fused en masse to the C-terminus of variant Upf3 proteins using loxP sites recognized by bacterial cre-recombinase. Finally, variant Upf3-Rev proteins that were functional in NMD were selected and examined for the types of amino acid substitutions present in NES-A. The mutational analysis revealed that amino acid substitutions in the Upf3 NES impair both nuclear export and the Upf2p-Upf3p interaction, both of which are required for Upf3p to function in NMD. The method described in this report could be modified for the genetic analysis of a variety of portable protein domains. [Abstract/Link to Full Text]

Li J, Sha B
Peptide substrate identification for yeast Hsp40 Ydj1 by screening the phage display library.
Biol Proced Online. 2004;6204-208.
We have identified a peptide substrate for molecular chaperone Hsp40 Ydj1 by utilizing the combination of phage display library screening and isothemol titration calirimetry (ITC). The initial peptide substrate screening for Hsp40 Ydj1 has been carried out by utilizing a 7-mer phage display library. The peptide sequences from the bio-panning were synthesized and object to the direct affinity measurement for Hsp40 Ydj1 by isothemol titration calirimetry studies. The peptide which has the measurable affinity with Ydj1 shows enriched hydrophobic residues in the middle of the substrate fragment. The peptide substrate specificity for molecular chaperone Hsp40 has been analyzed. [Abstract/Link to Full Text]

Lu Q, Richardson B
Methods for Analyzing the Role of DNA Methylation and Chromatin Structure in Regulating T Lymphocyte Gene Expression.
Biol Proced Online. 2004;6189-203.
Chromatin structure, determined in part by DNA methylation, is established during differentiation and prevents expression of genes unnecessary for the function of a given cell type. We reported that DNA methylation and chromatin structure contributes to lymphoid-specific ITGAL (CD11a) and PRF1 (perforin) expression. We used bisulfite sequencing to compare methylation patterns in the ITGAL promoter and 5' flanking region of T cells and fibroblasts, and in the PRF1 promoter and upstream enhancer of CD4+ and CD8+ T cells with fibroblasts. The effects of methylation on promoter function were tested using regional methylation of reporter constructs, and confirmed by DNA methyltransferase inhibition. The relationship between DNA methylation and chromatin structure was analyzed by DNaseI hypersensitivity. Herein we described the methods and results in greater detail. [Abstract/Link to Full Text]

Krishnan NM, Raina SZ, Pollock DD
Analysis of among-site variation in substitution patterns.
Biol Proced Online. 2004;6180-188.
Substitution patterns among nucleotides are often assumed to be constant in phylogenetic analyses. Although variation in the average rate of substitution among sites is commonly accounted for, variation in the relative rates of specific types of substitution is not. Here, we review details of methodologies used for detecting and analyzing differences in substitution processes among predefined groups of sites. We describe how such analyses can be performed using existing phylogenetic tools, and discuss how new phylogenetic analysis tools we have recently developed can be used to provide more detailed and sensitive analyses, including study of the evolution of mutation and substitution processes. As an example we consider the mitochondrial genome, for which two types of transition deaminations (C-->T and A-->G) are strongly affected by single-strandedness during replication, resulting in a strand asymmetric mutation process. Since time spent single-stranded varies along the mitochondrial genome, their differential mutational response results in very different substitution patterns in different regions of the genome. [Abstract/Link to Full Text]

Dunne JL, Goobic AP, Acton ST, Ley K
A novel method to analyze leukocyte rolling behavior in vivo.
Biol Proced Online. 2004;6173-179.
Leukocyte endothelial cell interaction is a fundamentally important process in many disease states. Current methods to analyze such interactions include the parallel-plate flow chamber and intravital microscopy. Here, we present an improvement of the traditional intravital microscopy that allows leukocyte-endothelial cell interaction to be studied from the time the leukocyte makes its initial contact with the endothelium until it adheres to or detaches from the endothelium. The leukocyte is tracked throughout the venular tree with the aid of a motorized stage and the rolling and adhesive behavior is measured off-line. Because this method can involve human error, methods to automate the tracking procedure have been developed. This novel tracking method allows for a more detailed examination of leukocyte-endothelial cell interactions. [Abstract/Link to Full Text]

Pinhero R, Liaw P, Yankulov K
A uniform procedure for the purification of CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1.
Biol Proced Online. 2004;6163-172.
We have established a uniform procedure for the expression and purification of the cyclin-dependent kinases CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. We attach a His(6)-tag to one of the subunits of each complex and then co-express it together with the other subunits in Spodoptera frugiperda insect cells. The CDK complexes are subsequently purified by Ni(2+)-NTA and Mono S chromatography. This approach generates large amounts of active recombinant kinases that are devoid of contaminating kinase activities. Importantly, the properties of these recombinant kinases are similar to their natural counterparts (Pinhero et al. 2004, Eur J Biochem 271:1004-14). Our protocol provides a novel systematic approach for the purification of these three (and possibly other) recombinant CDKs. [Abstract/Link to Full Text]

Vermeulen M, Stunnenberg HG
An in vitro assay to study the recruitment and substrate specificity of chromatin modifying enzymes.
Biol Proced Online. 2004;6157-162.
Post-translational modifications of core histones play an important role in regulating fundamental biological processes such as DNA repair, transcription and replication. In this paper, we describe a novel assay that allows sequential targeting of distinct histone modifying enzymes to immobilized nucleosomal templates using recombinant chimeric targeting molecules. The assay can be used to study the histone substrate specificity of chromatin modifying enzymes as well as whether and how certain enzymes affect each other's histone modifying activities. As such the assay can help to understand how a certain histone code is established and interpreted. [Abstract/Link to Full Text]

Gerhart J, Baytion M, Perlman J, Neely C, Hearon B, Nilsen T, Getts R, Kadushin J, George-Weinstein M
Visualizing the Needle in the Haystack: In Situ Hybridization With Fluorescent Dendrimers.
Biol Proced Online. 2004;6149-156.
In situ hybridization with 3DNA trade mark dendrimers is a novel tool for detecting low levels of mRNA in tissue sections and whole embryos. Fluorescently labeled dendrimers were used to identify cells that express mRNA for the skeletal muscle transcription factor MyoD in the early chick embryo. A small population of MyoD mRNA positive cells was found in the epiblast prior to the initiation of gastrulation, two days earlier than previously detected using enzymatic or radiolabeled probes for mRNA. When isolated from the epiblast and placed in culture, the MyoD mRNA positive cells were able to differentiate into skeletal muscle cells. These results demonstrate that DNA dendrimers are sensitive and precise tools for identifying low levels of mRNA in single cells and tissues. [Abstract/Link to Full Text]

Kienberger F, Zhu R, Moser R, Rankl C, Blaas D, Hinterdorfer P
Dynamic force microscopy for imaging of viruses under physiological conditions.
Biol Proced Online. 2004;6120-128.
Dynamic force microscopy (DFM) allows imaging of the structure and the assessment of the function of biological specimens in their physiological environment. In DFM, the cantilever is oscillated at a given frequency and touches the sample only at the end of its downward movement. Accordingly, the problem of lateral forces displacing or even destroying bio-molecules is virtually inexistent as the contact time and friction forces are reduced. Here, we describe the use of DFM in studies of human rhinovirus serotype 2 (HRV2) weakly adhering to mica surfaces. The capsid of HRV2 was reproducibly imaged without any displacement of the virus. Release of the genomic RNA from the virions was initiated by exposure to low pH buffer and snapshots of the extrusion process were obtained. In the following, the technical details of previous DFM investigations of HRV2 are summarized. [Abstract/Link to Full Text]

Chaves-Pozo E, Mulero V, Meseguer J, Garc�a Ayala A
Flow cytometry based techniques to study testicular acidophilic granulocytes from the protandrous fish gilthead seabream (Sparus aurata L.).
Biol Proced Online. 2004;6129-136.
The gilthead seabream is a protandrous seasonal breeding teleost that is an excellent model for studying the testicular regression process which occurs in both seasonal testicular involution and sex reversion. Little is known about the cell types and the molecular mechanisms involved in such processes, mainly because of the lack of appropriate methods for testis dissociation, and testicular cell isolation, culture and functional characterization. We have previously reported that gilthead seabream acidophilic granulocytes infiltrate the testis at post-spawning stage, settle close to the spermatogonia and accumulate intracellular interleukin-1beta. In this paper, we report several flow cytometry based assays which allow to establish the role played by gilthead seabream testicular acidophilic granulocytes and permits their quantification. [Abstract/Link to Full Text]

Pomati F, Burns BP, Neilan BA
Use of Ion-Channel Modulating Agents to Study Cyanobacterial Na(+) - K(+) Fluxes.
Biol Proced Online. 2004;6137-143.
Here we describe an experimental design aimed to investigate changes in total cellular levels of Na(+) and K(+) ions in cultures of freshwater filamentous cyanobacteria. Ion concentrations were measured in whole cells by flame photometry. Cellular Na(+) levels increased exponentially with rising alkalinity, with K(+) levels being maximal for optimal growth pH (~8). At standardized pH conditions, the increase in cellular Na(+), as induced by NaCl at 10 mM, was coupled by the two sodium channel-modulating agents lidocaine hydrochloride at 1 microM and veratridine at 100 microM. Both the channel-blockers amiloride (1 mM) and saxitoxin (1 microM), decreased cell-bound Na(+) and K(+) levels. Results presented demonstrate the robustness of well-defined channel blockers and channel-activators in the study of cyanobacterial Na(+)- K(+) fluxes. [Abstract/Link to Full Text]

Iqbal Z, Siddiqui RT, Qureshi JA
Two different point mutations in ABL gene ATP-binding domain conferring Primary Imatinib resistance in a Chronic Myeloid Leukemia (CML) patient: A case report.
Biol Proced Online. 2004;6144-148.
Imatinib (Gleevec) is the effective therapy for BCR-ABL positive CML patients. Point mutations have been detected in ATP-binding domain of ABL gene which disturbs the binding of Gleevec to this target leading to resistance. Detection of mutations is helpful in clinical management of imatinib resistance. We established a very sensitive (ASO) PCR to detect mutations in an imatinib-resistant CML patient. Mutations C944T and T1052C were detected which cause complete partial imatinib resistance, respectively. This is the first report of multiple point mutations conferring primary imatinib resistance in same patient at the same time. Understanding the biological reasons of primary imatinib resistance is one of the emerging issues of pharmacogenomics and will be helpful in understanding primary resistance of molecularly-targeted cancer therapies. It will also be of great utilization in clinical management of imatinib resistance. Moreover, this ASO-PCR assay is very effective in detecting mutations related to imatinib resistance. [Abstract/Link to Full Text]

Andersen SS
Expression and purification of recombinant vesicular glutamate transporter VGLUT1 using PC12 cells and High Five insect cells.
Biol Proced Online. 2004;6105-112.
In synaptic vesicles, the estimated concentration of the excitatory amino acid glutamate is 100-150 mM. It was recently discovered that VGLUT1, previously characterized as an inorganic phosphate transporter (BNPI) with 9-11 predicted transmembrane spanning domains, is capable of transporting glutamate. The expression and His-tag based purification of recombinant VGLUT1 from PC12 cells and High Five insect cells is described. Significantly better virus and protein expression was obtained using High Five rather than Sf9 insect cells. PC12 cell expressed VGLUT1 is functional but not the Baculovirus expressed protein. The lack of functionality of the Baculovirus expressed VGLUT1 is discussed. The data indicate that VGLUT1 readily oligomerizes/dimerizes. The data are discussed in the context of developing this system further in order to reconstitute vesicular glutamate uptake in vitro using lipid-detergent vesicles. [Abstract/Link to Full Text]

Davies AG, McIntire SL
Using C. elegans to screen for targets of ethanol and behavior-altering drugs.
Biol Proced Online. 2004;6113-119.
Caenorhabditis elegans is an attractive model system for determining the targets of neuroactive compounds. Genetic screens in C. elegans provide a relatively unbiased approach to the identification of genes that are essential for behavioral effects of drugs and neuroactive compounds such as alcohol. Much work in vertebrate systems has identified multiple potential targets of ethanol but which, if any, of those candidates are responsible for the behavioral effects of alcohol is uncertain. Here we provide detailed methodology for a genetic screen for mutants of C. elegans that are resistant to the depressive effects of ethanol on locomotion and for the subsequent behavioral analysis of those mutants. The methods we describe should also be applicable for use in screening for mutants that are resistant or hypersensitive to many neuroactive compounds and for identifying the molecular targets or biochemical pathways mediating drug responses. [Abstract/Link to Full Text]

Li J, Zhang WB, McManus DP
Recombinant antigens for immunodiagnosis of cystic echinococcosis.
Biol Proced Online. 2004;667-77.
Three cDNAs, termed EpC1, TPxEg and EgG5, were isolated by immunoscreening from an Echinococcus granulosus cDNA library. The recombinant phages exhibited strong reactivity with sera from humans with confirmed cystic echinococcosis (CE) and with sera from mice infected with E. granulosus oncospheres. The cDNAs were subcloned into a pET vector, expressed as fusion proteins tagged with GST and affinity purified against the GST tag. Of the three recombinant proteins, EpC1 achieved the highest performance for serodiagnosis of CE in Western blot analysis using a panel of clinically defined human sera to initially address the sensitivity and specificity of the molecules. The protein yielded an overall sensitivity of 92.2% and specificity of 95.6%, levels unprecedented taking into account the large panel of 896 human sera that were tested. The strategy used may also prove suitable for improved immunodiagnosis of other parasitic infections. [Abstract/Link to Full Text]

Centonze VE, Firulli BA, Firulli AB
Fluorescence Resonance Energy Transfer (FRET) as a method to calculate the dimerization strength of basic Helix-Loop-Helix (bHLH) proteins.
Biol Proced Online. 2004;678-82.
Post-translational modifications such as phosphorylation play a vital role in the regulation of protein function. In our study of the basic Helix-loop-Helix (bHLH) transcription factor HAND1, we show that HAND1 is phosphorylated during the trophoblast giant cell differentiation on residues residing in Helix I of the bHLH domain. Our hypothesis is that these modifications result in changes in HAND1 dimerization affinities with other bHLH factors. To test this idea, we employed FRET to measure the protein-protein interactions of HAND1 and HAND1 point mutants in HEK293 cells using YFP and CFP fusion proteins and laser scanning confocal microscopy. [Abstract/Link to Full Text]

Manna PR, Huhtaniemi IT, Stocco DM
Detection of hCG Responsive Expression of the Steroidogenic Acute Regulatory Protein in Mouse Leydig Cells.
Biol Proced Online. 2004;683-93.
The steroidogenic acute regulatory (StAR) protein, a novel mitochondrial protein, is involved in the regulation of steroid hormone biosynthesis through its mediation of the intramitochondrial transport of the steroid substrate, cholesterol, to the cytochrome P450 cholesterol side chain cleavage (P450scc) enzyme. The expression of StAR protein is regulated by cAMP-dependent signaling in steroidogenic cells. During the course of our studies in mouse Leydig cells, we employ several methods for studying the regulation of StAR protein expression by human chorionic gonadotropin (hCG). A sensitive quantitative reverse transcription and polymerase chain reaction (RT-PCR) was utilized for determining StAR mRNA expression. Stimulation of mLTC-1 mouse Leydig tumor cells with hCG resulted in the coordinate regulation of StAR mRNA expression and progesterone accumulation in a time-response manner. The validity and accuracy of quantitative RT-PCR results in mLTC-1 cells were verified by a competitive PCR approach and were further confirmed in primary cultures of isolated mouse Leydig cells. Immunoblotting studies demonstrated an increase in the levels of the StAR protein in a concentration dependent manner following hCG stimulation in mLTC-1 cells. Northern hybridization analysis revealed three StAR transcripts, all of which were of sufficient size to encode functional StAR protein, and which were coordinately expressed in response to hCG. Collectively, the experimental approaches utilized in the present investigation allow for the demonstration and characterization of hCG mediated regulation of StAR mRNA and StAR protein expression in mouse Leydig cells. [Abstract/Link to Full Text]

Munir S, Singh S, Kaur K, Kapur V
Suppression subtractive hybridization coupled with microarray analysis to examine differential expression of genes in virus infected cells.
Biol Proced Online. 2004;694-104.
High throughput detection of differential expression of genes is an efficient means of identifying genes and pathways that may play a role in biological systems under certain experimental conditions. There exist a variety of approaches that could be used to identify groups of genes that change in expression in response to a particular stimulus or environment. We here describe the application of suppression subtractive hybridization (SSH) coupled with cDNA microarray analysis for isolation and identification of chicken transcripts that change in expression on infection of host cells with a paramyxovirus. SSH was used for initial isolation of differentially expressed transcripts, a large-scale validation of which was accomplished by microarray analysis. The data reveals a large group of regulated genes constituting many biochemical pathways that could serve as targets for future investigations to explore their role in paramyxovirus pathogenesis. The detailed methods described herein could be useful and adaptable to any biological system for studying changes in gene expression. [Abstract/Link to Full Text]

Corsini J, Hacker C, Bare C
Serum-Free Cryopreservation of Five Mammalian Cell Lines in Either a Pelleted or Suspended State.
Biol Proced Online. 2004;661-66.
Herein we have explored two practical aspects of cryopreserving cultured mammalian cells during routine laboratory maintenance. First, we have examined the possibility of using a serum-free, hence more affordable, cryopreservative. Using five mammalian lines (Crandell Feline Kidney, MCF7, A72, WI 38 and NB324K), we found that the serum-free alternative preserves nearly as efficiently as the serum-containing preservatives. Second, we compared cryostorage of those cells in suspended versus a pellet form using both aforementioned cryopreservatives. Under our conditions, cells were in general recovered equally well in a suspended versus a pellet form. [Abstract/Link to Full Text]

Stark HJ, Szabowski A, Fusenig NE, Maas-Szabowski N
Organotypic cocultures as skin equivalents: A complex and sophisticated in vitro system.
Biol Proced Online. 2004;655-60.
To assess the role of genes required for skin organogenesis, tissue regeneration and homeostasis, we have established in vitro skin equivalents composed of primary cells or cell lines, respectively. In these organotypic cocultures keratinocytes generate a normal epidermis irrespective of the species and tissue origin of fibroblasts. The combination of cells derived from mouse and human tissues facilitates the identification of the origin of compounds involved in epidermal tissue reconstitution and thus the precise analysis of growth regulatory mechanisms. [Abstract/Link to Full Text]

Antoun A, Pavlov MY, Tenson T, Ehrenberg M
Ribosome formation from subunits studied by stopped-flow and Rayleigh light scattering.
Biol Proced Online. 2004;635-54.
Light scattering and standard stopped-flow techniques were used to monitor rapid association of ribosomal subunits during initiation of eubacterial protein synthesis. The effects of the initiation factors IF1, IF2, IF3 and buffer conditions on subunit association were studied along with the role of GTP in this process. The part of light scattering theory that is essential for kinetic measurements is high-lighted in the main text and a more general treatment of Rayleigh scattering from macromolecules is given in an appendix. [Abstract/Link to Full Text]

M, Li Y, Cheng Y, Walz T
Negative Staining and Image Classification - Powerful Tools in Modern Electron Microscopy.
Biol Proced Online. 2004;623-34.
Vitrification is the state-of-the-art specimen preparation technique for molecular electron microscopy (EM) and therefore negative staining may appear to be an outdated approach. In this paper we illustrate the specific advantages of negative staining, ensuring that this technique will remain an important tool for the study of biological macromolecules. Due to the higher image contrast, much smaller molecules can be visualized by negative staining. Also, while molecules prepared by vitrification usually adopt random orientations in the amorphous ice layer, negative staining tends to induce preferred orientations of the molecules on the carbon support film. Combining negative staining with image classification techniques makes it possible to work with very heterogeneous molecule populations, which are difficult or even impossible to analyze using vitrified specimens. [Abstract/Link to Full Text]

Firulli BA, Virshup DM, Firulli AB
Phosphopeptide mapping of proteins ectopically expressed in tissue culture cell lines.
Biol Proced Online. 2004;616-22.
Post-translational modifications such as phosphorylation play a vital role in the regulation of protein function. In our study of the basic Helix-loop-Helix (bHLH) transcription factor HAND1, it was suspected that HAND1 was being phosphorylated during trophoblast giant cell differentiation and that coexpression of a constitutively active kinase with HAND1 resulted in changes in the proteins dimerization profile. In order to accurately document HAND1 phosphorylation and identify the resides being modified, we employed metabolic cell labeling with (32)P of tissue culture cells coexpressing a Flag-epitope tagged HAND1 along with a number of active kinases and phosphatase subunits. We generated phosphopeptide maps of the phosphorylated HAND1 using the methods described below and linked these modifications to changes in HAND1 biological function. [Abstract/Link to Full Text]

Zaragoza O, Casadevall A
Experimental modulation of capsule size in Cryptococcus neoformans.
Biol Proced Online. 2004;610-15.
Experimental modulation of capsule size is an important technique for the study of the virulence of the encapsulated pathogen Cryptococcus neoformans. In this paper, we summarize the techniques available for experimental modulation of capsule size in this yeast and describe improved methods to induce capsule size changes. The response of the yeast to the various stimuli is highly dependent on the cryptococcal strain. A high CO(2) atmosphere and a low iron concentration have been used classically to increase capsule size. Unfortunately, these stimuli are not reliable for inducing capsular enlargement in all strains. Recently we have identified new and simpler conditions for inducing capsule enlargement that consistently elicited this effect. Specifically, we noted that mammalian serum or diluted Sabouraud broth in MOPS buffer pH 7.3 efficiently induced capsule growth. Media that slowed the growth rate of the yeast correlated with an increase in capsule size. Finally, we summarize the most commonly used media that induce capsule growth in C. neoformans. [Abstract/Link to Full Text]

Wojcikiewicz EP, Zhang X, Moy VT
Force and Compliance Measurements on Living Cells Using Atomic Force Microscopy (AFM).
Biol Proced Online. 2004;61-9.
We describe the use of atomic force microscopy (AFM) in studies of cell adhesion and cell compliance. Our studies use the interaction between leukocyte function associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) as a model system. The forces required to unbind a single LFA-1/ICAM-1 bond were measured at different loading rates. This data was used to determine the dynamic strength of the LFA-1/ICAM-1 complex and characterize the activation potential that this complex overcomes during its breakage. Force measurements acquired at the multiple- bond level provided insight about the mechanism of cell adhesion. In addition, the AFM was used as a microindenter to determine the mechanical properties of cells. The applications of these methods are described using data from a previous study. [Abstract/Link to Full Text]

Pridgeon JW, Geetha T, Wooten MW
A Method to Identify p62's UBA Domain Interacting Proteins.
Biol Proced Online. 2003;5228-237.
The UBA domain is a conserved sequence motif among polyubiquitin binding proteins. For the first time, we demonstrate a systematic, high throughput approach to identification of UBA domain-interacting proteins from a proteome-wide perspective. Using the rabbit reticulocyte lysate in vitro expression cloning system, we have successfully identified eleven proteins that interact with p62's UBA domain, and the majority of the eleven proteins are associated with neurodegenerative disorders, such as Alzheimer's disease. Therefore, p62 may play a novel regulatory role through its UBA domain. Our approach provides an easy route to the characterization of UBA domain interacting proteins and its application will unfold the important roles that the UBA domain plays. [Abstract/Link to Full Text]

Recent Articles in BMC Developmental Biology

Panchal H, Wansbury O, Parry S, Ashworth A, Howard B
Neuregulin3 alters cell fate in the epidermis and mammary gland.
BMC Dev Biol. 2007;7105.
BACKGROUND: The Neuregulin family of ligands and their receptors, the Erbb tyrosine kinases, have important roles in epidermal and mammary gland development as well as during carcinogenesis. Previously, we demonstrated that Neuregulin3 (Nrg3) is a specification signal for mammary placode formation in mice. Nrg3 is a growth factor, which binds and activates Erbb4, a receptor tyrosine kinase that regulates cell proliferation and differentiation. To understand the role of Neuregulin3 in epidermal morphogenesis, we have developed a transgenic mouse model that expresses Nrg3 throughout the basal layer (progenitor/stem cell compartment) of mouse epidermis and the outer root sheath of developing hair follicles. RESULTS: Transgenic females formed supernumerary nipples and mammary glands along and adjacent to the mammary line providing strong evidence that Nrg3 has a role in the initiation of mammary placodes along the body axis. In addition, alterations in morphogenesis and differentiation of other epidermal appendages were observed, including the hair follicles. The transgenic epidermis is hyperplastic with excessive sebaceous differentiation and shows striking similarities to mouse models in which c-Myc is activated in the basal layer including decreased expression levels of the adhesion receptors, alpha6-integrin and beta1-integrin. CONCLUSION: These results indicate that the epidermis is sensitive to Nrg3 signaling, and that this growth factor can regulate cell fate of pluripotent epidermal cell populations including that of the mammary gland. Nrg3 appears to act, in part, by inducing c-Myc, altering the proliferation and adhesion properties of the basal epidermis, and may promote exit from the stem cell compartment. The results we describe provide significant insight into how growth factors, such as Nrg3, regulate epidermal homeostasis by influencing the balance between stem cell renewal, lineage selection and differentiation. [Abstract/Link to Full Text]

La Salle S, Oakes CC, Neaga OR, Bourc'his D, Bestor TH, Trasler JM
Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L.
BMC Dev Biol. 2007 Sep 18;7(1):104.
ABSTRACT: BACKGROUND: Formation of haploid spermatozoa capable of fertilization requires proper programming of epigenetic information. Exactly how DNMT3L (DNA methyltransferase 3-Like), a postulated regulator of DNA methyltransferase activity, contributes to DNA methylation pattern acquisition during gametogenesis remains unclear. Here we report on the role of DNMT3L in male germ cell development. RESULTS: A developmental study covering the first 12 days following birth was conducted on a Dnmt3L mutant mouse model; lower germ cell numbers and delayed entry into meiosis were observed in Dnmt3L-/- males, pointing to a mitotic defect. A temporal expression study showed that expression of Dnmt3L is highest in prenatal gonocytes but is also detected and developmentally regulated during spermatogenesis. Using a restriction enzyme qPCR assay (qAMP), DNA methylation analyses were conducted on postnatal primitive type A spermatogonia lacking DNMT3L. Methylation levels along 61 sites across chromosomes 4 and X decreased significantly by approximately 50% compared to the levels observed in Dnmt3L+/+ germ cells, suggesting that many loci throughout the genome are marked for methylation by DNMT3L. More so, hypomethylation was more pronounced in regions of lower GC content than in regions of higher GC content. CONCLUSIONS: Taken together, these data suggest that DNMT3L plays a more global role in genomic methylation patterning than previously believed. [Abstract/Link to Full Text]

Garcia-Frigola C, Carreres MI, Vegar C, Herrera E
Gene delivery into mouse retinal ganglion cells by in utero electroporation.
BMC Dev Biol. 2007;7103.
BACKGROUND: The neural retina is a highly structured tissue of the central nervous system that is formed by seven different cell types that are arranged in layers. Despite much effort, the genetic mechanisms that underlie retinal development are still poorly understood. In recent years, large-scale genomic analyses have identified candidate genes that may play a role in retinal neurogenesis, axon guidance and other key processes during the development of the visual system. Thus, new and rapid techniques are now required to carry out high-throughput analyses of all these candidate genes in mammals. Gene delivery techniques have been described to express exogenous proteins in the retina of newborn mice but these approaches do not efficiently introduce genes into the only retinal cell type that transmits visual information to the brain, the retinal ganglion cells (RGCs). RESULTS: Here we show that RGCs can be targeted for gene expression by in utero electroporation of the eye of mouse embryos. Accordingly, using this technique we have monitored the morphology of electroporated RGCs expressing reporter genes at different developmental stages, as well as their projection to higher visual targets. CONCLUSION: Our method to deliver ectopic genes into mouse embryonic retinas enables us to follow the course of the entire retinofugal pathway by visualizing RGC bodies and axons. Thus, this technique will permit to perform functional studies in vivo focusing on neurogenesis, axon guidance, axon projection patterning or neural connectivity in mammals. [Abstract/Link to Full Text]

Barraud-Lange V, Naud-Barriant N, Saffar L, Gattegno L, Ducot B, Drillet AS, Bomsel M, Wolf JP, Ziyyat A
Alpha6beta1 integrin expressed by sperm is determinant in mouse fertilization.
BMC Dev Biol. 2007;7102.
BACKGROUND: Based on inhibition tests, the alpha6beta1 integrin was suggested to be a sperm receptor, but further experiments using gene deletion techniques have shown that neither oocyte alpha6, nor beta1 integrin subunits were essential for mouse fertilization. RESULTS: Using Western blot analysis and immunofluorescence, we showed that the mouse sperm expresses the alpha6beta1 integrin. As for oocyte, binding of GoH3 anti-alpha6 antibody to sperm induces a specific inhibition of sperm fertilizing ability. Comparing zona-intact and zona-free eggs in fusion tests, we showed that the removal of the zona pellucida by acid treatment bypasses fertilizing oocyte alpha6beta1 integrin's function in the adhesion/fusion process. CONCLUSION: These findings show that alpha6beta1 integrin is expressed by both gametes and is functional in their membranes interaction. These results and previous reports, about fertilization of alpha6 or beta1 integrin subunits deleted oocytes by wild type sperm, suggest that the presence of alpha6beta1 integrin on one of the two gamete membranes can rescue the fertilization process. This hypothesis is further supported by the exchange of membrane fragments occurring between gametes prior to fusion that we recently reported. [Abstract/Link to Full Text]

Ciche TA, Sternberg PW
Postembryonic RNAi in Heterorhabditis bacteriophora: a nematode insect parasite and host for insect pathogenic symbionts.
BMC Dev Biol. 2007;7101.
BACKGROUND: Heterorhabditis bacteriophora is applied throughout the world for the biological control of insects and is an animal model to study interspecies interactions, e.g. mutualism, parasitism and vector-borne disease. H. bacteriophora nematodes are mutually associated with the insect pathogen, Photorhabdus luminescens. The developmentally arrested infective juvenile (IJ) stage nematode (vector) specifically transmits Photorhabdus luminescens bacteria (pathogen) in its gut mucosa to the haemocoel of insects (host). The nematode vector and pathogen alone are not known to cause insect disease. RNA interference is an excellent reverse genetic tool to study gene function in C. elegans, and it would be useful in H. bacteriophora to exploit the H. bacteriophora genome project, currently in progress. RESULTS: Soaking L1 stage H. bacteriophora with seven dsRNAs of genes whose C. elegans orthologs had severe RNAi phenotypes resulted in highly penetrant and obvious developmental and reproductive abnormalities. The efficacy of postembryonic double strand RNA interference (RNAi) was evident by abnormal gonad morphology and sterility of adult H. bacteriophora and C. elegans presumable due to defects in germ cell proliferation and gonad development. The penetrance of RNAi phenotypes in H. bacteriophora was high for five genes (87-100%; Hba-cct-2, Hba-daf-21, Hba-icd-1; Hba-nol-5, and Hba-W01G7.3) and moderate for two genes (usually 30-50%; Hba-rack-1 and Hba-arf-1). RNAi of three additional C. elegans orthologs for which RNAi phenotypes were not previously detected in C. elegans, also did not result in any apparent phenotypes in H. bacteriophora. Specific and severe reduction in transcript levels in RNAi treated L1s was determined by quantitative real-time RT-PCR. These results suggest that postembryonic RNAi by soaking is potent and specific. CONCLUSION: Although RNAi is conserved in animals and plants, RNAi using long dsRNA is not. These results demonstrate that RNAi can be used effectively in H. bacteriophora and can be applied for analyses of nematode genes involved in symbiosis and parasitism. It is likely that RNAi will be an important tool for functional genomics utilizing the high quality draft H. bacteriophora genome sequence. [Abstract/Link to Full Text]

Woolfe A, Goode DK, Cooke J, Callaway H, Smith S, Snell P, McEwen GK, Elgar G
CONDOR: a database resource of developmentally associated conserved non-coding elements.
BMC Dev Biol. 2007;7100.
BACKGROUND: Comparative genomics is currently one of the most popular approaches to study the regulatory architecture of vertebrate genomes. Fish-mammal genomic comparisons have proved powerful in identifying conserved non-coding elements likely to be distal cis-regulatory modules such as enhancers, silencers or insulators that control the expression of genes involved in the regulation of early development. The scientific community is showing increasing interest in characterizing the function, evolution and language of these sequences. Despite this, there remains little in the way of user-friendly access to a large dataset of such elements in conjunction with the analysis and the visualization tools needed to study them. DESCRIPTION: Here we present CONDOR (COnserved Non-coDing Orthologous Regions) available at: http://condor.fugu.biology.qmul.ac.uk. In an interactive and intuitive way the website displays data on > 6800 non-coding elements associated with over 120 early developmental genes and conserved across vertebrates. The database regularly incorporates results of ongoing in vivo zebrafish enhancer assays of the CNEs carried out in-house, which currently number approximately 100. Included and highlighted within this set are elements derived from duplication events both at the origin of vertebrates and more recently in the teleost lineage, thus providing valuable data for studying the divergence of regulatory roles between paralogs. CONDOR therefore provides a number of tools and facilities to allow scientists to progress in their own studies on the function and evolution of developmental cis-regulation. CONCLUSION: By providing access to data with an approachable graphics interface, the CONDOR database presents a rich resource for further studies into the regulation and evolution of genes involved in early development. [Abstract/Link to Full Text]

Herpin A, Schindler D, Kraiss A, Hornung U, Winkler C, Schartl M
Inhibition of primordial germ cell proliferation by the medaka male determining gene Dmrt I bY.
BMC Dev Biol. 2007;799.
BACKGROUND: Dmrt I is a highly conserved gene involved in the determination and early differentiation phase of the primordial gonad in vertebrates. In the fish medaka dmrt I bY, a functional duplicate of the autosomal dmrt I a gene on the Y-chromosome, has been shown to be the master regulator of male gonadal development, comparable to Sry in mammals. In males mRNA and protein expression was observed before morphological sex differentiation in the somatic cells surrounding primordial germ cells (PGCs) of the gonadal anlage and later on exclusively in Sertoli cells. This suggested a role for dmrt I bY during male gonad and germ cell development. RESULTS: We provide functional evidence that expression of dmrt I bY leads to negative regulation of PGC proliferation. Flow cytometric measurements revealed a G2 arrest of dmrt I bY expressing cells. Interestingly, also non-transfected cells displayed a significantly lower fraction of proliferating cells, pointing to a possible non-cell autonomous action of dmrt I bY. Injection of antisense morpholinos led to an increase of PGCs in genetically male embryos due to loss of proliferation inhibition. CONCLUSION: In medaka, dmrt I bY mediates a mitotic arrest of PGCs in males prior to testes differentiation at the sex determination stage. This occurs possibly via a cross-talk of Sertoli cells and PGCs. [Abstract/Link to Full Text]

Pasterkamp RJ, Kolk SM, Hellemons AJ, Kolodkin AL
Expression patterns of semaphorin7A and plexinC1 during rat neural development suggest roles in axon guidance and neuronal migration.
BMC Dev Biol. 2007;798.
BACKGROUND: Although originally identified as embryonic axon guidance cues, semaphorins are now known to regulate multiple, distinct, processes crucial for neuronal network formation including axon growth and branching, dendritic morphology, and neuronal migration. Semaphorin7A (Sema7A), the only glycosylphosphatidylinositol-anchored semaphorin, promotes axon growth in vitro and is required for the proper growth of the mouse lateral olfactory tract in vivo. Sema7A has been postulated to signal through two unrelated receptors, an RGD-dependent alpha1beta1-integrin and a member of the plexin family, plexinC1. beta1-integrins underlie Sema7A-mediated axon growth and Sema7A function in the immune system. Sema7A-plexinC1 interactions have also been implicated in immune system function, but the neuronal role of this ligand-receptor pair remains to be explored. To gain further insight into the function(s) of Sema7A and plexinC1 during neural development, we present here a detailed analysis of Sema7A and plexinC1 expression in the developing rat nervous system. RESULTS: In situ hybridization revealed select expression of Sema7A and plexinC1 in multiple neuronal systems including: the olfactory system, the hypothalamo-hypophysial system, the hippocampus, the meso-diencephalic dopamine system, and the spinal cord. Within these systems, Sema7A and plexinC1 are often expressed in specific neuronal subsets. In general, Sema7A transcript levels increase significantly towards adulthood, whereas plexinC1 expression decreases as development proceeds.PlexinC1, but not Sema7A, is strongly expressed by distinct populations of migrating neurons. In addition to neuronal expression, Sema7A and plexinC1 transcripts were detected in oligodendrocytes and ependymal cells, respectively. CONCLUSION: Sema7A and plexinC1 expression patterns are consistent with these proteins serving both cooperative and separate functions during neural development. The prominent expression of plexinC1 in several distinct populations of migrating neurons suggests a novel role for this plexin family member in neuronal migration. [Abstract/Link to Full Text]

Yang Z, Jiang H, Zhao F, Shankar DB, Sakamoto KM, Zhang MQ, Lin S
A highly conserved regulatory element controls hematopoietic expression of GATA-2 in zebrafish.
BMC Dev Biol. 2007;797.
BACKGROUND: GATA-2 is a transcription factor required for hematopoietic stem cell survival as well as for neuronal development in vertebrates. It has been shown that specific expression of GATA-2 in blood progenitor cells requires distal cis-acting regulatory elements. Identification and characterization of these elements should help elucidating transcription regulatory mechanisms of GATA-2 expression in hematopoietic lineage. RESULTS: By pair-wise alignments of the zebrafish genomic sequences flanking GATA-2 to orthologous regions of fugu, mouse, rat and human genomes, we identified three highly conserved non-coding sequences in the genomic region flanking GATA-2, two upstream of GATA-2 and another downstream. Using both transposon and bacterial artificial chromosome mediated germline transgenic zebrafish analyses, one of the sequences was established as necessary and sufficient to direct hematopoietic GFP expression in a manner that recapitulates that of GATA-2. In addition, we demonstrated that this element has enhancer activity in mammalian myeloid leukemia cell lines, thus validating its functional conservation among vertebrate species. Further analysis of potential transcription factor binding sites suggested that integrity of the putative HOXA3 and LMO2 sites is required for regulating GATA-2/GFP hematopoietic expression. CONCLUSION: Regulation of GATA-2 expression in hematopoietic cells is likely conserved among vertebrate animals. The integrated approach described here, drawing on embryological, transgenesis and computational methods, should be generally applicable to analyze tissue-specific gene regulation involving distal DNA cis-acting elements. [Abstract/Link to Full Text]

Perea-Gomez A, Meilhac SM, Piotrowska-Nitsche K, Gray D, Collignon J, Zernicka-Goetz M
Regionalization of the mouse visceral endoderm as the blastocyst transforms into the egg cylinder.
BMC Dev Biol. 2007;796.
BACKGROUND: Reciprocal interactions between two extra-embryonic tissues, the extra-embryonic ectoderm and the visceral endoderm, and the pluripotent epiblast, are required for the establishment of anterior-posterior polarity in the mouse. After implantation, two visceral endoderm cell types can be distinguished, in the embryonic and extra-embryonic regions of the egg cylinder. In the embryonic region, the specification of the anterior visceral endoderm (AVE) is central to the process of anterior-posterior patterning. Despite recent advances in our understanding of the molecular interactions underlying the differentiation of the visceral endoderm, little is known about how cells colonise the three regions of the tissue. RESULTS: As a first step, we performed morphological observations to understand how the extra-embryonic region of the egg cylinder forms from the blastocyst. Our analysis suggests a new model for the formation of this region involving cell rearrangements such as folding of the extra-embryonic ectoderm at the early egg cylinder stage. To trace visceral endoderm cells, we microinjected mRNAs encoding fluorescent proteins into single surface cells of the inner cell mass of the blastocyst and analysed the distribution of labelled cells at E5.0, E5.5 and E6.5. We found that at E5.0 the embryonic and extra-embryonic regions of the visceral endoderm do not correspond to distinct cellular compartments. Clusters of labelled cells may span the junction between the two regions even after the appearance of histological and molecular differences at E5.5. We show that in the embryonic region cell dispersion increases after the migration of the AVE. At this time, visceral endoderm cell clusters tend to become oriented parallel to the junction between the embryonic and extra-embryonic regions. Finally we investigated the origin of the AVE and demonstrated that this anterior signalling centre arises from more than a single precursor between E3.5 and E5.5. CONCLUSION: We propose a new model for the formation of the extra-embryonic region of the egg cylinder involving a folding of the extra-embryonic ectoderm. Our analyses of the pattern of labelled visceral endoderm cells indicate that distinct cell behaviour in the embryonic and extra-embryonic regions is most apparent upon AVE migration. We also demonstrate the polyclonal origin of the AVE. Taken together, these studies lead to further insights into the formation of the extra-embryonic tissues as they first develop after implantation. [Abstract/Link to Full Text]

Lehnert SA, Reverter A, Byrne KA, Wang Y, Nattrass GS, Hudson NJ, Greenwood PL
Gene expression studies of developing bovine longissimus muscle from two different beef cattle breeds.
BMC Dev Biol. 2007;795.
BACKGROUND: The muscle fiber number and fiber composition of muscle is largely determined during prenatal development. In order to discover genes that are involved in determining adult muscle phenotypes, we studied the gene expression profile of developing fetal bovine longissimus muscle from animals with two different genetic backgrounds using a bovine cDNA microarray. Fetal longissimus muscle was sampled at 4 stages of myogenesis and muscle maturation: primary myogenesis (d 60), secondary myogenesis (d 135), as well as beginning (d 195) and final stages (birth) of functional differentiation of muscle fibers. All fetuses and newborns (total n = 24) were from Hereford dams and crossed with either Wagyu (high intramuscular fat) or Piedmontese (GDF8 mutant) sires, genotypes that vary markedly in muscle and compositional characteristics later in postnatal life. RESULTS: We obtained expression profiles of three individuals for each time point and genotype to allow comparisons across time and between sire breeds. Quantitative reverse transcription-PCR analysis of RNA from developing longissimus muscle was able to validate the differential expression patterns observed for a selection of differentially expressed genes, with one exception. We detected large-scale changes in temporal gene expression between the four developmental stages in genes coding for extracellular matrix and for muscle fiber structural and metabolic proteins. FSTL1 and IGFBP5 were two genes implicated in growth and differentiation that showed developmentally regulated expression levels in fetal muscle. An abundantly expressed gene with no functional annotation was found to be developmentally regulated in the same manner as muscle structural proteins. We also observed differences in gene expression profiles between the two different sire breeds. Wagyu-sired calves showed higher expression of fatty acid binding protein 5 (FABP5) RNA at birth. The developing longissimus muscle of fetuses carrying the Piedmontese mutation shows an emphasis on glycolytic muscle biochemistry and a large-scale up-regulation of the translational machinery at birth. We also document evidence for timing differences in differentiation events between the two breeds. CONCLUSION: Taken together, these findings provide a detailed description of molecular events accompanying skeletal muscle differentiation in the bovine, as well as gene expression differences that may underpin the phenotype differences between the two breeds. In addition, this study has highlighted a non-coding RNA, which is abundantly expressed and developmentally regulated in bovine fetal muscle. [Abstract/Link to Full Text]

Domanski D, Helbing CC
Analysis of the Rana catesbeiana tadpole tail fin proteome and phosphoproteome during T3-induced apoptosis: identification of a novel type I keratin.
BMC Dev Biol. 2007;794.
BACKGROUND: Thyroid hormones (THs) are vital in the maintenance of homeostasis and in the control of development. One postembryonic developmental process that is principally regulated by THs is amphibian metamorphosis. This process has been intensively studied at the genomic level yet very little information at the proteomic level exists. In addition, there is increasing evidence that changes in the phosphoproteome influence TH action. RESULTS: Here we identify components of the proteome and phosphoproteome in the tail fin that changed within 48 h of exposure of premetamorphic Rana catesbeiana tadpoles to 10 nM 3,5,3'-triiodothyronine (T3). To this end, we developed a cell and protein fractionation method combined with two-dimensional gel electrophoresis and phosphoprotein-specific staining. Altered proteins were identified using mass spectrometry (MS). We identified and cloned a novel Rana larval type I keratin, RLK I, which may be a target for caspase-mediated proteolysis upon exposure to T3. In addition, the RLK I transcript is reduced during T3-induced and natural metamorphosis which is consistent with a larval keratin. Furthermore, GILT, a protein involved in the immune system, is changed in phosphorylation state which is linked to its activation. Using a complementary MS technique for the analysis of differentially-expressed proteins, isobaric tags for relative and absolute quantitation (iTRAQ) revealed 15 additional proteins whose levels were altered upon T3 treatment. The success of identifying proteins whose levels changed upon T3 treatment with iTRAQ was enhanced through de novo sequencing of MS data and homology database searching. These proteins are involved in apoptosis, extracellular matrix structure, immune system, metabolism, mechanical function, and oxygen transport. CONCLUSION: We have demonstrated the ability to derive proteomics-based information from a model species for postembryonic development for which no genome information is currently available. The present study identifies proteins whose levels and/or phosphorylation states are altered within 48 h of the induction of tadpole tail regression prior to overt remodeling of the tail. In particular, we have identified a novel keratin that is a target for T3-mediated changes in the tail that can serve as an indicator of early response to this hormone. [Abstract/Link to Full Text]

Lee HC, Tsai JN, Liao PY, Tsai WY, Lin KY, Chuang CC, Sun CK, Chang WC, Tsai HJ
Glycogen synthase kinase 3 alpha and 3 beta have distinct functions during cardiogenesis of zebrafish embryo.
BMC Dev Biol. 2007;793.
BACKGROUND: Glycogen synthase kinase 3 (GSK3) encodes a serine/threonine protein kinase, is known to play roles in many biological processes. Two closely related GSK3 isoforms encoded by distinct genes: GSK3alpha (51 kDa) and GSK3beta (47 kDa). In previously studies, most GSK3 inhibitors are not only inhibiting GSK3, but are also affecting many other kinases. In addition, because of highly similarity in amino acid sequence between GSK3alpha and GSK3beta, making it difficult to identify an inhibitor that can be selective against GSK3alpha or GSK3beta. Thus, it is relatively difficult to address the functions of GSK3 isoforms during embryogenesis. At this study, we attempt to specifically inhibit either GSK3alpha or GSK3beta and uncover the isoform-specific roles that GSK3 plays during cardiogenesis. RESULTS: We blocked gsk3alpha and gsk3beta translations by injection of morpholino antisense oligonucleotides (MO). Both gsk3alpha- and gsk3beta-MO-injected embryos displayed similar morphological defects, with a thin, string-like shaped heart and pericardial edema at 72 hours post-fertilization. However, when detailed analysis of the gsk3alpha- and gsk3beta-MO-induced heart defects, we found that the reduced number of cardiomyocytes in gsk3alpha morphants during the heart-ring stage was due to apoptosis. On the contrary, gsk3beta morphants did not exhibit significant apoptosis in the cardiomyocytes, and the heart developed normally during the heart-ring stage. Later, however, the heart positioning was severely disrupted in gsk3beta morphants. bmp4 expression in gsk3beta morphants was up-regulated and disrupted the asymmetry pattern in the heart. The cardiac valve defects in gsk3beta morphants were similar to those observed in axin1 and apcmcr mutants, suggesting that GSK3beta might play a role in cardiac valve development through the Wnt/beta-catenin pathway. Finally, the phenotypes of gsk3alpha mutant embryos cannot be rescued by gsk3beta mRNA, and vice versa, demonstrating that GSK3alpha and GSK3beta are not functionally redundant. CONCLUSION: We conclude that (1) GSK3alpha, but not GSK3beta, is necessary in cardiomyocyte survival; (2) the GSK3beta plays important roles in modulating the left-right asymmetry and affecting heart positioning; and (3) GSK3alpha and GSK3beta play distinct roles during zebrafish cardiogenesis. [Abstract/Link to Full Text]

Hou J, Charters AM, Lee SC, Zhao Y, Wu MK, Jones SJ, Marra MA, Hoodless PA
A systematic screen for genes expressed in definitive endoderm by Serial Analysis of Gene Expression (SAGE).
BMC Dev Biol. 2007;792.
BACKGROUND: The embryonic definitive endoderm (DE) gives rise to organs of the gastrointestinal and respiratory tract including the liver, pancreas and epithelia of the lung and colon. Understanding how DE progenitor cells generate these tissues is critical to understanding the cause of visceral organ disorders and cancers, and will ultimately lead to novel therapies including tissue and organ regeneration. However, investigation into the molecular mechanisms of DE differentiation has been hindered by the lack of early DE-specific markers. RESULTS: We describe the identification of novel as well as known genes that are expressed in DE using Serial Analysis of Gene Expression (SAGE). We generated and analyzed three longSAGE libraries from early DE of murine embryos: early whole definitive endoderm (0-6 somite stage), foregut (8-12 somite stage), and hindgut (8-12 somite stage). A list of candidate genes enriched for expression in endoderm was compiled through comparisons within these three endoderm libraries and against 133 mouse longSAGE libraries generated by the Mouse Atlas of Gene Expression Project encompassing multiple embryonic tissues and stages. Using whole mount in situ hybridization, we confirmed that 22/32 (69%) genes showed previously uncharacterized expression in the DE. Importantly, two genes identified, Pyy and 5730521E12Rik, showed exclusive DE expression at early stages of endoderm patterning. CONCLUSION: The high efficiency of this endoderm screen indicates that our approach can be successfully used to analyze and validate the vast amount of data obtained by the Mouse Atlas of Gene Expression Project. Importantly, these novel early endoderm-expressing genes will be valuable for further investigation into the molecular mechanisms that regulate endoderm development. [Abstract/Link to Full Text]

Chapman SC, Matsumoto K, Cai Q, Schoenwolf GC
Specification of germ layer identity in the chick gastrula.
BMC Dev Biol. 2007;791.
BACKGROUND: Chick definitive endoderm is an important source of signals that pattern the early embryo forming a central structure around which the body plan is constructed. Although the origin of definitive endoderm has been mapped in the chick, arising principally from rostral streak at elongating streak stages, it is not known when this layer first becomes fully committed to its germ layer fate, an important issue to resolve in light of its critical role in subsequent patterning of the early embryo. RESULTS: Through gene expression screening of chick gastrula, we identified molecular markers of definitive endoderm restricted to rostral (Sox17) and caudal (Gata5/6) regions, suggesting that at least two subpopulations of definitive endodermal cells exist during ingression. We show (1) that presumptive mesoderm cells migrate to the middle layer and remain mesenchymal when transplanted to rostral primitive streak, and prospective endoderm cells enter the lower layer and become epithelial when transplanted to caudal primitive streak; and (2) that presumptive endoderm cells and mesoderm cells lose normal gene expression (Sox17 and Wnt8c, respectively) when transplanted outside of their normal position of origin. Moreover, when rostral or caudal primitive streak segments are transplanted into rostral blastoderm isolates (RBIs), both types of transplants express Sox17 4-6 hours later--consistent with their new position, regardless of their presumptive germ layer origin--and prospective mesoderm transplants, which normally express Wnt8c, turn off expression, suggesting that signals within the rostral blastoderm induce endoderm gene expression, and repress mesoderm gene expression, during gastrulation. CONCLUSION: Our results demonstrate that germ layer identity is fixed at the time populations of endoderm and mesoderm cells ingress through the primitive streak, whereas their gene expression patterns remain labile. In addition, our results show that inductive and repressive signals are present, and that these signals regulate gene expression of both ingressed endoderm and mesoderm cells. Thus, gastrula cells display elements of both pre-patterning and plasticity, with endoderm the first germ layer becoming committed to its fate during early gastrulation stages. [Abstract/Link to Full Text]

Ghanem N, H�lker M, Rings F, Jennen D, Tholen E, Sirard MA, Torner H, Kanitz W, Schellander K, Tesfaye D
Alterations in transcript abundance of bovine oocytes recovered at growth and dominance phases of the first follicular wave.
BMC Dev Biol. 2007;790.
BACKGROUND: Oocyte developmental competence is highly affected by the phase of ovarian follicular wave. Previous studies have shown that oocytes from subordinate follicles recovered at growth phase (day 3 after estrus) are developmentally more competent than those recovered at dominance phase (day 7 after estrus). However, the molecular mechanisms associated with these differences are not well elucidated. Therefore, the objective of this study was to investigate transcript abundance of bovine oocytes retrieved from small follicles at growth and dominance phases of the first follicular wave and to identify candidate genes related to oocyte developmental competence using cDNA microarray. RESULTS: Comparative gene expression analysis of oocytes from growth and dominance phases and subsequent data analysis using Significant Analysis of Microarray (SAM) revealed a total of 51 differentially regulated genes, including 36 with known function, 6 with unknown function and 9 novel transcripts. Real-time PCR has validated 10 transcripts revealed by microarray analysis and quantified 5 genes in cumulus cells derived from oocytes of both phases. The expression profile of 8 (80%) transcripts (ANAXA2, FL396, S100A10, RPL24, PP, PTTG1, MSX1 and BMP15) was in agreement with microarray data. Transcript abundance of five candidate genes in relation to oocyte developmental competence was validated using Brilliant Cresyl Blue (BCB) staining as an independent model. Furthermore, localization of mRNA and protein product of the candidate gene MSX1 in sections of ovarian follicles at days 0, 1, 3 and 7 of estrous cycle showed a clear fluorescent signal in both oocytes and cumulus cells with higher intensity in the former. Moreover, the protein product was detected in bovine oocytes and early cleavage embryos after fertilization with higher intensity around the nucleus. CONCLUSION: This study has identified distinct sets of differentially regulated transcripts between bovine oocytes recovered from small follicles at growth and dominance phases of the first follicular wave. The validation with independent model supports our notion that many of the transcripts identified here may represent candidate genes associated with oocyte developmental competence. Further specific functional analysis will provide insights into the exact role of these transcripts in oocyte competence and early embryonic development. [Abstract/Link to Full Text]

Mahler J, Driever W
Expression of the zebrafish intermediate neurofilament Nestin in the developing nervous system and in neural proliferation zones at postembryonic stages.
BMC Dev Biol. 2007;789.
BACKGROUND: The intermediate filament Nestin has been reported as a marker for stem cells and specific precursor cell populations in the developing mammalian central nervous system (CNS). Nestin expressing precursors may give rise to neurons and glia. Mouse nestin expression starts at the onset of neurulation in the neuroectodermal cells and is dramatically down regulated when progenitor cells differentiate and become postmitotic. It has been reported that in the adult zebrafish (Danio rerio) active neurogenesis continues in all major subdivisions of the CNS, however few markers for zebrafish precursors cells are known, and Nestin has not been described in zebrafish. RESULTS: We cloned a zebrafish nestin gDNA fragment in order to find a marker for precursor cells in the developing and postembryonic brain. Phylogenetic tree analysis reveals that this zebrafish ortholog clusters with Nestin sequences from other vertebrates but not with other intermediate filament proteins. We analyzed nestin expression from gastrula stage to 4 day larvae, and in post-embryonic brains. We found broad expression in the neuroectoderm during somitogenesis. In the larvae, nestin expression progressively becomes restricted to all previously described proliferative zones of the developing and postembryonic central nervous system. nestin expressing cells of the forebrain also express PCNA during late embryogenesis, identifying them as proliferating precursor or neural stem cells. nestin is also expressed in the cranial ganglia, in mesodermal precursors of muscle cells, and in cranial mesenchymal tissue. CONCLUSION: Our data demonstrate that in zebrafish, like in mammals, the expression of the intermediated neurofilament nestin gene may serve as a marker for stem cells and proliferating precursors in the developing embryonic nervous system as well as in the postembryonic brain. [Abstract/Link to Full Text]

Liu S, Xia Q, Zhao P, Cheng T, Hong K, Xiang Z
Characterization and expression patterns of let-7 microRNA in the silkworm (Bombyx mori).
BMC Dev Biol. 2007;788.
BACKGROUND: lin-4 and let-7, the two founding members of heterochronic microRNA genes, are firstly confirmed in Caenorhabditis elegans to control the proper timing of developmental programs in a heterochronic pathway. let-7 has been thought to trigger the onset of adulthood across animal phyla. Ecdysone and Broad-Complex are required for the temporal expression of let-7 in Drosophila melanogaster. For a better understanding of the conservation and functions of let-7, we seek to explore how it is expressed in the silkworm (Bombyx mori). RESULTS: One member of let-7 family has been identified in silkworm computationally and experimentally. All known members of this family share the same nucleotides at ten positions within the mature sequences. Sequence logo and phylogenetic tree show that they are not only conserved but diversify to some extent among some species. The bmo-let-7 was very lowly expressed in ova harvested from newborn unmated female adult and in individuals from the first molt to the early third instar, highly expressed after the third molt, and the most abundant expression was observed after mounting, particularly after pupation. The expression levels were higher at the end of each instar and at the beginning of each molt than at other periods, coinciding with the pulse of ecdysone and BR-C as a whole. Using cultured ovary cell line, BmN-SWU1, we examined the effect of altered ecdysone levels on bmo-let-7 expression. The expression was also detected in various tissues of day 3 of the fifth instar and of from day 7 of the fifth to pupa, suggesting a wide distributing pattern with various signal intensities. CONCLUSION: bmo-let-7 is stage- and tissue-specifically expressed in the silkworm. Although no signals were detected during embryonic development and first larval instar stages, the expression of bmo-let-7 was observed from the first molt, suggesting that it might also function at early larval stage of the silkworm. The detailed expression profiles in the whole life cycle and cultured cell line of silkworm showed a clear association with ecdysone pulse and a variety of biological processes. [Abstract/Link to Full Text]

Ghafari F, Gutierrez CG, Hartshorne GM
Apoptosis in mouse fetal and neonatal oocytes during meiotic prophase one.
BMC Dev Biol. 2007;787.
BACKGROUND: The vast majority of oocytes formed in the fetal ovary do not survive beyond birth. Possible reasons for their loss include the elimination of non-viable genetic constitutions arising through meiosis, however, the precise relationship between meiotic stages and prenatal apoptosis of oocytes remains elusive. We studied oocytes in mouse fetal and neonatal ovaries, 14.5-21 days post coitum, to examine the relationship between oocyte development and programmed cell death during meiotic prophase I. RESULTS: Microspreads of fetal and neonatal ovarian cells underwent immunocytochemistry for meiosis- and apoptosis-related markers. COR-1 (meiosis-specific) highlighted axial elements of the synaptonemal complex and allowed definitive identification of the stages of meiotic prophase I. Labelling for cleaved poly-(ADP-ribose) polymerase (PARP-1), an inactivated DNA repair protein, indicated apoptosis. The same oocytes were then labelled for DNA double strand breaks (DSBs) using TUNEL. 1960 oocytes produced analysable results. Oocytes at all stages of meiotic prophase I stained for cleaved PARP-1 and/or TUNEL, or neither. Oocytes with fragmented (19.8%) or compressed (21.2%) axial elements showed slight but significant differences in staining for cleaved PARP-1 and TUNEL to those with intact elements. However, fragmentation of axial elements alone was not a good indicator of cell demise. Cleaved PARP-1 and TUNEL staining were not necessarily coincident, showing that TUNEL is not a reliable marker of apoptosis in oocytes. CONCLUSION: Our data indicate that apoptosis can occur throughout meiotic prophase I in mouse fetal and early postnatal oocytes, with greatest incidence at the diplotene stage. Careful selection of appropriate markers for oocyte apoptosis is essential. [Abstract/Link to Full Text]

Parmar M, Li M
Early specification of dopaminergic phenotype during ES cell differentiation.
BMC Dev Biol. 2007;786.
BACKGROUND: Understanding how lineage choices are made during embryonic stem (ES) cell differentiation is critical for harnessing strategies for controlled production of therapeutic somatic cell types for cell transplantation and pharmaceutical drug screens. The in vitro generation of dopaminergic neurons, the type of cells lost in Parkinson's disease patients' brains, requires the inductive molecules sonic hedgehog and FGF8, or an unknown stromal cell derived inducing activity (SDIA). However, the exact identity of the responding cells and the timing of inductive activity that specify a dopaminergic fate in neural stem/progenitors still remain elusive. RESULTS: Using ES cells carrying a neuroepithelial cell specific vital reporter (Sox1-GFP) and FACS purification of Sox1-GFP neural progenitors, we have investigated the temporal aspect of SDIA mediated dopaminergic neuron specification during ES cell differentiation. Our results establish that SDIA induces a dopaminergic neuron fate in nascent neural stem or progenitor cells at, or prior to, Sox1 expression and does not appear to have further instructive role or neurotrophic activity during late neuronal differentiation of neural precursors. Furthermore, we show that dopaminergic neurons could be produced efficiently in a monolayer differentiation paradigm independent of SDIA activity or exogenous signalling molecules. In this case, the competence for dopaminergic neuron differentiation is also established at the level of Sox1 expression. CONCLUSION: Dopaminergic neurons are specified early during mouse ES cell differentiation. The subtype specification seems to be tightly linked with the acquisition of a pan neuroectoderm fate. [Abstract/Link to Full Text]

French CR, Erickson T, Callander D, Berry KM, Koss R, Hagey DW, Stout J, Wuennenberg-Stapleton K, Ngai J, Moens CB, Waskiewicz AJ
Pbx homeodomain proteins pattern both the zebrafish retina and tectum.
BMC Dev Biol. 2007;785.
BACKGROUND: Pbx genes encode TALE class homeodomain transcription factors that pattern the developing neural tube, pancreas, and blood. Within the hindbrain, Pbx cooperates with Hox proteins to regulate rhombomere segment identity. Pbx cooperates with Eng to regulate midbrain-hindbrain boundary maintenance, and with MyoD to control fast muscle cell differentiation. Although previous results have demonstrated that Pbx is required for proper eye size, functions in regulating retinal cell identity and patterning have not yet been examined. RESULTS: Analysis of retinal ganglion cell axon pathfinding and outgrowth in pbx2/4 null embryos demonstrated a key role for pbx genes in regulating neural cell behavior. To identify Pbx-dependent genes involved in regulating retino-tectal pathfinding, we conducted a microarray screen for Pbx-dependent transcripts in zebrafish, and detected genes that are specifically expressed in the eye and tectum. A subset of Pbx-dependent retinal transcripts delineate specific domains in the dorso-temporal lobe of the developing retina. Furthermore, we determined that some Pbx-dependent transcripts also require Meis1 and Gdf6a function. Since gdf6a expression is also dependent on Pbx, we propose a model in which Pbx proteins regulate expression of the growth factor gdf6a, which in turn regulates patterning of the dorso-temporal lobe of the retina. This, in concert with aberrant tectal patterning in pbx2/4 null embryos, may lead to the observed defects in RGC outgrowth. CONCLUSION: These data define a novel role for Pbx in patterning the vertebrate retina and tectum in a manner required for proper retinal ganglion cell axon outgrowth. [Abstract/Link to Full Text]

Pozner A, Lotem J, Xiao C, Goldenberg D, Brenner O, Negreanu V, Levanon D, Groner Y
Developmentally regulated promoter-switch transcriptionally controls Runx1 function during embryonic hematopoiesis.
BMC Dev Biol. 2007;784.
BACKGROUND: Alternative promoters usage is an important paradigm in transcriptional control of mammalian gene expression. However, despite the growing interest in alternative promoters and their role in genome diversification, very little is known about how and on what occasions those promoters are differentially regulated. Runx1 transcription factor is a key regulator of early hematopoiesis and a frequent target of chromosomal translocations in acute leukemias. Mice deficient in Runx1 lack definitive hematopoiesis and die in mid-gestation. Expression of Runx1 is regulated by two functionally distinct promoters designated P1 and P2. Differential usage of these two promoters creates diversity in distribution and protein-coding potential of the mRNA transcripts. While the alternative usage of P1 and P2 likely plays an important role in Runx1 biology, very little is known about the function of the P1/P2 switch in mediating tissue and stage specific expression of Runx1 during development. RESULTS: We employed mice bearing a hypomorphic Runx1 allele, with a largely diminished P2 activity, to investigate the biological role of alternative P1/P2 usage. Mice homozygous for the hypomorphic allele developed to term, but died within a few days after birth. During embryogenesis the P1/P2 activity is spatially and temporally modulated. P2 activity is required in early hematopoiesis and when attenuated, development of liver hematopoietic progenitor cells (HPC) was impaired. Early thymus development and thymopoiesis were also abrogated as reflected by thymic hypocellularity and loss of corticomedullary demarcation. Differentiation of CD4/CD8 thymocytes was impaired and their apoptosis was enhanced due to altered expression of T-cell receptors. CONCLUSION: The data delineate the activity of P1 and P2 in embryogenesis and describe previously unknown functions of Runx1. The findings show unequivocally that the role of P1/P2 during development is non redundant and underscore the significance of alternative promoter usage in Runx1 biology. [Abstract/Link to Full Text]

Sheeba CJ, Palmeirim I, Andrade RP
Chick Hairy1 protein interacts with Sap18, a component of the Sin3/HDAC transcriptional repressor complex.
BMC Dev Biol. 2007;783.
BACKGROUND: The vertebrate adult axial skeleton, trunk and limb skeletal muscles and dermis of the back all arise from early embryonic structures called somites. Somites are symmetrically positioned flanking the embryo axial structures (neural tube and notochord) and are periodically formed in a anterior-posterior direction from the presomitic mesoderm. The time required to form a somite pair is constant and species-specific. This extraordinary periodicity is proposed to depend on an underlying somitogenesis molecular clock, firstly evidenced by the cyclic expression of the chick hairy1 gene in the unsegmented presomitic mesoderm with a 90 min periodicity, corresponding to the time required to form a somite pair in the chick embryo. The number of hairy1 oscillations at any given moment is proposed to provide the cell with both temporal and positional information along the embryo's anterior-posterior axis. Nevertheless, how this is accomplished and what biological processes are involved is still unknown. Aiming at understanding the molecular events triggered by the somitogenesis clock Hairy1 protein, we have employed the yeast two-hybrid system to identify Hairy1 interaction partners. RESULTS: Sap18, an adaptor molecule of the Sin3/HDAC transcriptional repressor complex, was found to interact with the C-terminal portion of the Hairy1 protein in a yeast two-hybrid assay and the Hairy1/Sap18 interaction was independently confirmed by co-immunoprecipitation experiments. We have characterized the expression patterns of both sap18 and sin3a genes during chick embryo development, using in situ hybridization experiments. We found that both sap18 and sin3a expression patterns co-localize in vivo with hairy1 expression domains in chick rostral presomitic mesoderm and caudal region of somites. CONCLUSION: Hairy1 belongs to the hairy-enhancer-of-split family of transcriptional repressor proteins. Our results indicate that during chick somitogenesis Hairy1 may mediate gene transcriptional repression by recruiting the Sin3/HDAC complex, through a direct interaction with the Sap18 adaptor molecule. Moreover, since sap18 and sin3a are not expressed in the PSM territory where hairy1 presents cyclic expression, our study strongly points to different roles for Hairy1 throughout the PSM and in the prospective somite and caudal region of already formed somites. [Abstract/Link to Full Text]

Copeland JM, Bosdet I, Freeman JD, Guo M, Gorski SM, Hay BA
echinus, required for interommatidial cell sorting and cell death in the Drosophila pupal retina, encodes a protein with homology to ubiquitin-specific proteases.
BMC Dev Biol. 2007;782.
BACKGROUND: Programmed cell death is used to remove excess cells between ommatidia in the Drosophila pupal retina. This death is required to establish the crystalline, hexagonal packing of ommatidia that characterizes the adult fly eye. In previously described echinus mutants, interommatidial cell sorting, which precedes cell death, occurred relatively normally. Interommatidial cell death was partially suppressed, resulting in adult eyes that contained excess pigment cells, and in which ommatidia were mildly disordered. These results have suggested that echinus functions in the pupal retina primarily to promote interommatidial cell death. RESULTS: We generated a number of new echinus alleles, some likely null mutants. Analysis of these alleles provides evidence that echinus has roles in cell sorting as well as cell death. echinus encodes a protein with homology to ubiquitin-specific proteases. These proteins cleave ubiquitin-conjugated proteins at the ubiquitin C-terminus. The echinus locus encodes multiple splice forms, including two proteins that lack residues thought to be critical for deubiquitination activity. Surprisingly, ubiquitous expression in the eye of versions of Echinus that lack residues critical for ubiquitin specific protease activity, as well as a version predicted to be functional, rescue the echinus loss-of-function phenotype. Finally, genetic interactions were not detected between echinus loss and gain-of-function and a number of known apoptotic regulators. These include Notch, EGFR, the caspases Dronc, Drice, Dcp-1, Dream, the caspase activators, Rpr, Hid, and Grim, the caspase inhibitor DIAP1, and Lozenge or Klumpfuss. CONCLUSION: The echinus locus encodes multiple splice forms of a protein with homology to ubiquitin-specific proteases, but protease activity is unlikely to be required for echinus function, at least when echinus is overexpressed. Characterization of likely echinus null alleles and genetic interactions suggests that echinus acts at a novel point(s) to regulate interommatidial cell sorting and/or cell death in the fly eye. [Abstract/Link to Full Text]

Magnol L, Chevallier MC, Nalesso V, Retif S, Fuchs H, Klempt M, Pereira P, Riottot M, Andrzejewski S, Doan BT, Panthier JJ, Puech A, Beloeil JC, de Angelis MH, H�rault Y
KIT is required for hepatic function during mouse post-natal development.
BMC Dev Biol. 2007;781.
BACKGROUND: The Kit gene encodes a receptor tyrosine kinase involved in various biological processes including melanogenesis, hematopoiesis and gametogenesis in mice and human. A large number of Kit mutants has been described so far showing the pleiotropic phenotypes associated with partial loss-of-function of the gene. Hypomorphic mutations can induce a light coat color phenotype while complete lack of KIT function interferes with embryogenesis. Interestingly several intermediate hypomorphic mutations induced in addition growth retardation and post-natal mortality. RESULTS: In this report we investigated the post-natal role of Kit by using a panel of chemically-induced hypomorphic mutations recently isolated in the mouse. We found that, in addition to the classical phenotypes, mutations of Kit induced juvenile steatosis, associated with the downregulation of the three genes, VldlR, Lpin1 and Lpl, controlling lipid metabolism in the post-natal liver. Hence, Kit loss-of-functions mimicked the inactivation of genes controlling the hepatic metabolism of triglycerides, the major source of energy from maternal milk, leading to growth and viability defects during neonatal development. CONCLUSION: This is a first report involving KIT in the control of lipid metabolism in neonates and opening new perspectives for understanding juvenile steatosis. Moreover, it reinforces the role of Kit during development of the liver and underscores the caution that should be exerted in using KIT inhibitors during anti-cancer treatment. [Abstract/Link to Full Text]

Shimosato D, Shiki M, Niwa H
Extra-embryonic endoderm cells derived from ES cells induced by GATA factors acquire the character of XEN cells.
BMC Dev Biol. 2007;780.
BACKGROUND: Three types of cell lines have been established from mouse blastocysts: embryonic stem (ES) cells, trophoblast stem (TS) cells, and extra-embryonic endoderm (XEN) cells, which have the potential to differentiate into their respective cognate lineages. ES cells can differentiate in vitro not only into somatic cell lineages but into extra-embryonic lineages, including trophectoderm and extra-embryonic endoderm (ExEn) as well. TS cells can be established from ES cells by the artificial repression of Oct3/4 or the upregulation of Cdx2 in the presence of FGF4 on feeder cells. The relationship between these embryo-derived XEN cells and ES cell-derived ExEn cell lines remains unclear, although we have previously reported that overexpression of Gata4 or Gata6 induces differentiation of mouse ES cells into extra-embryonic endoderm in vitro. RESULTS: A system in which GATA factors were conditionally activated revealed that the cells continue to proliferate while expressing a set of extra-embryonic endoderm markers, and, following injection into blastocysts, contribute only to the extra-embryonic endoderm lineage in vivo. Although the in vivo contribution is limited to cells of parietal endoderm lineage, Gata-induced extra-embryonic endoderm cells (gExEn) can be induced to differentiate into visceral endoderm-like cells in vitro by repression of Gata6. During early passage, the propagation of gExEn cells is dependent on the expression of the Gata6 transgene. These cells, however, lose this dependency following establishment of endogenous Gata6 expression. CONCLUSION: We show here that Gata-induced extra-embryonic endoderm cells derived from ES cells mimic the character of XEN cells. These findings indicate that Gata transcription factors are sufficient for the derivation and propagation of XEN-like extra-embryonic endoderm cells from ES cells. [Abstract/Link to Full Text]

Giroux SJ, Alves-Leiva C, L�cluse Y, Martin P, Albagli O, Godin I
Gene transfer to pre-hematopoietic and committed hematopoietic precursors in the early mouse yolk sac: a comparative study between in situ electroporation and retroviral transduction.
BMC Dev Biol. 2007;779.
BACKGROUND: Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated. While intra-embryonic precursors harbour the features of hematopoietic stem cells (HSC), precursors formed earlier in the yolk sac (YS) display limited differentiation and self-renewal potentials. The mechanisms leading to the generation of the precursors in both sites are still largely unknown, as are the molecular basis underlying their different potential. A possible approach to assess the role of candidate genes is to transfer or modulate their expression/activity in both sites. We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors. RESULTS: One transduction protocol involves transient modification of gene expression through in situ electroporation of the prospective blood islands, which allows the evolution of transfected mesodermal cells in their "normal" environment, upon organ culture. Following in situ electroporation of a GFP reporter construct into the YS cavity of embryos at post-streak (mesodermal/pre-hematopoietic precursors) or early somite (hematopoietic precursors) stages, high GFP expression levels as well as a good preservation of cell viability is observed in YS explants. Moreover, the erythro-myeloid progeny typical of the YS arises from GFP+ mesodermal cells or hematopoietic precursors, even if the number of targeted precursors is low. The second approach, based on retroviral transduction allows a very efficient transduction of large precursor numbers, but may only be used to target 8 dpc YS hematopoietic precursors. Again, transduced cells generate a progeny quantitatively and qualitatively similar to that of control YS. CONCLUSION: We thus provide two protocols whose combination may allow a thorough study of both early and late events of hematopoietic development in the murine YS. In situ electroporation constitutes the only possible gene transfer method to transduce mesodermal/pre-hematopoietic precursors and analyze the earliest steps of hematopoietic development. Both in situ electroporation and retroviral transduction may be used to target early hematopoietic precursors, but the latter appears more convenient if a large pool of stably transduced cells is required. We discuss the assets and limitation of both methods, which may be alternatively chosen depending on scientific constraints. [Abstract/Link to Full Text]

Chen B, Cepko CL
Requirement of histone deacetylase activity for the expression of critical photoreceptor genes.
BMC Dev Biol. 2007;778.
BACKGROUND: Histone deacetylases (HDACs) play a major role in the regulation of gene transcription, often leading to transcriptional repression, as well as other effects following deacetylation of non-histone proteins. RESULTS: To investigate the role of HDACs in the developing mammalian retina, a general inhibitor of HDACs, trichostatin-A (TSA), was used to treat newborn murine retinae in explant cultures. Inhibition of HDAC activity resulted in a reduction in RNA levels for genes that regulate retinal development, as well as cell cycle regulators. Several of the genes encode transcription factors essential for rod photoreceptor development, Otx2, Nrl, and Crx. Using luciferase reporter assays, the promoter activity of both Nrl and Crx was found to be compromised by HDAC inhibition. Furthermore, downregulation of gene expression by HDAC inhibition didn't require de novo protein synthesis, and was associated with hyperacetylation of histones and non-histone proteins. Finally, HDAC inhibition in retinal explant cultures resulted in increased cell death, reduction in proliferation, a complete loss of rod photoreceptors and M�ller glial cells, and an increase in bipolar cells. CONCLUSION: HDAC activity is required for the expression of critical pro-rod transcription factors and the development of rod photoreceptor cells. [Abstract/Link to Full Text]

Amadio S, Vacca F, Martorana A, Sancesario G, Volont� C
P2Y1 receptor switches to neurons from glia in juvenile versus neonatal rat cerebellar cortex.
BMC Dev Biol. 2007;777.
BACKGROUND: In the CNS, several P2 receptors for extracellular nucleotides are identified on neurons and glial cells to participate to neuron-neuron, glia-glia and glia-neuron communication. RESULTS: In this work, we describe the cellular and subcellular presence of metabotropic P2Y1 receptor in rat cerebellum at two distinct developmental ages, by means of immunofluorescence-confocal and electron microscopy as well as western blotting and direct membrane separation techniques. At postnatal day 21, we find that P2Y1 receptor in addition to Purkinje neurons, is abundant on neuronal specializations identified as noradrenergic by anatomical, morphological and biochemical features. P2Y1 receptor immunoreactivity colocalizes with dopamine beta-hydroxylase, tyrosine hydroxylase, neurofilament light chain, synaptophysin and flotillin, but not with glial fibrillary acidic protein for astrocytes. P2Y1 receptor is found enriched in membrane microdomains such as lipid rafts, in cerebellar synaptic vesicles, and is moreover visualized on synaptic varicosities by electron microscopy analysis. When examined at postnatal day 7, P2Y1 receptor immunoreactivity is instead predominantly expressed only on Bergmann and astroglial cells, as shown by colocalization with glial fibrillary acidic protein rather then neuronal markers. At this age, we moreover identify that P2Y1 receptor-positive Bergmann fibers wrap up doublecortin-positive granule cells stretching along them, while migrating through the cerebellar layers. CONCLUSION: Membrane components including purinergic receptors are already known to mediate cellular contact and aggregation in platelets. Our results suggesting a potential role for P2Y1 protein in cell junction/communication and development, are totally innovative for the CNS. [Abstract/Link to Full Text]

Wang JL, Jiang XJ, Wang Q, Hou LJ, Xu DW, Wang JX, Zhao XF
Identification and expression profile of a putative basement membrane protein gene in the midgut of Helicoverpa armigera.
BMC Dev Biol. 2007;776.
BACKGROUND: The midgut undergoes histolysis and remodeling during the larval to adult transition in holometabolous insects, but the molecular mechanisms underlying this process are not well understood. RESULTS: Using Suppression Subtractive Hybridization (SSH), we identified a 531 bp cDNA predicted to encode a 176 amino acid protein, which we call hmg176. Northern and western blot analysis suggested that high levels of hmg176 are expressed in the midgut during molting, but not during metamorphosis. HMG176 protein was detected by immunofluorescence within the membrane of fat bodies and the basement membrane of the midgut of both molting and feeding larvae, but not in metamorphically committed larvae. In situ hybridization revealed that hmg176 transcripts mainly localized to the columnar cells of the midgut. Interestingly, a non-steroidal ecdysone agonist, RH-2485, significantly upregulated expression of hmg176. CONCLUSION: These observations suggest that hmg176 encodes a larval-specific protein that may participate in sustaining larval midgut during larval development, possibly in response to ecdysteroid in vivo. This study will enlighten our understanding of the molecular mechanisms of tissue histolysis during metamorphosis. [Abstract/Link to Full Text]

Recent Articles in BMC Evolutionary Biology

Koene JM, Ter Maat A
Coolidge effect in pond snails: male motivation in a simultaneous hermaphrodite.
BMC Evol Biol. 2007 Nov 6;7(1):212.
ABSTRACT: BACKGROUND: The simultaneously hermaphroditic pond snail, Lymnaea stagnalis, can mate in the male and female role, but within one copulation only one sexual role is performed at a time. Previous work has shown that male motivation is determined by the availability of seminal fluid in the prostate gland, which is detected via a nervous connection by the brain area controlling male behaviour. Based on this knowledge, patterns of sexual role alternations within mating pairs can be explained. RESULTS: The data presented here reveal that these snails can donate and receive sperm several times within 24 hours, and that they have increased mating rates in larger groups (i.e. more mating opportunities). For mating pairs we show, by introducing novel mating partners after copulation, that animals do inseminate new partners, while they are no longer motivated to inseminate their original partners. CONCLUSIONS: Our findings provide the first direct evidence for higher motivation in a hermaphrodite to copulate when a new partner is encountered. This Coolidge effect seems to be attenuated when mucus trails are excluded, which suggests that a chemical or textural cue may be responsible for mediating this response to sperm competition. [Abstract/Link to Full Text]

Kouyos RD, Salathe M, Bonhoeffer S
The Red Queen and the persistence of linkage-disequilibrium oscillations in finite and infinite populations.
BMC Evol Biol. 2007 Nov 6;7(1):211.
ABSTRACT: BACKGROUND: The Red Queen Hypothesis (RQH) suggests that the coevolutionary dynamics of host-parasite systems can generate selection for increased host recombination. Since host-parasite interactions often have a strong genetic basis, recombination between different hosts can increase the fraction of novel and potentially resistant offspring genotypes. A prerequisite for this mechanism is that host-parasite interactions generate persistent oscillations of linkage disequilibria (LD). RESULTS: We use deterministic and stochastic models to investigate the persistence of LD oscillations and its impact on the RQH. The standard models of the Red Queen dynamics exhibit persistent LD oscillations under most circumstances. Here, we show that altering the standard model from discrete to continuous time or from simultaneous to sequential updating results in damped LD oscillations. This suggests that LD oscillations are structurally not robust. We then show that in a stochastic regime, drift can counteract this dampening and maintain the oscillations. In addition, we show that the amplitude of the oscillations and therefore the strength of the resulting selection for or against recombination are inversely proportional to the size of the (host) population. CONCLUSION: We find that host parasite-interactions cannot generally maintain oscillations in the absence of drift. As a consequence, the RQH can strongly depend on population size and should therefore not be interpreted as a purely deterministic hypothesis. [Abstract/Link to Full Text]

De Mita S, Santoni S, Ronfort J, Bataillon T
Adaptive evolution of the symbiotic gene NORK is not correlated with shifts of rhizobial specificity in the genus Medicago.
BMC Evol Biol. 2007 Nov 6;7(1):210.
ABSTRACT: BACKGROUND: The NODULATION RECEPTOR KINASE (NORK) gene encodes a Leucine-Rich Repeat (LRR)-containing receptor-like protein and controls the infection by symbiotic rhizobia and endomycorrhizal fungi in Legumes. The occurrence of numerous amino acid changes driven by directional selection has been reported in this gene, using a limited number of messenger RNA sequences, but the functional reason of these changes remains obscure. The Medicago genus, where changes in rhizobial associations have been previously examined, is a good model to test whether the evolution of NORK is influenced by rhizobial interactions. RESULTS: We sequenced a region of 3610 nucleotides (encoding a 392 amino acid-long region of the NORK protein) in 32 Medicago species. We confirm that positive selection in NORK has occurred within the Medicago genus and find that the amino acid positions targeted by selection occur in sites outside of solvent-exposed regions in LRRs, and other sites in the N-terminal region of the protein. We tested if branches of the Medicago phylogeny where changes of rhizobial symbionts occurred displayed accelerated rates of amino acid substitutions. Only one branch out of five tested, leading to M. noeana, displays such a pattern. Among other branches, the most likely for having undergone positive selection is not associated with documented shift of rhizobial specificity. CONCLUSIONS: Adaptive changes in the sequence of the NORK receptor have involved the LRRs, but targeted different sites than in most previous studies of LRR proteins evolution. The fact that positive selection in NORK tends not to be associated to changes in rhizobial specificity indicates that this gene was probably not involved in evolving rhizobial preferences. Other explanations (e.g. coevolutionary arms race) must be tested to explain the adaptive evolution of NORK. [Abstract/Link to Full Text]

Kawahara R, Nishida M
Extensive lineage-specific gene duplication and evolution of the spiggin multi-gene family in stickleback.
BMC Evol Biol. 2007 Nov 4;7(1):209.
ABSTRACT: BACKGROUND: The threespine stickleback (Gasterosteus aculeatus) has a characteristic reproductive mode; mature males build nests using a secreted glue-like protein called spiggin. Although recent studies reported multiple occurrences of genes that encode this glue-like protein spiggin in threespine and ninespine sticklebacks, it is still unclear how many genes compose the spiggin multi-gene family. RESULTS: Genome sequence analysis of threespine stickleback showed that there are at least five spiggin genes and two pseudogenes, whereas a single spiggin homolog occurs in the genomes of other fishes. Comparative genome sequence analysis demonstrated that Muc19, a single-copy mucous gene in human and mouse, is an ortholog of spiggin. Phylogenetic and molecular evolutionary analyses of these sequences suggested that an ancestral spiggin gene originated from a member of the mucin gene family as a single gene in the common ancestor of teleosts, and gene duplications of spiggin have occurred in the stickleback lineage. There was inter-population variation in the copy number of spiggin genes and positive selection on some codons, indicating that additional gene duplication/deletion events and adaptive evolution at some amino acid sites may have occurred in each stickleback population. CONCLUSION: A number of spiggin genes exist in the threespine stickleback genome. Our results provide insight into the origin and dynamic evolutionary process of the spiggin multi-gene family in the threespine stickleback lineage. The dramatic evolution of genes for mucous substrates may have contributed to the generation of distinct characteristics such as "bio-glue" in vertebrates. [Abstract/Link to Full Text]

Guo X, Wang Y, Keightley PD, Fan L
Patterns of selective constraints in noncoding DNA of rice.
BMC Evol Biol. 2007 Nov 1;7(1):208.
ABSTRACT: BACKGROUND: Several studies have investigated the relationships between selective constraints in introns and their length, GC content and location within genes. To date, however, no such investigation has been done in plants. Studies of selective constraints in noncoding DNA have generally involved interspecific comparisons, under the assumption of the same selective pressures acting in each lineage. Such comparisons are limited to cases in which the noncoding sequences are not too strongly diverged so that reliable sequence alignments can be obtained. Here, we investigate selective constraints in a recent segmental duplication that includes 605 paralogous intron pairs that occurred about 7 million years ago in rice (O. sativa). RESULTS: Our principal findings are: (1) intronic divergence is negatively correlated with intron length, a pattern that has previously been described in Drosophila and mammals; (2) there is a signature of strong purifying selection at splice control sites; (3) first introns are significantly longer and have a higher GC content than other introns; (4) the divergences of first and non-first introns are not significantly different from one another, a pattern that differs from Drosophila and mammals. (5) Short introns are more diverged than four-fold degenerate sites suggesting that selection reduces divergence at four-fold sites. CONCLUSION: Our observation of stronger selective constraints in long introns suggests that functional elements subject to purifying selection may be concentrated within long introns. Our results are consistent with the presence of strong purifying selection at splicing control sites. Selective constraints are not significantly stronger in first introns of rice, as they are in other species. [Abstract/Link to Full Text]

Jacob A, Nussle S, Britschgi A, Evanno G, Muller R, Wedekind C
Male dominance linked to size and age, but not to 'good genes' in brown trout (Salmo trutta).
BMC Evol Biol. 2007 Nov 1;7(1):207.
ABSTRACT: BACKGROUND: Males that are successful in intra-sexual competition are often assumed to be of superior quality. In the mating system of most salmonid species, intensive dominance fights are common and the winners monopolise most mates and sire most offspring. We drew a random sample of mature male brown trout (Salmo trutta) from two wild populations and determined their dominance hierarchy or traits linked to dominance. The fish were then stripped and their sperm was used for in vitro fertilisations in two full-factorial breeding designs. We recorded embryo viability until hatching in both experiments, and juvenile survival during 20 months after release into a natural streamlet in the second experiment. Since offspring of brown trout get only genes from their fathers, we used offspring survival as a quality measure to test (i) whether males differ in their genetic quality, and if so, (ii) whether dominance or traits linked to dominance reveal 'good genes'. RESULTS: We found significant additive genetic variance on embryo survival, i.e. males differed in their genetic quality. Older, heavier and larger males were more successful in intra-sexual selection. However, neither dominance nor dominance indicators like body length, weight or age were significantly linked to genetic quality measured as embryo or juvenile survival. CONCLUSION: We found no evidence that females can improve their offspring's genetic viability by mating with large and dominant males. If there still were advantages of mating with dominant males, they may be linked to non-genetic benefits or to genetic advantages that are context dependent and therefore possibly not revealed under our experimental conditions - even if we found significant additive genetic variation for embryo viability under such conditions. [Abstract/Link to Full Text]

Zhou Y, Rodrigue N, Lartillot N, Philippe H
Evaluation of the models handling heterotachy in phylogenetic inference.
BMC Evol Biol. 2007 Nov 1;7(1):206.
ABSTRACT: BACKGROUND: The evolutionary rate at a given homologous position varies across time. When sufficiently pronounced, this phenomenon-also called heterotachy, may produce artefactual phylogenetic reconstructions under the commonly used models of sequence evolution. These observations have motivated the development of models that explicitly recognize heterotachy, with research directions proposed along two main axes: 1) the covarion approach, where sites switch from variable to invariable states; and 2) the mixture of branch lengths (MBL) approach, where alignment patterns are assumed to arise from one of several sets of branch lengths, under a given phylogeny. RESULTS: Here, we report the first statistical comparisons contrasting the performance of covarion and MBL modeling strategies. Using simulations under heterotachous conditions, we explore the properties of three model comparison methods: the Akaike information criterion, the Bayesian information criterion, and cross validation. Although more time consuming, cross validation is the most reliable of the three methods as it directly measures the predictive power of a model on 'future' data. We also analyze three large datasets (nuclear proteins of animals, mitochondrial proteins of mammals, and plastid proteins of plants), and find the optimal number of components of the MBL model to be two for all datasets, indicating that this model is preferred over the standard homogeneous model. However, the covarion model is always favored over the optimal MBL model. CONCLUSION: We demonstrated, using three large datasets, that the covarion model is more efficient at handling heterotachy than the MBL model. This is probably due to the fact that the MBL model requires a drastic increase in the number of parameters, as compared to two supplementary parameters of the covarion approach. Further improvements of the both the mixture and the covarion approaches might be obtained by modeling heterogeneous behavior both along time and across sites. [Abstract/Link to Full Text]

Soyer OS
Emergence and maintenance of functional modules in signaling pathways.
BMC Evol Biol. 2007 Oct 31;7(1):205.
ABSTRACT: BACKGROUND: While detection and analysis of functional modules in biological systems have received great attention in recent years, we still lack a complete understanding of how such modules emerge. One theory is that systems must encounter a varying selection (i.e. environment) in order for modularity to emerge. Here, we provide an alternative and simpler explanation using a realistic model of biological signaling pathways and simulating their evolution. RESULTS: These evolutionary simulations start with a homogenous population of a minimal pathway containing two effectors coupled to two signals via a single receptor. This population is allowed to evolve under a constant selection pressure for mediating two separate responses. Results of these evolutionary simulations show that under such a selective pressure, mutational processes easily lead to the emergence of pathways with two separate sub-pathways (i.e. modules) each mediating a distinct response only to one of the signals. Such functional modules are maintained as long as mutations leading to new interactions among existing proteins in the pathway are rare. CONCLUSIONS: While supporting a neutralistic view for the emergence of modularity in biological systems, these findings highlight the relevant rate of different mutational processes and the distribution of functional pathways in the topology space as key factors for its maintenance. [Abstract/Link to Full Text]

Sato Y, Nishida M
Post-duplication charge evolution of phosphoglucose isomerases in teleost fishes through weak selection on many amino acid sites.
BMC Evol Biol. 2007 Oct 29;7(1):204.
ABSTRACT: BACKGROUND: The partitioning of ancestral functions among duplicated genes by neutral evolution, or subfunctionalization, has been considered the primary process for the evolution of novel proteins (neofunctionalization). Nonetheless, how a subfunctionalized protein can evolve into a more adaptive protein is poorly understood, mainly due to the limitations of current analytical methods, which can detect only strong selection for amino acid substitutions involved in adaptive molecular evolution. In this study, we employed a comparative evolutionary approach to this question, focusing on differences in the structural properties of a protein, specifically the electric charge, encoded by fish-specific duplicated phosphoglucose isomerase (Pgi) genes. RESULTS: Full-length cDNA cloning, RT-PCR based gene expression analyses, and comparative sequence analyses showed that after subfunctionalization with respect to the expression organ of duplicate Pgi genes, the net electric charge of the PGI-1 protein expressed mainly in internal tissues became more negative, and that of PGI-2 expressed mainly in muscular tissues became more positive. The difference in net protein charge was attributable not to specific amino acid sites but to the sum of various amino acid sites located on the surface of the PGI molecule. CONCLUSIONS: This finding suggests that the surface charge evolution of PGI proteins was not driven by strong selection on individual amino acid sites leading to permanent fixation of a particular residue, but rather was driven by weak selection on a large number of amino acid sites and consequently by steady directional and/or purifying selection on the overall structural properties of the protein, which is derived from many modifiable sites. The mode of molecular evolution presented here may be relevant to various cases of adaptive modification in proteins, such as hydrophobic properties, molecular size, and electric charge. [Abstract/Link to Full Text]

Brandvain Y, Wade MJ
The evolution of competition and policing: opposing selection within and among groups.
BMC Evol Biol. 2007 Oct 25;7(1):203.
ABSTRACT: BACKGROUND: Although selection favors exploitative competition within groups, a group of hyper-competitive individuals may be less productive than a cooperative group. When competition is costly for group fitness, among-group selection can favor groups with 'policing' individuals who reduce within-group competition at a cost to their own fitness, or groups of individuals who restrain their competitive intensity ('self policing'). We examine these possibilities in a series of explicit population-genetic models. RESULTS: By comparing results from models of half and full-sib structured populations, we find that increased relatedness increases the strength of among-group selection against competition genotypes, and increases the strength of among group selection favoring policing genotypes. However, the strength of selection favoring costly policing behavior also increases with increased levels of competition. When levels of competition and policing feedback on one another, groups with lower levels of relatedness can favor higher levels of costly policing. CONCLUSIONS: The result of the joint selection on policing and competition leads to results different from those based on the evolution of policing alone. Our model makes 'long term' predictions equivalent to those of optimization models, but we also show the existence of protected polymorphisms of police and civilians, as well as competitors and non-competitors. [Abstract/Link to Full Text]

Singh ND, Macpherson JM, Jensen JD, Petrov DA
Similar Levels of X-linked and Autosomal Nucleotide Variation in African and non-African populations of Drosophila melanogaster.
BMC Evol Biol. 2007 Oct 25;7(1):202.
ABSTRACT: BACKGROUND: Levels of molecular diversity in Drosophila have repeatedly been shown to be higher in ancestral, African populations than in derived, non-African populations. This pattern holds for both coding and noncoding regions for a variety of molecular markers including single nucleotide polymorphisms and microsatellites. Comparisons of X-linked and autosomal diversity have yielded results largely dependent on population of origin. RESULTS: In an attempt to further elucidate patterns of sequence diversity in Drosophila melanogaster, we studied nucleotide variation at putatively nonfunctional X-linked and autosomal loci in sub-Saharan African and North American strains of D. melanogaster. We combine our experimental results with data from previous studies of molecular polymorphism in this species. We confirm that levels of diversity are consistently higher in African versus North American strains. The relative reduction of diversity for X-linked and autosomal loci in the derived, North American strains depends heavily on the studied loci. While the compiled dataset, comprised primarily of regions within or in close proximity to genes, shows a much more severe reduction of diversity on the X chromosome compared to autosomes in derived strains, the dataset consisting of intergenic loci located far from genes shows very similar reductions of diversities for X-linked and autosomal loci in derived strains. In addition, levels of diversity at X-linked and autosomal loci in the presumably ancestral African population are more similar than expected under an assumption of neutrality and equal numbers of breeding males and females. CONCLUSIONS: We show that simple demographic scenarios under assumptions of neutral theory cannot explain all of the observed patterns of molecular diversity. We suggest that the simplest model is a population bottleneck that retains an ancestral female-biased sex ratio, coupled with higher rates of positive selection at X-linked loci in close proximity to genes specifically in derived, non-African populations. [Abstract/Link to Full Text]

Aruga J, Odaka YS, Kamiya A, Furuya H
Dicyema Pax6 and Zic: tool-kit genes in a highly simplified bilaterian.
BMC Evol Biol. 2007 Oct 25;7(1):201.
ABSTRACT: BACKGROUND: Dicyemid mesozoans (Phylum Dicyemida) are simple (8-40-cell) cephalopod endoparasites. They have neither body cavities nor differentiated organs, such as nervous and gastrointestinal systems. Whether dicyemids are intermediate between Protozoa and Metazoa (as represented by their "Mesozoa" classification) or degenerate species of more complex metazoans is controversial. Recent molecular phylogenetic studies suggested that they are simplified bilaterians belonging to the Lophotrochozoa. We cloned two genes developmentally critical in bilaterian animals (Pax6 and Zic), together with housekeeping genes (actin, fructose-bisphosphate aldolase, and ATP synthase beta subunit) from a dicyemid to reveal whether their molecular phylogeny supported the "simplification" hypothesis, and to clarify evolutionary changes in dicyemid gene structure and expression profiles. RESULTS: Genomic/cDNA sequence analysis showed that 1) the Pax6 molecular phylogeny and Zic intron positions supported the idea of dicyemids as reduced bilaterians; 2) the aa sequences deduced from the five genes were highly divergent; and 3) Dicyema genes contained very short introns of uniform length. In situ hybridization analyses revealed that Zic genes were expressed in hermaphroditic gonads, and Pax6 was expressed weakly throughout the developmental stages of the two types of embryo and in the hermaphroditic gonads. CONCLUSIONS: The accelerated evolutionary rates and very short and uniform intron may represent a part of Dicyema genomic features. The presence and expression of the two tool-kit genes (Pax6 and Zic) in Dicyema suggests that they can be very versatile genes even required for the highly reduced bilaterian like Dicyema. Dicyemids may be useful models of evolutionary body plan simplification. [Abstract/Link to Full Text]

Bailes HJ, Davies WL, Trezise AE, Collin SP
Visual pigments in a living fossil, the Australian lungfish Neoceratodus forsteri.
BMC Evol Biol. 2007 Oct 25;7(1):200.
ABSTRACT: BACKGROUND: One of the greatest challenges facing the early land vertebrates was the need to effectively interpret a terrestrial environment. Interpretation was based on ocular adaptations evolved for an aquatic environment millions of years earlier. The Australian lungfish Neoceratodus forsteri is thought to be the closest living relative to the first terrestrial vertebrate, and yet nothing is known about the visual pigments present in lungfish or the early tetrapods. RESULTS: Here we identify and characterise five visual pigments (rh1, rh2, lws, sws1 and sws2) expressed in the retina of N. forsteri. Phylogenetic analysis of the molecular evolution of lungfish and other vertebrate visual pigment genes indicates a closer relationship between lungfish and amphibian pigments than to pigments in teleost fishes. However, the relationship between lungfish, the coelacanth and tetrapods could not be absolutely determined from opsin phylogeny, supporting an unresolved trichotomy between the three groups. CONCLUSIONS: The presence of four cone pigments in Australian lungfish suggests that the earliest tetrapods would have had a colorful view of their terrestrial environment. [Abstract/Link to Full Text]

Ruiz-Herrera A, Robinson TJ
Chromosomal instability in Afrotheria: fragile sites, evolutionary breakpoints and phylogenetic inference from genome sequence assemblies.
BMC Evol Biol. 2007 Oct 24;7(1):199.
ABSTRACT: Backgound: Extant placental mammals are divided into four major clades (Laurasiatheria, Supraprimates, Xenarthra and Afrotheria). Given that Afrotheria is generally thought to root the eutherian tree in phylogenetic analysis of large nuclear gene data sets, the study of the organization of the genomes of afrotherian species provides new insights into the dynamics of mammalian chromosomal evolution. Here we test if there are loci with a high tendency to break and reorganize in Afrotheria, and by analyzing the expression of aphidicolin-induced common fragile sites in three afrotherian species, whether these are coincidental with recognized evolutionary breakpoints. RESULTS: We described 29 fragile sites in the aardvark genome, 27 in the golden mole, and 35 in the elephant-shrew genome. We show that fragile sites are conserved among afrotherian species and these are correlated with evolutionary breakpoints when compared to the human (HSA) genome. In addition, by computationally scanning the newly released opossum (Monodelphis domestica) and chicken (Gallus gallus) sequence assemblies for use as outgroups to Placentalia, we validate the HSA 3/21/5 chromosomal synteny as a rare genomic change that defines the monophyly of this ancient African clade of mammals. On the other hand, support for HSA 1/19p, thought to underpin Afrotheria, is currently ambiguous. CONCLUSIONS: We provide evidence that (i) the evolutionary breakpoints that characterise human syntenies detected in the basal Afrotheria correspond at the chromosomal band level with fragile sites, (ii) that HSA 3p/21 was in the amniote ancestor (i.e., common to turtles, lepidosaurs, crocodilians, birds and mammals), and was subsequently disrupted in the lineage leading to marsupials. Its expansion to include HSA 5 in Afrotheria is unique and (iii) that its fragmentation to HSA 3p/21 + HSA 5/21 in elephant and manatee was due to a fission within HSA 21 that is probably shared by all Paenungulata. [Abstract/Link to Full Text]

Yu L, Li YW, Ryder OA, Zhang YP
Analysis of complete mitochondrial genome sequences increases phylogenetic resolution of bears (Ursidae), a mammalian family that experienced rapid speciation.
BMC Evol Biol. 2007 Oct 24;7(1):198.
ABSTRACT: BACKGROUND: Despite the small number of ursid species, bear phylogeny has long been a focus of study due to their conservation value, as all bear genera have been classified as endangered at either the species or subspecies level. The Ursidae family represents a typical example of rapid evolutionary radiation. Previous analyses with a single mitochondrial (mt) gene or a small number of mt genes either provide weak support or a large unresolved polytomy for ursids. We revisit the contentious relationships within Ursidae by analyzing complete mt genome sequences and evaluating the performance of both entire mt genomes and constituent mtDNA genes in recovering a phylogeny of extremely recent speciation events. RESULTS: This mitochondrial genome-based phylogeny provides strong evidence that the spectacled bear diverged first, while within the genus Ursus, the sloth bear is the sister taxon of all the other five ursines. The latter group is divided into the brown bear/polar bear and the two black bears/sun bear assemblages. These findings resolve the previous conflicts between trees using partial mt genes. The ability of different categories of mt protein coding genes to recover the correct phylogeny is concordant with previous analyses for taxa with deep divergence times. This study provides a robust Ursidae phylogenetic framework for future validation by additional independent evidence, and also has significant implications for assisting in the resolution of other similarly difficult phylogenetic investigations. CONCLUSIONS: Identification of base composition bias and utilization of the combined data of whole mitochondrial genome sequences has allowed recovery of a strongly supported phylogeny that is upheld when using multiple alternative outgroups for the Ursidae, a mammalian family that underwent a rapid radiation since the mid- to late Pliocene. It remains to be seen if the reliability of mt genome analysis will hold up in studies of other difficult phylogenetic issues. Although the whole mitochondrial DNA sequence based phylogeny is robust, it remains in conflict with phylogenetic relationships suggested by analysis of limited nuclear-encoded data, a situation that will require gathering more nuclear DNA sequence information. [Abstract/Link to Full Text]

Gonzalez EG, Zardoya R
Relative role of life-history traits and historical factors in shaping genetic population structure of sardines (Sardina pilchardus).
BMC Evol Biol. 2007 Oct 22;7(1):197.
ABSTRACT: BACKGROUND: Marine pelagic fishes exhibit rather complex patterns of genetic differentiation, which are the result of both historical processes and present day gene flow. Comparative multi-locus analyses based on both nuclear and mitochondrial genetic markers are probably the most efficient and informative approach to discerning the relative role of historical events and life-history traits in shaping genetic heterogeneity. The European sardine (Sardina pilchardus) is a small pelagic fish with a relatively high migratory capability that is expected to show low levels of genetic differentiation among populations. Previous genetic studies based on meristic and mitochondrial control region haplotype frequency data supported the existence of two sardine subspecies (S. p. pilchardus and S. p. sardina). RESULTS: We investigated genetic structure of sardine among nine locations in the Atlantic Ocean and Mediterranean Sea using allelic size variation of eight specific microsatellite loci. Bayesian clustering and assignment tests, maximum likelihood estimates of migration rates, as well as classical genetic-variance-based methods (hierarchical AMOVA test and RST pairwise comparisons) supported a single evolutionary unit for sardines. These analyses only detected weak but significant genetic differentiation, which followed an isolation-by-distance pattern according to Mantel test. CONCLUSIONS: We suggest that the discordant genetic structuring patterns inferred based on mitochondrial and microsatellite data might indicate that the two different classes of molecular markers may be reflecting different and complementary aspects of the evolutionary history of sardine. Mitochondrial data might be reflecting past isolation of sardine populations into two distinct groupings during Pleistocene whereas microsatellite data reveal the existence of present day gene flow among populations, and a pattern of isolation by distance. [Abstract/Link to Full Text]

Kammerer R, Popp T, H�rtle S, Singer BB, Zimmermann W
Species-specific evolution of immune receptor tyrosine based activation motif-containing CEACAM1-related immune receptors in the dog.
BMC Evol Biol. 2007;7196.
BACKGROUND: Although the impact of pathogens on the evolution of the mammalian immune system is still under debate, proteins, which both regulate immune responses and serve as cellular receptors for pathogens should be at the forefront of pathogen-driven host evolution. The CEA (carcinoembryonic antigen) gene family codes for such proteins and indeed shows tremendous species-specific variation between human and rodents. Since little is known about the CEA gene family in other lineages of placental mammals, we expected to gain new insights into the evolution of the rapidly diverging CEA family by analyzing the CEA family of the dog. RESULTS: Here we describe the complete CEA gene family in the dog. We found that the gene coding for the ITIM-bearing immunoregulatory molecule CEACAM1 gave rise to a recent expansion of the canine CEA gene family by gene duplication, similar to that previously found in humans and mice. However, while the murine and human CEACAMs (carcinoembryonic antigen-related cell adhesion molecules) are predominantly secreted and GPI-anchored, respectively, in the dog, most of the CEACAMs represent ITAM-bearing transmembrane proteins. One of these proteins, CEACAM28, exhibits nearly complete sequence identity with the ligand-binding N domain of CEACAM1, but antagonizing signaling motifs in the cytoplasmic tail. Comparison of nonsynonymous and synonymous substitutions indicates that the CEACAM28 N domain is under the strongest purifying selection of all canine CEACAM1-related CEACAMs. In addition, CEACAM28 shows a similar expression pattern in resting immune cells and tissues as CEACAM1. However, upon activation CEACAM28 mRNA and CEACAM1 mRNA are differentially regulated. CONCLUSION: Thus, CEACAM1 and CEACAM28 are the first paired immune receptors identified within the CEA gene family, which are expressed on T cells and are most likely involved in the fine-tuning of T cell responses. The direction of gene conversion accompanied by purifying selection and expression in immune cells suggests the possibility that CEACAM28 evolved in response to selective pressure imposed by species-specific pathogens. [Abstract/Link to Full Text]

Takahashi R, Watanabe K, Nishida M, Hori M
Evolution of feeding specialization in Tanganyikan scale-eating cichlids: a molecular phylogenetic approach.
BMC Evol Biol. 2007 Oct 18;7(1):195.
ABSTRACT: BACKGROUND: Cichlid fishes in Lake Tanganyika exhibit remarkable diversity in their feeding habits. Among them, seven species in the genus Perissodus are known for their unique feeding habit of scale eating with specialized feeding morphology and behaviour. Although the origin of the scale-eating habit has long been questioned, its evolutionary process is still unknown. In the present study, we conducted interspecific phylogenetic analyses for all nine known species in the tribe Perissodini (seven Perissodus and two Haplotaxodon species) using amplified fragment length polymorphism (AFLP) analyses of the nuclear DNA. On the basis of the resultant phylogenetic frameworks, the evolution of their feeding habits was traced using data from analyses of stomach contents, habitat depths, and observations of oral jaw tooth morphology. RESULTS: AFLP analyses resolved the phylogenetic relationships of the Perissodini, strongly supporting monophyly for each species. The character reconstruction of feeding ecology based on the AFLP tree suggested that scale eating evolved from general carnivorous feeding to highly specialized scale eating. Furthermore, scale eating is suggested to have evolved in deepwater habitats in the lake. Oral jaw tooth shape was also estimated to have diverged in step with specialization for scale eating. CONCLUSIONS: The present evolutionary analyses of feeding ecology and morphology based on the obtained phylogenetic tree demonstrate for the first time the evolutionary process leading from generalised to highly specialized scale eating, with diversification in feeding morphology and behaviour among species. [Abstract/Link to Full Text]

Teuschl Y, Hosken DJ, Blanckenhorn WU
Is reduced female survival after mating a by-product of male-male competition in the dung fly Sepsis cynipsea?
BMC Evol Biol. 2007 Oct 17;7(1):194.
ABSTRACT: BACKGROUND: In a number of species males damage females during copulation, but the reasons for this remain unclear. It may be that males are trying to manipulate female mating behaviour or their life histories. Alternatively, damage may be a side-effect of male-male competition. In the black scavenger or dung fly Sepsis cynipsea (Diptera: Sepsidae) mating reduces female survival, apparently because males wound females during copulation. However, this damage does not seem to relate to attempted manipulation of female reproduction by males. Here we tested the hypothesis that harming females during mating is an incidental by-product of characters favoured during pre-copulatory male-male competition. We assessed whether males and their sons vary genetically in their ability to obtain matings and harm females, and whether more successful males were also more damaging. We did this by ranking males' mating success in paired competitions across several females whose longevity under starvation was subsequently measured. RESULTS: As previously reported, our results show mating is costly for female S. cynipsea. However, variance in female longevity was not explained by male identity, family, body size, number of previous copulations, or copulation duration. Nevertheless, there was a positive correlation between the harm fathers inflicted on their mates (affecting female longevity) and the harm sons inflicted on theirs. Additionally, family identity significantly influenced male copulation success. CONCLUSION: Our results indicate a heritable component of some yet unspecified male trait(s) that influence harm and mating success. However, there was no relationship between copulation success of fathers or sons and the mean longevity of their mates. We therefore found no support for harm being a side effect of traits favoured in pre-copulatory male-male competition. [Abstract/Link to Full Text]

Makalowska I, Lin CF, Hernandez K
Birth and death of gene overlaps in vertebrates.
BMC Evol Biol. 2007 Oct 16;7(1):193.
ABSTRACT: BACKGROUND: Between five and fourteen per cent of genes in the vertebrate genomes do overlap sharing some intronic and/or exonic sequence. It was observed that majority of these overlaps are not conserved among vertebrate lineages. Although several mechanisms have been proposed to explain gene overlap origin the evolutionary basis of this phenomenon is still not well understood. Here, we present results of the comparative analysis of several vertebrate genomes. The purpose of this study was to examine overlapping genes in the context of their evolution and mechanisms leading to their origin. RESULTS: Based on the presence and arrangement of human overlapping genes orthologs in rodent and fish genomes we developed 15 theoretical scenarios of overlapping genes evolution. Analysis of these theoretical scenarios and close examination of genomic sequences revealed new mechanisms leading to the overlaps evolution and confirmed that many of the vertebrate gene overlaps are not conserved. This study also demonstrates that repetitive elements contribute to the overlapping genes origination and, for the first time, that evolutionary events could lead to the loss of an ancient overlap. CONCLUSIONS: Birth as well as most probably death of gene overlaps occurred over the entire time of vertebrate evolution and there wasn't any rapid origin or big bang in the course of overlapping genes evolution. The major forces in the gene overlaps origination are transposition and exaptation. Our results also imply that origin of overlapping genes is not an issue of saving space and contracting genomes size. [Abstract/Link to Full Text]

Carmel L, Rogozin IB, Wolf YI, Koonin EV
Patterns of intron gain and conservation in eukaryotic genes.
BMC Evol Biol. 2007 Oct 12;7(1):192.
ABSTRACT: BACKGROUND: The presence of introns in protein-coding genes is a universal feature of eukaryotic genome organization, and the genes of multicellular eukaryotes, typically, contain multiple introns, a substantial fraction of which share position in distant taxa, such as plants and animals. Depending on the methods and data sets used, researchers have reached opposite conclusions on the causes of the high fraction of shared introns in orthologous genes from distant eukaryotes. Some studies conclude that shared intron positions reflect, almost entirely, a remarkable evolutionary conservation, whereas others attribute it to parallel gain of introns. To resolve these contradictions, it is crucial to analyze the evolution of introns by using a model that minimally relies on arbitrary assumptions. RESULTS: We developed a probabilistic model of evolution that allows for variability of intron gain and loss rates over branches of the phylogenetic tree, individual genes, and individual sites. Applying this model to an extended set of conserved eukaryotic genes, we find that parallel gain, on average, accounts for only ~8% of the shared intron positions. However, the distribution of parallel gains over the phylogenetic tree of eukaryotes is highly non-uniform. There are, practically, no parallel gains in closely related lineages, whereas for distant lineages, such as animals and plants, parallel gains appear to contribute up to 20% of the shared intron positions. In accord with these findings, we estimated that ancestral introns have a high probability to be retained in extant genomes, and conversely, that a substantial fraction of extant introns have retained their positions since the early stages of eukaryotic evolution. In addition, the density of sites that are available for intron insertion is estimated to be, approximately, one in seven basepairs. CONCLUSIONS: We obtained robust estimates of the contribution of parallel gain to the observed sharing of intron positions between eukaryotic species separated by different evolutionary distances. The results indicate that, although the contribution of parallel gains varies across the phylogenetic tree, the high level of intron position sharing is due, primarily, to evolutionary conservation. Accordingly, numerous introns appear to persist in the same position over hundreds of millions of years of evolution. This is compatible with recent observations of a negative correlation between the rate of intron gain and coding sequence evolution rate of a gene, suggesting that at least some of the introns are functionally relevant. [Abstract/Link to Full Text]

de la Chaux N, Messer PW, Arndt PF
DNA indels in coding regions reveal selective constraints on protein evolution in the human lineage.
BMC Evol Biol. 2007 Oct 12;7(1):191.
ABSTRACT: BACKGROUND: Insertions and deletions of DNA segments (indels) are together with substitutions the major mutational processes that generate genetic variation. Here we focus on recent DNA insertions and deletions in protein coding regions of the human genome to investigate selective constraints on indels in protein evolution. RESULTS: Frequencies of inserted and deleted amino acids differ from background amino acid frequencies in the human proteome. Small amino acids are overrepresented, while hydrophobic, aliphatic and aromatic amino acids are strongly suppressed. Indels are found to be preferentially located in protein regions that do not form important structural domains. Amino acid insertion and deletion rates in genes associated with elementary biochemical reactions (e.g. catalytic activity, ligase activity, electron transport, or catabolic process) are lower compared to those in other genes and are therefore subject to stronger purifying selection. CONCLUSIONS: Our analysis indicates that indels in human protein coding regions are subject to distinct levels of selective pressure with regard to their structural impact on the amino acid sequence, as well as to general properties of the genes they are located in. These findings confirm that many commonly accepted characteristics of selective constraints for substitutions are also valid for amino acid insertions and deletions. [Abstract/Link to Full Text]

Kriegs JO, Matzke A, Churakov G, Kuritzin A, Mayr G, Brosius J, Schmitz J
Waves of genomic hitchhikers shed light on the evolution of gamebirds (Aves: Galliformes).
BMC Evol Biol. 2007 Oct 9;7(1):190.
ABSTRACT: BACKGROUND: The phylogenetic tree of Galliformes (gamebirds, including megapodes, currassows, guinea fowl, New and Old World quails, chicken, pheasants, grouse, and turkeys) has been considerably remodeled over the last decades as new data and analytical methods became available. Analyzing presence/absence patterns of retroposed elements avoids the problems of homoplastic characters inherent in other methodologies. In gamebirds, chicken repeats 1 (CR1) are the most prevalent retroposed elements, but little is known about the activity of their various subtypes over time. Ascertaining the fixation patterns of CR1 elements would help unravel the phylogeny of gamebirds and other poorly resolved avian clades. RESULTS: We analyzed 1,978 nested CR1 elements and developed a multidimensional approach taking advantage of their transposition in transposition character (TinT) to characterize the fixation patterns of all 22 known chicken CR1 subtypes. The presence/absence patterns of those elements that were active at different periods of gamebird evolution provided evidence for a clade (Cracidae + (Numididae + (Odontophoridae + Phasianidae))) not including Megapodiidae; and for Rollulus as the sister taxon of the other analyzed Phasianidae. Genomic trace sequences of the turkey genome further demonstrated that the endangered African Congo Peafowl (Afropavo congensis) is the sister taxon of the Asian Peafowl (Pavo), rejecting other predominantly morphology-based groupings, and that phasianids are monophyletic, including the sister taxa Tetraoninae and Meleagridinae. CONCLUSIONS: The TinT information concerning relative fixation times of CR1 subtypes enabled us to efficiently investigate gamebird phylogeny and to reconstruct an unambiguous tree topology. This method should provide a useful tool for investigations in other taxonomic groups as well. [Abstract/Link to Full Text]

Salvaudon L, Heraudet V, Shykoff JA
Genotype-specific interactions and the trade-off between host and parasite fitness.
BMC Evol Biol. 2007 Oct 5;7(1):189.
ABSTRACT: BACKGROUND: Evolution of parasite traits is inextricably linked to their hosts. For instance one common definition of parasite virulence is the reduction in host fitness due to infection. Thus, traits of infection must be viewed in both protagonists and may be under shared genetic and physiological control. We investigated these questions on the oomycete Hyaloperonospora arabidopsis (=parasitica), a natural pathogen of the Brassicaceae Arabidopsis thaliana. RESULTS: We performed a controlled cross inoculation experiment confronting six lines of the host plant with seven strains of the parasite in order to evaluate genetic variation for phenotypic traits of infection among hosts, parasites, and distinct combinations. Parasite infection intensity and transmission were highly variable among parasite strains and host lines but depended also on the interaction between particular genotypes of the protagonists, and genetic variation for the infection phenotype of parasites from natural populations was found even at a small spatial scale within population. Furthermore, increased parasite fitness led to a significant decrease in host fitness only on a single host line (Gb), although a trade-off between these two traits was expected because host and parasite share the same resource pool for their respective reproduction. We propose that different levels of compatibility dependent on genotype by genotype interactions might lead to different amounts of resources available for host and parasite reproduction. This variation in compatibility could thus mask the expected negative relationship between host and parasite fitness, as the total resource pool would not be constant. CONCLUSIONS: These results highlight the importance of host variation in the determination of parasite fitness traits. This kind of interaction may in turn decouple the relationship between parasite transmission and its negative effect on host fitness, altering theoretical predictions of parasite evolution. [Abstract/Link to Full Text]

Irimia M, Rukov JL, Penny D, Roy SW
Functional and evolutionary analysis of alternatively spliced genes is consistent with an early eukaryotic origin of alternative splicing.
BMC Evol Biol. 2007;7188.
BACKGROUND: Alternative splicing has been reported in various eukaryotic groups including plants, apicomplexans, diatoms, amoebae, animals and fungi. However, whether widespread alternative splicing has evolved independently in the different eukaryotic groups or was inherited from their last common ancestor, and may therefore predate multicellularity, is still unknown. To better understand the origin and evolution of alternative splicing and its usage in diverse organisms, we studied alternative splicing in 12 eukaryotic species, comparing rates of alternative splicing across genes of different functional classes, cellular locations, intron/exon structures and evolutionary origins. RESULTS: For each species, we find that genes from most functional categories are alternatively spliced. Ancient genes (shared between animals, fungi and plants) show high levels of alternative splicing. Genes with products expressed in the nucleus or plasma membrane are generally more alternatively spliced while those expressed in extracellular location show less alternative splicing. We find a clear correspondence between incidence of alternative splicing and intron number per gene both within and between genomes. In general, we find several similarities in patterns of alternative splicing across these diverse eukaryotes. CONCLUSION: Along with previous studies indicating intron-rich genes with weak intron boundary consensus and complex spliceosomes in ancestral organisms, our results suggest that at least a simple form of alternative splicing may already have been present in the unicellular ancestor of plants, fungi and animals. A role for alternative splicing in the evolution of multicellularity then would largely have arisen by co-opting the preexisting process. [Abstract/Link to Full Text]

Fumasoni I, Meani N, Rambaldi D, Scafetta G, Alcalay M, Ciccarelli FD
Family expansion and gene rearrangements contributed to the functional specialization of PRDM genes in vertebrates.
BMC Evol Biol. 2007;7187.
BACKGROUND: Progressive diversification of paralogs after gene expansion is essential to increase their functional specialization. However, mode and tempo of this divergence remain mostly unclear. Here we report the comparative analysis of PRDM genes, a family of putative transcriptional regulators involved in human tumorigenesis. RESULTS: Our analysis assessed that the PRDM genes originated in metazoans, expanded in vertebrates and further duplicated in primates. We experimentally showed that fast-evolving paralogs are poorly expressed, and that the most recent duplicates, such as primate-specific PRDM7, acquire tissue-specificity. PRDM7 underwent major structural rearrangements that decreased the number of encoded Zn-Fingers and modified gene splicing. Through internal duplication and activation of a non-canonical splice site (GC-AG), PRDM7 can acquire a novel intron. We also detected an alternative isoform that can retain the intron in the mature transcript and that is predominantly expressed in human melanocytes. CONCLUSION: Our findings show that (a) molecular evolution of paralogs correlates with their expression pattern; (b) gene diversification is obtained through massive genomic rearrangements; and (c) splicing modification contributes to the functional specialization of novel genes. [Abstract/Link to Full Text]

Kumar S, Nagarajan M, Sandhu JS, Kumar N, Behl V
Phylogeography and domestication of Indian river buffalo.
BMC Evol Biol. 2007 Oct 4;7(1):186.
ABSTRACT: BACKGROUND: The water buffalo- Bubalus bubalis holds tremendous potential in livestock sector in many Asian countries, particularly India. The origin, domestication and genetic structure of the Indian river buffalo are poorly understood. Therefore, to understand the relationship among the maternal lineages of Indian river buffalo breeds and their domestication process, we analysed mitochondrial D-loop region of 217 animals representing eight breeds from eight different locations in India along with published sequences of Mediterranean buffalo. RESULTS: The maximum parsimony tree showed one major clade with six internal branches. Reduced median network revealed expansion from more than one set of haplotypes indicating complex domestication events for this species. In addition, we found several singleton haplotypes. Using rho statistics, we obtained a time estimate of 6300 years BP for the expansion of one set of hapltoypes of the Indian domestic buffalo. A few breed specific branches in the network indicated an ancient time depth of differentiation of some of the maternal lineages of river buffalo breeds. The multidimensional display of breed pairwise FST values showed significant breed differentiation. CONCLUSIONS: Present day river buffalo is the result of complex domestication processes involving more than one maternal lineage and a significant maternal gene flow from the wild populations after the initial domestication events. Our data are consistent with the available archaeological information in supporting the proposition that the river buffalo was likely to be domesticated in the Western region of the Indian subcontinent, specifically the present day breeding tracts of the Mehsana, Surati and Pandharpuri breeds. [Abstract/Link to Full Text]

Pike N, Whitfield JA, Foster WA
Ecological correlates of sociality in Pemphigus aphids, with a partial phylogeny of the genus.
BMC Evol Biol. 2007 Oct 3;7(1):185.
ABSTRACT: BACKGROUND: Because the systems of social organisation in the various species of Pemphigus aphids span the continuum from asociality through to advanced sociality (typified by the possession of morphologically specialised soldiers), the genus is an ideal model clade in which to study the influence of ecology on the origins of eusociality. We made detailed study of the ecology of three gall-dwelling species that show clear differences in their levels of social behaviour. To elucidate evolutionary relationships and to attempt to estimate the number of origins of sociality, we also created a phylogeny based on sequences spanning the mitochondrial genes Cytochrome Oxidase I and II for nine species of Pemphigus. RESULTS: P. spyrothecae, a highly social species with aggressive morphologically-specialised soldiers, has the longest galling phase, unsynchronised development of a large number of individuals in a densely-populated gall, and an extended period over which alates emerge. P. populi, a species with no soldiers, has the shortest galling phase, synchronised development of a small number of individuals in a sparsely-populated gall, and an extremely brief emergence period. The ecology of P. bursarius, which has behavioural soldiers that are not morphologically specialised, is intermediate between these two extremes. The galls of P. spyrothecae and P. bursarius form small openings during the course of the season and predation-related mortality is relatively high in these two species. Conversely, predation does not occur during the galling phase of P. populi, which has no soldiers but makes an entirely-sealed gall. The phylogeny allowed us to infer one likely point of origin of basic social defence and two independent origins of enhanced defence. Based on current knowledge of behaviour, the phylogeny also suggests that the defence trait may have been lost at least once. CONCLUSIONS: The life-history strategy of P. spyrothecae appears to be geared towards defending the colony against the constant threat of predation that faces the inhabitants of a long-lived, open gall. The life-history strategy of P. populi, on the other hand, is to avoid predation in the closed gall fortress and flee for the secondary host at the earliest opportunity. The life-history strategy of P. bursarius appears to represent a compromise between these strategies. [Abstract/Link to Full Text]

Larsen J, Kuhnert P, Frey J, Christensen H, Bisgaard M, Olsen JE
Analysis of gene order data supports vertical inheritance of the leukotoxin operon and genome rearrangements in the 5' flanking region in genus Mannheimia.
BMC Evol Biol. 2007 Oct 3;7(1):184.
ABSTRACT: BACKGROUND: The Mannheimia subclades belong to the same bacterial genus, but have taken divergent paths toward their distinct lifestyles. For example, M. haemolytica + M. glucosida are potential pathogens of the respiratory tract in the mammalian suborder Ruminantia, whereas M. ruminalis, the supposed sister group, lives as a commensal in the ovine rumen. We have tested the hypothesis that vertical inheritance of the leukotoxin (lktCABD) operon has occurred from the last common ancestor of genus Mannheimia to any ancestor of the diverging subclades by exploring gene order data. RESULTS: We examined the gene order in the 5' flanking region of the leukotoxin operon and found that the 5' flanking gene strings, hslVU-lapB-artJ-lktC and xylAB-lktC, are peculiar to M. haemolytica + M. glucosida and M. granulomatis, respectively, whereas the gene string hslVU-lapB-lktC is present in M. ruminalis, the supposed sister group of M. haemolytica + M. glucosida, and in the most ancient subclade M. varigena. In M. granulomatis, we found remnants of the gene string hslVU-lapB-lktC in the xylB-lktC intergenic region. CONCLUSIONS: These observations indicate that the gene string hslVU-lapB-lktC is more ancient than the hslVU-lapB-artJ-lktC and xylAB-lktC gene strings. The presence of (remnants of) the ancient gene string hslVU-lapB-lktC among any subclades within genus Mannheimia supports that it has been vertically inherited from the last common ancestor of genus Mannheimia to any ancestor of the diverging subclades, thus reaffirming the hypothesis of vertical inheritance of the leukotoxin operon. The presence of individual 5' flanking regions in M. haemolytica + M. glucosida and M. granulomatis reflects later genome rearrangements within each subclade. The evolution of the novel 5' flanking region in M. haemolytica + M. glucosida resulted in transcriptional coupling between the divergently arranged artJ and lkt promoters. We propose that the chimeric promoter have led to high level expression of the leukotoxin operon which could explain the increased potential of certain M. haemolytica + M. glucosida strains to cause a particular type of infection. [Abstract/Link to Full Text]

Fewer DP, Rouhiainen L, Jokela J, Wahlsten M, Laakso K, Wang H, Sivonen K
Recurrent adenylation domain replacement in the microcystin synthetase gene cluster.
BMC Evol Biol. 2007 Oct 1;7(1):183.
ABSTRACT: BACKGROUND: Microcystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. Microcystins are synthesized on large NRPS-PKS enzyme complexes. Many structural variants of microcystins are produced simulatenously. A recombination event between the first module of mcyB (mcyB1) and mcyC in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus Microcystis. RESULTS: Here we undertook a phylogenetic study to investigate the order and timing of recombination between the mcyB1 and mcyC genes in a diverse selection of microcystin producing cyanobacteria. Our results provide support for complex evolutionary processes taking place at the mcyB1 and mcyC adenylation domains which recognize and activate the amino acids found at X and Z positions. We find evidence for recent recombination between mcyB1 and mcyC in strains of the genera Anabaena, Microcystis, and Hapalosiphon. We also find clear evidence for independent adenylation domain conversion of mcyB1 by unrelated peptide synthetase modules in strains of the genera Nostoc and Microcystis. The recombination events replace only the adenylation domain in each case and the condensation domains of mcyB1 and mcyC are not transferred together with the adenylation domain. Our findings demonstrate that the mcyB1 and mcyC adenylation domains are recombination hotspots in the microcystin synthetase gene cluster. CONCLUSIONS: Recombination is thought to be one of the main mechanisms driving the diversification of NRPSs. However, there is very little information on how recombination takes place in nature. This study demonstrates that functional peptide synthetases are created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains. [Abstract/Link to Full Text]

Recent Articles in BMC Structural Biology

Delatorre P, Rocha BA, Souza EP, Oliveira TM, Bezerra GA, Moreno FB, Freitas BT, Santi-Gadelha T, Sampaio AH, Azevedo WF, Cavada BS
Structure of a lectin from Canavalia gladiata seeds: new structural insights for old molecules.
BMC Struct Biol. 2007;752.
BACKGROUND: Lectins are mainly described as simple carbohydrate-binding proteins. Previous studies have tried to identify other binding sites, which possible recognize plant hormones, secondary metabolites, and isolated amino acid residues. We report the crystal structure of a lectin isolated from Canavalia gladiata seeds (CGL), describing a new binding pocket, which may be related to pathogen resistance activity in ConA-like lectins; a site where a non-protein amino-acid, alpha-aminobutyric acid (Abu), is bound. RESULTS: The overall structure of native CGL and complexed with alpha-methyl-mannoside and Abu have been refined at 2.3 A and 2.31 A resolution, respectively. Analysis of the electron density maps of the CGL structure shows clearly the presence of Abu, which was confirmed by mass spectrometry. CONCLUSION: The presence of Abu in a plant lectin structure strongly indicates the ability of lectins on carrying secondary metabolites. Comparison of the amino acids composing the site with other legume lectins revealed that this site is conserved, providing an evidence of the biological relevance of this site. This new action of lectins strengthens their role in defense mechanisms in plants. [Abstract/Link to Full Text]

Acharya R, Gupta M, Ramakumar S, Ramagopal UA, Chauhan VS
Observation of glycine zipper and unanticipated occurrence of ambidextrous helices in the crystal structure of a chiral undecapeptide.
BMC Struct Biol. 2007;751.
BACKGROUND: The de novo design of peptides and proteins has recently surfaced as an approach for investigating protein structure and function. This approach vitally tests our knowledge of protein folding and function, while also laying the groundwork for the fabrication of proteins with properties not precedented in nature. The success of these studies relies heavily on the ability to design relatively short peptides that can espouse stable secondary structures. To this end, substitution with alpha, beta-dehydroamino acids, especially alpha, beta-dehydrophenylalanine (Delta Phe) comes in use for spawning well-defined structural motifs. Introduction of Delta Phe induces beta-bends in small and 3(10)-helices in longer peptide sequences. RESULTS: The present report is an investigation of the effect of incorporating two glycines in the middle of a DeltaPhe containing undecapeptide. A de novo designed undecapeptide, Ac-Gly1-Ala2-Delta Phe3-Leu4-Gly5-Delta Phe6-Leu7-Gly8-Delta Phe9-Ala10-Gly11-NH2, was synthesized and characterized using X-ray diffraction and Circular Dichroism spectroscopic methods. Crystallographic studies suggest that, despite the presence of L-amino acid (L-Ala and L-Leu) residues in the middle of the sequence, the peptide adopts a 3(10)-helical conformation of ambidextrous screw sense, one of them a left-handed (A) and the other a right-handed (B) 3(10)-helix with A and B being antiparallel to each other. However, CD studies reveal that the undecapeptide exclusively adopts a right-handed 3(10)-helical conformation. In the crystal packing, three different interhelical interfaces, Leu-Leu, Gly-Gly and Delta Phe-Delta Phe are observed between the helices A and B. A network of C-H...O hydrogen bonds are observed at Delta Phe-Delta Phe and Gly-Gly interhelical interfaces. An important feature observed is the occurrence of glycine zipper motif at Gly-Gly interface. At this interface, the geometric pattern of interhelical interactions seems to resemble those observed between helices in transmembrane (TM) proteins. CONCLUSION: The present design strategy can thus be exploited in future work on de novo design of helical bundles of higher order and compaction utilizing Delta Phe residues along with GXXG motif. [Abstract/Link to Full Text]

Mayr G, Domingues FS, Lackner P
Comparative analysis of protein structure alignments.
BMC Struct Biol. 2007;750.
BACKGROUND: Several methods are currently available for the comparison of protein structures. These methods have been analysed regarding the performance in the identification of structurally/evolutionary related proteins, but so far there has been less focus on the objective comparison between the alignments produced by different methods. RESULTS: We analysed and compared the structural alignments obtained by different methods using three sets of pairs of structurally related proteins. The first set corresponds to 355 pairs of remote homologous proteins according to the SCOP database (ASTRAL40 set). The second set was derived from the SISYPHUS database and includes 69 protein pairs (SISY set). The third set consists of 40 pairs that are challenging to align (RIPC set). The alignment of pairs of this set requires indels of considerable number and size and some of the proteins are related by circular permutations, show extensive conformational variability or include repetitions. Two standard methods (CE and DALI) were applied to align the proteins in the ASTRAL40 set. The extent of structural similarity identified by both methods is highly correlated and the alignments from the two methods agree on average in more than half of the aligned positions. CE, DALI, as well as four additional methods (FATCAT, MATRAS, Calpha-match and SHEBA) were then compared using the SISY and RIPC sets. The accuracy of the alignments was assessed by comparison to reference alignments. The alignments generated by the different methods on average match more than half of the reference alignments in the SISY set. The alignments obtained in the more challenging RIPC set tend to differ considerably and match reference alignments less successfully than the SISY set alignments. CONCLUSION: The alignments produced by different methods tend to agree to a considerable extent, but the agreement is lower for the more challenging pairs. The results for the comparison to reference alignments are encouraging, but also indicate that there is still room for improvement. [Abstract/Link to Full Text]

Schmidt B, Hogg PJ
Search for allosteric disulfide bonds in NMR structures.
BMC Struct Biol. 2007;749.
BACKGROUND: Allosteric disulfide bonds regulate protein function when they break and/or form. They typically have a -RHStaple configuration, which is defined by the sign of the five chi angles that make up the disulfide bond. RESULTS: All disulfides in NMR and X-ray protein structures as well as in refined structure datasets were compared and contrasted for configuration and strain energy. CONCLUSION: The mean dihedral strain energy of 55,005 NMR structure disulfides was twice that of 42,690 X-ray structure disulfides. Moreover, the energies of all twenty types of disulfide bond was higher in NMR structures than X-ray structures, where there was an exponential decrease in the mean strain energy as the incidence of the disulfide type increased. Evaluation of protein structures for which there are X-ray and NMR models shows that the same disulfide bond can exist in different configurations in different models. A disulfide bond configuration that is rare in X-ray structures is the -LHStaple. In NMR structures, this disulfide is characterised by a particularly high potential energy and very short alpha-carbon distance. The HIV envelope glycoprotein gp120, for example, is regulated by thiol/disulfide exchange and contains allosteric -RHStaple bonds that can exist in the -LHStaple configuration. It is an open question which form of the disulfide is the functional configuration. [Abstract/Link to Full Text]

Ibryashkina EM, Zakharova MV, Baskunov VB, Bogdanova ES, Nagornykh MO, Den'mukhamedov MM, Melnik BS, Kolinski A, Gront D, Feder M, Solonin AS, Bujnicki JM
Type II restriction endonuclease R.Eco29kI is a member of the GIY-YIG nuclease superfamily.
BMC Struct Biol. 2007;748.
BACKGROUND: The majority of experimentally determined crystal structures of Type II restriction endonucleases (REases) exhibit a common PD-(D/E)XK fold. Crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI), and bioinformatics analyses supported by mutagenesis suggested that some REases belong to the HNH fold. Our previous bioinformatic analysis suggested that REase R.Eco29kI shares sequence similarities with one more unrelated nuclease superfamily, GIY-YIG, however so far no experimental data were available to support this prediction. The determination of a crystal structure of the GIY-YIG domain of homing endonuclease I-TevI provided a template for modeling of R.Eco29kI and prompted us to validate the model experimentally. RESULTS: Using protein fold-recognition methods we generated a new alignment between R.Eco29kI and I-TevI, which suggested a reassignment of one of the putative catalytic residues. A theoretical model of R.Eco29kI was constructed to illustrate its predicted three-dimensional fold and organization of the active site, comprising amino acid residues Y49, Y76, R104, H108, E142, and N154. A series of mutants was constructed to generate amino acid substitutions of selected residues (Y49A, R104A, H108F, E142A and N154L) and the mutant proteins were examined for their ability to bind the DNA containing the Eco29kI site 5'-CCGCGG-3' and to catalyze the cleavage reaction. Experimental data reveal that residues Y49, R104, E142, H108, and N154 are important for the nuclease activity of R.Eco29kI, while H108 and N154 are also important for specific DNA binding by this enzyme. CONCLUSION: Substitutions of residues Y49, R104, H108, E142 and N154 predicted by the model to be a part of the active site lead to mutant proteins with strong defects in the REase activity. These results are in very good agreement with the structural model presented in this work and with our prediction that R.Eco29kI belongs to the GIY-YIG superfamily of nucleases. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD-(D/E)XK or HNH superfamilies of nucleases, and is instead a member of the unrelated GIY-YIG superfamily. [Abstract/Link to Full Text]

Byun JS, Rhee JK, Kim ND, Yoon J, Kim DU, Koh E, Oh JW, Cho HS
Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties.
BMC Struct Biol. 2007;747.
BACKGROUND: EstE1 is a hyperthermophilic esterase belonging to the hormone-sensitive lipase family and was originally isolated by functional screening of a metagenomic library constructed from a thermal environmental sample. Dimers and oligomers may have been evolutionally selected in thermophiles because intersubunit interactions can confer thermostability on the proteins. The molecular mechanisms of thermostabilization of this extremely thermostable esterase are not well understood due to the lack of structural information. RESULTS: Here we report for the first time the 2.1-A resolution crystal structure of EstE1. The three-dimensional structure of EstE1 exhibits a classic alpha/beta hydrolase fold with a central parallel-stranded beta sheet surrounded by alpha helices on both sides. The residues Ser154, Asp251, and His281 form the catalytic triad motif commonly found in other alpha/beta hydrolases. EstE1 exists as a dimer that is formed by hydrophobic interactions and salt bridges. Circular dichroism spectroscopy and heat inactivation kinetic analysis of EstE1 mutants, which were generated by structure-based site-directed mutagenesis of amino acid residues participating in EstE1 dimerization, revealed that hydrophobic interactions through Val274 and Phe276 on the beta8 strand of each monomer play a major role in the dimerization of EstE1. In contrast, the intermolecular salt bridges contribute less significantly to the dimerization and thermostability of EstE1. CONCLUSION: Our results suggest that intermolecular hydrophobic interactions are essential for the hyperthermostability of EstE1. The molecular mechanism that allows EstE1 to endure high temperature will provide guideline for rational design of a thermostable esterase/lipase using the lipolytic enzymes showing structural similarity to EstE1. [Abstract/Link to Full Text]

Shaw N, Cheng C, Tempel W, Chang J, Ng J, Wang XY, Perrett S, Rose J, Rao Z, Wang BC, Liu ZJ
(NZ)CH...O contacts assist crystallization of a ParB-like nuclease.
BMC Struct Biol. 2007;746.
BACKGROUND: The major bottleneck for determination of 3 D structures of proteins using X-rays is the production of diffraction quality crystals. Often proteins are subjected to chemical modification to improve the chances of crystallization RESULTS: Here, we report the successful crystallization of a nuclease employing a reductive methylation protocol. The key to crystallization was the successful introduction of 44 new cohesive (NZ) CH...O contacts (3.2-3.7 A) by the addition of 2 methyl groups to the side chain amine nitrogen (NZ) of 9 lysine residues of the nuclease. The new contacts dramatically altered the crystallization properties of the protein, resulting in crystals that diffracted to 1.2 A resolution. Analytical ultracentrifugation analysis and thermodynamics results revealed a more compact protein structure with better solvent exclusion of buried Trp residues in the folded state of the methylated protein, assisting crystallization. CONCLUSION: In this study, introduction of novel cohesive (NZ)CH...O contacts by reductive methylation resulted in the crystallization of a protein that had previously resisted crystallization in spite of extensive purification and crystallization space screening. Introduction of (NZ)CH...O contacts could provide a solution to crystallization problems for a broad range of protein targets. [Abstract/Link to Full Text]

Schormann N, Grigorian A, Samal A, Krishnan R, DeLucas L, Chattopadhyay D
Crystal structure of vaccinia virus uracil-DNA glycosylase reveals dimeric assembly.
BMC Struct Biol. 2007;745.
BACKGROUND: Uracil-DNA glycosylases (UDGs) catalyze excision of uracil from DNA. Vaccinia virus, which is the prototype of poxviruses, encodes a UDG (vvUDG) that is significantly different from the UDGs of other organisms in primary, secondary and tertiary structure and characteristic motifs. It adopted a novel catalysis-independent role in DNA replication that involves interaction with a viral protein, A20, to form the processivity factor. UDG:A20 association is essential for assembling of the processive DNA polymerase complex. The structure of the protein must have provisions for such interactions with A20. This paper provides the first glimpse into the structure of a poxvirus UDG. RESULTS: Results of dynamic light scattering experiments and native size exclusion chromatography showed that vvUDG is a dimer in solution. The dimeric assembly is also maintained in two crystal forms. The core of vvUDG is reasonably well conserved but the structure contains one additional beta-sheet at each terminus. A glycerol molecule is found in the active site of the enzyme in both crystal forms. Interaction of this glycerol molecule with the protein possibly mimics the enzyme-substrate (uracil) interactions. CONCLUSION: The crystal structures reveal several distinctive features of vvUDG. The new structural features may have evolved for adopting novel functions in the replication machinery of poxviruses. The mode of interaction between the subunits in the dimers suggests a possible model for binding to its partner and the nature of the processivity factor in the polymerase complex. [Abstract/Link to Full Text]

Lomize AL, Pogozheva ID, Lomize MA, Mosberg HI
The role of hydrophobic interactions in positioning of peripheral proteins in membranes.
BMC Struct Biol. 2007;744.
BACKGROUND: Three-dimensional (3D) structures of numerous peripheral membrane proteins have been determined. Biological activity, stability, and conformations of these proteins depend on their spatial positions with respect to the lipid bilayer. However, these positions are usually undetermined. RESULTS: We report the first large-scale computational study of monotopic/peripheral proteins with known 3D structures. The optimal translational and rotational positions of 476 proteins are determined by minimizing energy of protein transfer from water to the lipid bilayer, which is approximated by a hydrocarbon slab with a decadiene-like polarity and interfacial regions characterized by water-permeation profiles. Predicted membrane-binding sites, protein tilt angles and membrane penetration depths are consistent with spin-labeling, chemical modification, fluorescence, NMR, mutagenesis, and other experimental studies of 53 peripheral proteins and peptides. Experimental membrane binding affinities of peripheral proteins were reproduced in cases that did not involve a helix-coil transition, specific binding of lipids, or a predominantly electrostatic association. Coordinates of all examined peripheral proteins and peptides with the calculated hydrophobic membrane boundaries, subcellular localization, topology, structural classification, and experimental references are available through the Orientations of Proteins in Membranes (OPM) database. CONCLUSION: Positions of diverse peripheral proteins and peptides in the lipid bilayer can be accurately predicted using their 3D structures that represent a proper membrane-bound conformation and oligomeric state, and have membrane binding elements present. The success of the implicit solvation model suggests that hydrophobic interactions are usually sufficient to determine the spatial position of a protein in the membrane, even when electrostatic interactions or specific binding of lipids are substantial. Our results demonstrate that most peripheral proteins not only interact with the membrane surface, but penetrate through the interfacial region and reach the hydrocarbon interior, which is consistent with published experimental studies. [Abstract/Link to Full Text]

Kmiecik S, Gront D, Kolinski A
Towards the high-resolution protein structure prediction. Fast refinement of reduced models with all-atom force field.
BMC Struct Biol. 2007;743.
BACKGROUND: Although experimental methods for determining protein structure are providing high resolution structures, they cannot keep the pace at which amino acid sequences are resolved on the scale of entire genomes. For a considerable fraction of proteins whose structures will not be determined experimentally, computational methods can provide valuable information. The value of structural models in biological research depends critically on their quality. Development of high-accuracy computational methods that reliably generate near-experimental quality structural models is an important, unsolved problem in the protein structure modeling. RESULTS: Large sets of structural decoys have been generated using reduced conformational space protein modeling tool CABS. Subsequently, the reduced models were subject to all-atom reconstruction. Then, the resulting detailed models were energy-minimized using state-of-the-art all-atom force field, assuming fixed positions of the alpha carbons. It has been shown that a very short minimization leads to the proper ranking of the quality of the models (distance from the native structure), when the all-atom energy is used as the ranking criterion. Additionally, we performed test on medium and low accuracy decoys built via classical methods of comparative modeling. The test placed our model evaluation procedure among the state-of-the-art protein model assessment methods. CONCLUSION: These test computations show that a large scale high resolution protein structure prediction is possible, not only for small but also for large protein domains, and that it should be based on a hierarchical approach to the modeling protocol. We employed Molecular Mechanics with fixed alpha carbons to rank-order the all-atom models built on the scaffolds of the reduced models. Our tests show that a physic-based approach, usually considered computationally too demanding for large-scale applications, can be effectively used in such studies. [Abstract/Link to Full Text]

Asojo OA, Homma K, Sedlacek M, Ngamelue M, Goud GN, Zhan B, Deumic V, Asojo O, Hotez PJ
X-ray structures of Na-GST-1 and Na-GST-2 two glutathione S-transferase from the human hookworm Necator americanus.
BMC Struct Biol. 2007;742.
BACKGROUND: Human hookworm infection is a major cause of anemia and malnutrition of adults and children in the developing world. As part of on-going efforts to control hookworm infection, The Human Hookworm Vaccine Initiative has identified candidate vaccine antigens from the infective L3 larval stages and adult stages of the parasite. Adult stage antigens include the cytosolic glutathione-S-transferases (GSTs). Nematode GSTs facilitate the inactivation and degradation of a variety of electrophilic substrates (drugs) via the nucleophilic addition of reduced glutathione. Parasite GSTs also play significant roles in multi-drug resistance and the modulation of host-immune defense mechanisms. RESULTS: The crystal structures of Na-GST-1 and Na-GST-2, two major GSTs from Necator americanus the main human hookworm parasite, have been solved at the resolution limits of 2.4 A and 1.9 A respectively. The structure of Na-GST-1 was refined to R-factor 18.9% (R-free 28.3%) while that of Na-GST-2 was refined to R-factor 17.1% (R-free 21.7%). Glutathione usurped during the fermentation process in bound in the glutathione binding site (G-site) of each monomer of Na-GST-2. Na-GST-1 is uncomplexed and its G-site is abrogated by Gln 50. These first structures of human hookworm parasite GSTs could aid the design of novel hookworm drugs. CONCLUSION: The 3-dimensional structures of Na-GST-1 and Na-GST-2 show two views of human hookworm GSTs. While the GST-complex structure of Na-GST-2 reveals a typical GST G-site that of Na-GST-1 suggests that there is some conformational flexibility required in order to bind the substrate GST. In addition, the overall binding cavities for both are larger, more open, as well as more accessible to diverse ligands than those of GSTs from organisms that have other major detoxifying mechanisms. The results from this study could aid in the design of novel drugs and vaccine antigens. [Abstract/Link to Full Text]

Dergez T, Lorinczy D, K�ncz�l F, Farkas N, Belagyi J
Differential scanning calorimetry study of glycerinated rabbit psoas muscle fibres in intermediate state of ATP hydrolysis.
BMC Struct Biol. 2007;741.
BACKGROUND: Thermal denaturation experiments were extended to study the thermal behaviour of the main motor proteins (actin and myosin) in their native environment in striated muscle fibres. The interaction of actin with myosin in the highly organized muscle structure is affected by internal forces; therefore their altered conformation and interaction may differ from those obtained in solution. The energetics of long functioning intermediate states of ATP hydrolysis cycle was studied in muscle fibres by differential scanning calorimetry (DSC). RESULTS: SETARAM Micro DSC-II was used to monitor the thermal denaturation of the fibre system in rigor and in the presence of nucleotide and nucleotide analogues. The AM.ADP.Pi state of the ATP hydrolysis cycle has a very short lifetime therefore, we mimicked the different intermediate states with AMP.PNP and/or inorganic phosphate analogues Vi and AlF4 or BeFx. Studying glycerol-extracted muscle fibres from the rabbit psoas muscle by DSC, three characteristic thermal transitions were detected in rigor. The thermal transitions can be assigned to myosin heads, myosin rods and actin with transition temperatures (Tm) of 52.9 +/- 0.7 degrees C, 57.9 +/- 0.7 degrees C, 63.7 +/- 1.0 degrees C. In different intermediate states of the ATP hydrolysis mimicked by nucleotide analogues a fourth thermal transition was also detected which is very likely connected with nucleotide binding domain of myosin and/or actin filaments. This transition temperature Tm4 depended on the mimicked intermediate states, and varied in the range of 66-77 degrees C. CONCLUSION: According to DSC measurements, strongly and weakly binding states of myosin to actin were significantly different. In the presence of ADP only a moderate change of the DSC pattern was detected in comparison with rigor, whereas in ADP.Pi state trapped by Vi, AlF4 or BeFx a remarkable stabilization was detected on the myosin head and actin filament which is reflected in a 3.0-10.0 degrees C shift in Tm to higher temperature. A similar effect was observed in the case of the nonhydrolyzable AMP.PNP analogue. Differential DSC measurements suggest that stabilization actin structure in the intermediate states of ATP hydrolysis may play an additional role in actin-myosin interaction. [Abstract/Link to Full Text]

Knizewski L, Kinch LN, Grishin NV, Rychlewski L, Ginalski K
Realm of PD-(D/E)XK nuclease superfamily revisited: detection of novel families with modified transitive meta profile searches.
BMC Struct Biol. 2007;740.
BACKGROUND: PD-(D/E)XK nucleases constitute a large and highly diverse superfamily of enzymes that display little sequence similarity despite retaining a common core fold and a few critical active site residues. This makes identification of new PD-(D/E)XK nuclease families a challenging task as they usually escape detection with standard sequence-based methods. We developed a modified transitive meta profile search approach and to consider the structural diversity of PD-(D/E)XK nuclease fold more thoroughly we analyzed also lower than threshold Meta-BASIC hits to select potentially correct predictions placed among unreliable or incorrect ones. RESULTS: Application of a modified transitive Meta-BASIC searches on updated PFAM families and PDB structures resulted in detection of five new PD-(D/E)XK nuclease families encompassing hundreds of so far uncharacterized and poorly annotated proteins. These include four families catalogued in PFAM database as domains of unknown function (DUF506, DUF524, DUF1626 and DUF1703) and YhgA-like family of putative transposases. Three of these families represent extremely distant homologs (DUF506, DUF524, and YhgA-like), while two are newly defined in updated database (DUF1626 and DUF1703). In addition, we also confidently identified an extended AAA-ATPase domain in the N-terminal region of DUF1703 family proteins. CONCLUSION: Obtained results suggest that detailed analysis of below threshold Meta-BASIC hits may push limits further for distant homology detection in the 'midnight zone' of homology. All identified families conserve the core evolutionary fold, secondary structure and hydrophobic patterns common to existing PD-(D/E)XK nucleases and maintain critical active site motifs that contribute to nucleic acid cleavage. Further experimental investigations should address the predicted activity and clarify potential substrates providing further insight into detailed biological role of these newly detected nucleases. [Abstract/Link to Full Text]

Singh K, Bhakuni V
Cation induced differential effect on structural and functional properties of Mycobacterium tuberculosis alpha-isopropylmalate synthase.
BMC Struct Biol. 2007;739.
BACKGROUND: Alpha-isopropylmalate synthase (MtalphaIPMS), an enzyme that catalyzes the first committed step of the leucine biosynthetic pathway of Mycobacterium tuberculosis is a potential drug target for the anti-tuberculosis drugs. Cations induce differential effect of activation and inhibition of MtalphaIPMS. To date no concrete mechanism for such an opposite effect of similarly charged cations on the functional activity of enzyme has been presented. RESULTS: Effect of cations on the structure and function of the MtalphaIPMS has been studied in detail. The studies for the first time demonstrate that different cations interact specifically at different sites in the enzyme and modulate the enzyme structure differentially. The inhibitors Zn2+ and Cd2+ ions interact directly with the catalytic domain of the enzyme and induce unfolding/denaturation of the domain. The activator K+ also interacts with the catalytic TIM barrel domain however, it does not induce any significant effect on the enzyme structure. Studies with isolated catalytic TIM barrel domain showed that it can carry out the catalytic function on its own but probably requires the non-catalytic C-terminal domain for optimum functioning. An important observation was that divalent cations induce significant interaction between the regulatory and the catalytic domain of MtalphaIPMS thus inducing structural cooperativity in the enzyme. This divalent cation induced structural cooperativity might result in modulation of activity of the catalytic domain by regulatory domain. CONCLUSION: The studies for the first time demonstrate that different cations bind at different sites in the enzyme leading to their differential effects on the structure and functional activity of the enzyme. [Abstract/Link to Full Text]

He W, Wang Y, Liu W, Zhou CZ
Crystal structure of Saccharomyces cerevisiae 6-phosphogluconate dehydrogenase Gnd1.
BMC Struct Biol. 2007;738.
BACKGROUND: As the third enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase (6PGDH) is the main generator of cellular NADPH. Both thioredoxin reductase and glutathione reductase require NADPH as the electron donor to reduce oxidized thioredoxin or glutathione (GSSG). Since thioredoxin and GSH are important antioxidants, it is not surprising that 6PGDH plays a critical role in protecting cells from oxidative stress. Furthermore the activity of 6PGDH is associated with several human disorders including cancer and Alzheimer's disease. The 3D structural investigation would be very valuable in designing small molecules that target this enzyme for potential therapeutic applications. RESULTS: The crystal structure of 6-phosphogluconate dehydrogenase (6PGDH/Gnd1) from Saccharomyces cerevisiae has been determined at 2.37 A resolution by molecular replacement. The overall structure of Gnd1 is a homodimer with three domains for each monomer, a Rossmann fold NADP+ binding domain, an all-alpha helical domain contributing the majority to hydrophobic interaction between the two subunits and a small C-terminal domain penetrating the other subunit. In addition, two citrate molecules occupied the 6PG binding pocket of each monomer. The intact Gnd1 had a Km of 50 +/- 9 microM for 6-phosphogluconate and of 35 +/- 6 microM for NADP+ at pH 7.5. But the truncated mutants without the C-terminal 35, 39 or 53 residues of Gnd1 completely lost their 6PGDH activity, despite remaining the homodimer in solution. CONCLUSION: The overall tertiary structure of Gnd1 is similar to those of 6PGDH from other species. The substrate and coenzyme binding sites are well conserved, either from the primary sequence alignment, or from the 3D structural superposition. Enzymatic activity assays suggest a sequential mechanism of catalysis, which is in agreement with previous studies. The C-terminal domain of Gnd1 functions as a hook to further tighten the dimer, but it is not necessary for the dimerization. This domain also works as a lid on the substrate binding pocket to control the binding of substrate and the release of product, so it is indispensable for the 6PGDH activity. Moreover, the co-crystallized citrate molecules, which mimic the binding mode of the substrate 6-phosphogluconate, provided us a novel strategy to design the 6PDGH inhibitors. [Abstract/Link to Full Text]

Dell'Orco D, De Benedetti PG, Fanelli F
In silico screening of mutational effects on enzyme-proteic inhibitor affinity: a docking-based approach.
BMC Struct Biol. 2007;737.
BACKGROUND: Molecular recognition between enzymes and proteic inhibitors is crucial for normal functioning of many biological pathways. Mutations in either the enzyme or the inhibitor protein often lead to a modulation of the binding affinity with no major alterations in the 3D structure of the complex. RESULTS: In this study, a rigid body docking-based approach has been successfully probed in its ability to predict the effects of single and multiple point mutations on the binding energetics in three enzyme-proteic inhibitor systems. The only requirement of the approach is an accurate structural model of the complex between the wild type forms of the interacting proteins, with the assumption that the architecture of the mutated complexes is almost the same as that of the wild type and no major conformational changes occur upon binding. The method was applied to 23 variants of the ribonuclease inhibitor-angiogenin complex, to 15 variants of the barnase-barstar complex, and to 8 variants of the bovine pancreatic trypsin inhibitor-beta Trypsin system, leading to thermodynamic and kinetic estimates consistent with in vitro data. Furthermore, simulations with and without explicit water molecules at the protein-protein interface suggested that they should be included in the simulations only when their positions are well defined both in the wild type and in the mutants and they result to be relevant for the modulation of mutational effects on the association process. CONCLUSION: The correlative models built in this study allow for predictions of mutational effects on the thermodynamics and kinetics of association of three substantially different systems, and represent important extensions of our computational approach to cases in which it is not possible to estimate the absolute free energies. Moreover, this study is the first example in the literature of an extensive evaluation of the correlative weights of the single components of the ZDOCK score on the thermodynamics and kinetics of binding of protein mutants compared to the native state.Finally, the results of this study corroborate and extend a previously developed quantitative model for in silico predictions of absolute protein-protein binding affinities spanning a wide range of values, i.e. from -10 up to -21 kcal/mol.The computational approach is simple and fast and can be used for structure-based design of protein-protein complexes and for in silico screening of mutational effects on protein-protein recognition. [Abstract/Link to Full Text]

Adam J, Pokorn� M, Sabin C, Mitchell EP, Imberty A, Wimmerov� M
Engineering of PA-IIL lectin from Pseudomonas aeruginosa - Unravelling the role of the specificity loop for sugar preference.
BMC Struct Biol. 2007;736.
BACKGROUND: Lectins are proteins of non-immune origin capable of binding saccharide structures with high specificity and affinity. Considering the high encoding capacity of oligosaccharides, this makes lectins important for adhesion and recognition. The present study is devoted to the PA-IIL lectin from Pseudomonas aeruginosa, an opportunistic human pathogen capable of causing lethal complications in cystic fibrosis patients. The lectin may play an important role in the process of virulence, recognizing specific saccharide structures and subsequently allowing the bacteria to adhere to the host cells. It displays high values of affinity towards monosaccharides, especially fucose--a feature caused by unusual binding mode, where two calcium ions participate in the interaction with saccharide. Investigating and understanding the nature of lectin-saccharide interactions holds a great potential of use in the field of drug design, namely the targeting and delivery of active compounds to the proper site of action. RESULTS: In vitro site-directed mutagenesis of the PA-IIL lectin yielded three single point mutants that were investigated both structurally (by X-ray crystallography) and functionally (by isothermal titration calorimetry). The mutated amino acids (22-23-24 triad) belong to the so-called specificity binding loop responsible for the monosaccharide specificity of the lectin. The mutation of the amino acids resulted in changes to the thermodynamic behaviour of the mutants and subsequently in their relative preference towards monosaccharides. Correlation of the measured data with X-ray structures provided the molecular basis for rationalizing the affinity changes. The mutations either prevent certain interactions to be formed or allow formation of new interactions--both of afore mentioned have strong effects on the saccharide preferences. CONCLUSION: Mutagenesis of amino acids forming the specificity binding loop allowed identification of one amino acid that is crucial for definition of the lectin sugar preference. Altering specificity loop amino acids causes changes in saccharide-binding preferences of lectins derived from PA-IIL, via creation or blocking possible binding interactions. This finding opens a gate towards protein engineering and subsequent protein design to refine the desired binding properties and preferences, an approach that could have strong potential for drug design. [Abstract/Link to Full Text]

Kim IK, Kim MK, Kim JH, Yim HS, Cha SS, Kang SO
High resolution crystal structure of PedB: a structural basis for the classification of pediocin-like immunity proteins.
BMC Struct Biol. 2007;735.
BACKGROUND: Pediocin-like bacteriocins, ribosomally-synthesized antimicrobial peptides, are generally coexpressed with cognate immunity proteins in order to protect the bacteriocin-producer from its own bacteriocin. As a step for understanding the mode of action of immunity proteins, we determined the crystal structure of PedB, a pediocin-like immunity protein conferring immunity to pediocin PP-1. RESULTS: The 1.6 A crystal structure of PedB reveals that PedB consists of an antiparallel four-helix bundle with a flexible C-terminal end. PedB shows structural similarity to an immunity protein against enterocin A (EntA-im) but some disparity to an immunity protein against carnobacteriocin B2 (ImB2) in both the C-terminal conformation and the local structure constructed by alpha3, alpha4, and their connecting loop. Structure-inspired mutational studies reveal that deletion of the last seven residues of the C-terminus of PedB almost abolished its immunity activity. CONCLUSION: The fact that PedB, EntA-im, and ImB2 share a four-helix bundle structure strongly suggests the structural conservation of this motif in the pediocin-like immunity proteins. The significant difference in the core structure and the C-terminal conformation provides a structural basis for the classification of pediocin-like immunity proteins. Our mutational study using C-terminal-shortened PedBs and the investigation of primary sequence of the C-terminal region, propose that several polar or charged residues in the extreme C-terminus of PedB which is crucial for the immunity are involved in the specific recognition of pediocin PP-1. [Abstract/Link to Full Text]

Kwan JJ, Donaldson LW
The NMR structure of the murine DLC2 SAM domain reveals a variant fold that is similar to a four-helix bundle.
BMC Struct Biol. 2007;734.
BACKGROUND: The tumor suppressor DLC2 (Deleted in Liver Cancer -2) participates in cell signaling at the mitochondrial membrane. DLC2 is characterized by a SAM (sterile alpha motif) domain, a Rho GTPase activating protein (GAP) domain, and a START lipid transfer domain. RESULTS: Towards understanding the function of DLC2, we have solved the NMR solution structure of the SAM domain. The DLC2-SAM domain structure reveals an atypical four-helix composition that is distinct from the five-helix SAM domain structures that have been determined to date. From structural alignments, helix 3 of the canonical SAM domain appears to be replaced by shorter, extended secondary structure that follows a similar path. Another difference is demonstrated by helices 1 and 2 that form a helical hairpin that is situated approximately parallel to the canonical helix 5. CONCLUSION: The DLC2-SAM domain adopts a structure that is topologically more similar to an anti-parallel four-helix bundle than a canonical SAM domain. This alternate topology may allow the DLC2-SAM domain to interact with a novel set of ligands. [Abstract/Link to Full Text]

Liu SQ, Meng ZH, Yang JK, Fu YX, Zhang KQ
Characterizing structural features of cuticle-degrading proteases from fungi by molecular modeling.
BMC Struct Biol. 2007;733.
BACKGROUND: Serine proteases secreted by nematode and insect pathogenic fungi are bio-control agents which have commercial potential for developing into effective bio-pesticides. A thorough understanding of the structural and functional features of these proteases would significantly assist with targeting the design of efficient bio-control agents. RESULTS: Structural models of serine proteases PR1 from entomophagous fungus, Ver112 and VCP1 from nematophagous fungi, have been modeled using the homology modeling technique based on the crystal coordinate of the proteinase K. In combination with multiple sequence alignment, these models suggest one similar calcium-binding site and two common disulfide bridges in the three cuticle-degrading enzymes. In addition, the predicted models of the three cuticle-degrading enzymes present an essentially identical backbone topology and similar geometric properties with the exception of a limited number of sites exhibiting relatively large local conformational differences only in some surface loops and the N-, C termini. However, they differ from each other in the electrostatic surface potential, in hydrophobicity and size of the S4 substrate-binding pocket, and in the number and distribution of hydrogen bonds and salt bridges within regions that are part of or in close proximity to the S2-loop. CONCLUSION: These differences likely lead to variations in substrate specificity and catalytic efficiency among the three enzymes. Amino acid polymorphisms in cuticle-degrading enzymes were discussed with respect to functional effects and host preference. It is hoped that these structural models would provide a further basis for exploitation of these serine proteases from pathogenic fungi as effective bio-control agents. [Abstract/Link to Full Text]

Ettrich R, Kopeck� V, Hofbauerov� K, Baumruk V, Nov�k P, Pompach P, Man P, Pl�hal O, Kut� M, Kulik N, Sklen�r J, Ryslav� H, Kren V, Bezouska K
Structure of the dimeric N-glycosylated form of fungal beta-N-acetylhexosaminidase revealed by computer modeling, vibrational spectroscopy, and biochemical studies.
BMC Struct Biol. 2007;732.
BACKGROUND: Fungal beta-N-acetylhexosaminidases catalyze the hydrolysis of chitobiose into its constituent monosaccharides. These enzymes are physiologically important during the life cycle of the fungus for the formation of septa, germ tubes and fruit-bodies. Crystal structures are known for two monomeric bacterial enzymes and the dimeric human lysosomal beta-N-acetylhexosaminidase. The fungal beta-N-acetylhexosaminidases are robust enzymes commonly used in chemoenzymatic syntheses of oligosaccharides. The enzyme from Aspergillus oryzae was purified and its sequence was determined. RESULTS: The complete primary structure of the fungal beta-N-acetylhexosaminidase from Aspergillus oryzae CCF1066 was used to construct molecular models of the catalytic subunit of the enzyme, the enzyme dimer, and the N-glycosylated dimer. Experimental data were obtained from infrared and Raman spectroscopy, and biochemical studies of the native and deglycosylated enzyme, and are in good agreement with the models. Enzyme deglycosylated under native conditions displays identical kinetic parameters but is significantly less stable in acidic conditions, consistent with model predictions. The molecular model of the deglycosylated enzyme was solvated and a molecular dynamics simulation was run over 20 ns. The molecular model is able to bind the natural substrate - chitobiose with a stable value of binding energy during the molecular dynamics simulation. CONCLUSION: Whereas the intracellular bacterial beta-N-acetylhexosaminidases are monomeric, the extracellular secreted enzymes of fungi and humans occur as dimers. Dimerization of the fungal beta-N-acetylhexosaminidase appears to be a reversible process that is strictly pH dependent. Oligosaccharide moieties may also participate in the dimerization process that might represent a unique feature of the exclusively extracellular enzymes. Deglycosylation had only limited effect on enzyme activity, but it significantly affected enzyme stability in acidic conditions. Dimerization and N-glycosylation are the enzyme's strategy for catalytic subunit stabilization. The disulfide bridge that connects Cys448 with Cys483 stabilizes a hinge region in a flexible loop close to the active site, which is an exclusive feature of the fungal enzymes, neither present in bacterial nor mammalian structures. This loop may play the role of a substrate binding site lid, anchored by a disulphide bridge that prevents the substrate binding site from being influenced by the flexible motion of the loop. [Abstract/Link to Full Text]

Keskin O
Binding induced conformational changes of proteins correlate with their intrinsic fluctuations: a case study of antibodies.
BMC Struct Biol. 2007;731.
BACKGROUND: How antibodies recognize and bind to antigens can not be totally explained by rigid shape and electrostatic complimentarity models. Alternatively, pre-existing equilibrium hypothesis states that the native state of an antibody is not defined by a single rigid conformation but instead with an ensemble of similar conformations that co-exist at equilibrium. Antigens bind to one of the preferred conformations making this conformation more abundant shifting the equilibrium. RESULTS: Here, two antibodies, a germline antibody of 36-65 Fab and a monoclonal antibody, SPE7 are studied in detail to elucidate the mechanism of antibody-antigen recognition and to understand how a single antibody recognizes different antigens. An elastic network model, Anisotropic Network Model (ANM) is used in the calculations. Pre-existing equilibrium is not restricted to apply to antibodies. Intrinsic fluctuations of eight proteins, from different classes of proteins, such as enzymes, binding and transport proteins are investigated to test the suitability of the method. The intrinsic fluctuations are compared with the experimentally observed ligand induced conformational changes of these proteins. The results show that the intrinsic fluctuations obtained by theoretical methods correlate with structural changes observed when a ligand is bound to the protein. The decomposition of the total fluctuations serves to identify the different individual modes of motion, ranging from the most cooperative ones involving the overall structure, to the most localized ones. CONCLUSION: Results suggest that the pre-equilibrium concept holds for antibodies and the promiscuity of antibodies can also be explained this hypothesis: a limited number of conformational states driven by intrinsic motions of an antibody might be adequate to bind to different antigens. [Abstract/Link to Full Text]

Burman JD, Stevenson CE, Sawers RG, Lawson DM
The crystal structure of Escherichia coli TdcF, a member of the highly conserved YjgF/YER057c/UK114 family.
BMC Struct Biol. 2007;730.
BACKGROUND: The YjgF/YER057c/UK114 family of proteins is widespread in nature, but has as yet no clearly defined biological role. Members of the family exist as homotrimers and are characterised by intersubunit clefts that are delineated by well-conserved residues; these sites are likely to be of functional significance, yet catalytic activity has never been detected for any member of this family. The gene encoding the TdcF protein of E. coli, a YjgF/YER057c/UK114 family member, resides in an operon that strongly suggests a role in the metabolism of 2-ketobutyrate for this protein. RESULTS: We have determined the crystal structure of E. coli TdcF by molecular replacement to a maximum resolution of 1.6 A. Structures are also presented of TdcF complexed with a variety of ligands. CONCLUSION: The TdcF structure closely resembles those of all YjgF/YER057c/UK114 family members determined thus far. It has the trimeric quaternary structure and intersubunit cavities characteristic of this family of proteins. We show that TdcF is capable of binding several low molecular weight metabolites bearing a carboxylate group, although the interaction with 2-ketobutyrate appears to be the most well defined. These observations may be indicative of a role for TdcF in sensing this potentially toxic metabolite. [Abstract/Link to Full Text]

Hou X, Chen M, Chen L, Meehan EJ, Xie J, Huang M
X-ray sequence and crystal structure of luffaculin 1, a novel type 1 ribosome-inactivating protein.
BMC Struct Biol. 2007;729.
BACKGROUND: Protein sequence can be obtained through Edman degradation, mass spectrometry, or cDNA sequencing. High resolution X-ray crystallography can also be used to derive protein sequence information, but faces the difficulty in distinguishing the Asp/Asn, Glu/Gln, and Val/Thr pairs. Luffaculin 1 is a new type 1 ribosome-inactivating protein (RIP) isolated from the seeds of Luffa acutangula. Besides rRNA N-glycosidase activity, luffaculin 1 also demonstrates activities including inhibiting tumor cells' proliferation and inducing tumor cells' differentiation. RESULTS: The crystal structure of luffaculin 1 was determined at 1.4 A resolution. Its amino-acid sequence was derived from this high resolution structure using the following criteria: 1) high resolution electron density; 2) comparison of electron density between two molecules that exist in the same crystal; 3) evaluation of the chemical environment of residues to break down the sequence assignment ambiguity in residue pairs Glu/Gln, Asp/Asn, and Val/Thr; 4) comparison with sequences of the homologous proteins. Using the criteria 1 and 2, 66% of the residues can be assigned. By incorporating with criterion 3, 86% of the residues were assigned, suggesting the effectiveness of chemical environment evaluation in breaking down residue ambiguity. In total, 94% of the luffaculin 1 sequence was assigned with high confidence using this improved X-ray sequencing strategy. Two N-acetylglucosamine moieties, linked respectively to the residues Asn77 and Asn84, can be identified in the structure. Residues Tyr70, Tyr110, Glu159 and Arg162 define the active site of luffaculin 1 as an RNA N-glycosidase. CONCLUSION: X-ray sequencing method can be effective to derive sequence information of proteins. The evaluation of the chemical environment of residues is a useful method to break down the assignment ambiguity in Glu/Gln, Asp/Asn, and Val/Thr pairs. The sequence and the crystal structure confirm that luffaculin 1 is a new type 1 RIP. [Abstract/Link to Full Text]

Jin AH, Brandstaetter H, Nevin ST, Tan CC, Clark RJ, Adams DJ, Alewood PF, Craik DJ, Daly NL
Structure of alpha-conotoxin BuIA: influences of disulfide connectivity on structural dynamics.
BMC Struct Biol. 2007;728.
BACKGROUND: Alpha-conotoxins have exciting therapeutic potential based on their high selectivity and affinity for nicotinic acetylcholine receptors. The spacing between the cysteine residues in alpha-conotoxins is variable, leading to the classification of sub-families. BuIA is the only alpha-conotoxin containing a 4/4 cysteine spacing and thus it is of significant interest to examine the structure of this conotoxin. RESULTS: In the current study we show the native globular disulfide connectivity of BuIA displays multiple conformations in solution whereas the non-native ribbon isomer has a single well-defined conformation. Despite having multiple conformations in solution the globular form of BuIA displays activity at the nicotinic acetylcholine receptor, contrasting with the lack of activity of the structurally well-defined ribbon isomer. CONCLUSION: These findings are opposite to the general trends observed for alpha-conotoxins where the native isomers have well-defined structures and the ribbon isomers are generally disordered. This study thus highlights the influence of the disulfide connectivity of BuIA on the dynamics of the three-dimensional structure. [Abstract/Link to Full Text]

Bansal A, Sankararamakrishnan R
Homology modeling of major intrinsic proteins in rice, maize and Arabidopsis: comparative analysis of transmembrane helix association and aromatic/arginine selectivity filters.
BMC Struct Biol. 2007;727.
BACKGROUND: The major intrinsic proteins (MIPs) facilitate the transport of water and neutral solutes across the lipid bilayers. Plant MIPs are believed to be important in cell division and expansion and in water transport properties in response to environmental conditions. More than 30 MIP sequences have been identified in Arabidopsis thaliana, maize and rice. Plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), Nod26-like intrinsic protein (NIPs) and small and basic intrinsic proteins (SIPs) are subfamilies of plant MIPs. Despite sequence diversity, all the experimentally determined structures belonging to the MIP superfamily have the same "hour-glass" fold. RESULTS: We have structurally characterized 39 rice and 31 maize MIPs and compared them with that of Arabidopsis. Homology models of 105 MIPs from all three plant species were built. Structure-based sequence alignments were generated and the residues in the helix-helix interfaces were analyzed. Small residues (Gly/Ala/Ser/Thr) are found to be highly conserved as a group in the helix-helix interface of MIP structures. Individual families sometimes prefer one or another of the residues from this group. The narrow aromatic/arginine (ar/R) selectivity filter in MIPs has been shown to provide an important constriction for solute permeability. Ar/R regions were analyzed and compared between the three plant species. Seventeen TIP, NIP and SIP members from rice and maize have ar/R signatures that are not found in Arabidopsis. A subgroup of rice and maize NIPs has small residues in three of the four positions in the ar/R tetrad, resulting in a wider constriction. These MIP members could transport larger solute molecules. CONCLUSION: Small residues are group-conserved in the helix-helix interface of MIP structures and they seem to be important for close helix-helix interactions. Such conservation might help to preserve the hour-glass fold in MIP structures. Analysis and comparison of ar/R selectivity filters suggest that rice and maize MIPs could transport more diverse solutes than Arabidopsis MIPs. Thus the MIP members show conservation in helix-helix interfaces and diversity in aromatic/arginine selectivity filters. The former is related to structural stability and the later can be linked to functional diversity. [Abstract/Link to Full Text]

Chyou DT, Rawle VL, Trotman CN
Quaternary structure of Artemia haemoglobin II: analysis of T and C polymer alignment and interpolymer interface.
BMC Struct Biol. 2007;726.
BACKGROUND: The brine shrimp Artemia expresses four different types of haemoglobin subunits namely C1, C2, T1 and T2. Two of these four subunits dimerize in different combinations to produce the three isoforms of the heterodimeric Artemia haemoglobin: HbI (C1 and C2), HbII (C1 and T2) and HbIII (T1 and T2). Previous biochemical, biophysical and computational analyses demonstrate that the T and C polymers are rings of nine concatenated globin domains, which are covalently joined by interdomain linkers. Two such rings stacked coaxially give the functional molecule. This research aimed to construct a quaternary structural model of Artemia HbII that shows the interpolymer interface and domain-domain alignment, using the MS3D (mass spectrometry for three dimensional analysis) approach. This involved introducing chemical crosslinks between the two polymers, cleaving with trypsin and analyzing the resulting products by mass spectrometry. This was followed by computational analysis of the mass spectrometry data using the program SearchXlinks to identify putatively crosslinked peptides. RESULTS: Six putative EGS (ethylene glycol bis [succinimidylsuccinate]) crosslinked tryptic peptides were identified. All of them support a model in which the EF helices of all domains are in contact along the interpolymer surface, and Domain 1 of the T-polymer aligns with Domain 1 of the C-polymer. Any two adjacent interpolymer domain pairs contact through the early Helix H and early Helix A. The orientation of domains is different from the subunit proposed model proposed previously by this group. Crosslinking with GMBS (N- [gamma-maleimidobutyryloxy]succinimide ester) was also performed, and the results show good agreement with this model. CONCLUSION: The interpolymer EF-contact allows the hydrophobic E and F helices to be buried in the interface and therefore allow the complex to solubilize readily to facilitate efficient oxygen transport. Furthermore the EF-contact is a common contact in cooperative haemoglobins and thus the model is consistent with the cooperative behaviour of Artemia HbII. [Abstract/Link to Full Text]

Chen K, Kurgan LA, Ruan J
Prediction of flexible/rigid regions from protein sequences using k-spaced amino acid pairs.
BMC Struct Biol. 2007;725.
BACKGROUND: Traditionally, it is believed that the native structure of a protein corresponds to a global minimum of its free energy. However, with the growing number of known tertiary (3D) protein structures, researchers have discovered that some proteins can alter their structures in response to a change in their surroundings or with the help of other proteins or ligands. Such structural shifts play a crucial role with respect to the protein function. To this end, we propose a machine learning method for the prediction of the flexible/rigid regions of proteins (referred to as FlexRP); the method is based on a novel sequence representation and feature selection. Knowledge of the flexible/rigid regions may provide insights into the protein folding process and the 3D structure prediction. RESULTS: The flexible/rigid regions were defined based on a dataset, which includes protein sequences that have multiple experimental structures, and which was previously used to study the structural conservation of proteins. Sequences drawn from this dataset were represented based on feature sets that were proposed in prior research, such as PSI-BLAST profiles, composition vector and binary sequence encoding, and a newly proposed representation based on frequencies of k-spaced amino acid pairs. These representations were processed by feature selection to reduce the dimensionality. Several machine learning methods for the prediction of flexible/rigid regions and two recently proposed methods for the prediction of conformational changes and unstructured regions were compared with the proposed method. The FlexRP method, which applies Logistic Regression and collocation-based representation with 95 features, obtained 79.5% accuracy. The two runner-up methods, which apply the same sequence representation and Support Vector Machines (SVM) and Na�ve Bayes classifiers, obtained 79.2% and 78.4% accuracy, respectively. The remaining considered methods are characterized by accuracies below 70%. Finally, the Na�ve Bayes method is shown to provide the highest sensitivity for the prediction of flexible regions, while FlexRP and SVM give the highest sensitivity for rigid regions. CONCLUSION: A new sequence representation that uses k-spaced amino acid pairs is shown to be the most efficient in the prediction of the flexible/rigid regions of protein sequences. The proposed FlexRP method provides the highest prediction accuracy of about 80%. The experimental tests show that the FlexRP and SVM methods achieved high overall accuracy and the highest sensitivity for rigid regions, while the best quality of the predictions for flexible regions is achieved by the Na�ve Bayes method. [Abstract/Link to Full Text]

Stanley WA, Pursiainen NV, Garman EF, Juffer AH, Wilmanns M, Kursula P
A previously unobserved conformation for the human Pex5p receptor suggests roles for intrinsic flexibility and rigid domain motions in ligand binding.
BMC Struct Biol. 2007;724.
BACKGROUND: The C-terminal tetratricopeptide (TPR) repeat domain of Pex5p recognises proteins carrying a peroxisomal targeting signal type 1 (PTS1) tripeptide in their C-terminus. Previously, structural data have been obtained from the TPR domain of Pex5p in both the liganded and unliganded states, indicating a conformational change taking place upon cargo protein binding. Such a conformational change would be expected to play a major role both during PTS1 protein recognition as well as in cargo release into the peroxisomal lumen. However, little information is available on the factors that may regulate such structural changes. RESULTS: We have used a range of biophysical and computational methods to further analyse the conformational flexibility and ligand binding of Pex5p. A new crystal form for the human Pex5p C-terminal domain (Pex5p(C)) was obtained in the presence of Sr2+ ions, and the structure presents a novel conformation, distinct from all previous liganded and apo crystal structures for Pex5p(C). The difference relates to a near-rigid body movement of two halves of the molecule, and this movement is different from that required to reach a ring-like conformation upon PTS1 ligand binding. The bound Sr2+ ion changes the dynamic properties of Pex5p(C) affecting its conformation, possibly by making the Sr2+-binding loop - located near the hinge region for the observed domain motions - more rigid. CONCLUSION: The current data indicate that Pex5p(C) is able to sample a range of conformational states in the absence of bound PTS1 ligand. The domain movements between various apo conformations are distinct from those involved in ligand binding, although the differences between all observed conformations so far can be characterised by the movement of the two halves of Pex5p(C) as near-rigid bodies with respect to each other. [Abstract/Link to Full Text]

Madej T, Panchenko AR, Chen J, Bryant SH
Protein homologous cores and loops: important clues to evolutionary relationships between structurally similar proteins.
BMC Struct Biol. 2007;723.
BACKGROUND: To discover remote evolutionary relationships and functional similarities between proteins, biologists rely on comparative sequence analysis, and when structures are available, on structural alignments and various measures of structural similarity. The measures/scores that have most commonly been used for this purpose include: alignment length, percent sequence identity, superposition RMSD and their different combinations. More recently, we have introduced the "Homologous core structure overlap score" (HCS) and the "Loop Hausdorff Measure" (LHM). Along with these we also consider the "gapped structural alignment score" (GSAS), which was introduced earlier by other researchers. RESULTS: We analyze the performance of these and other conventional measures at the task of ranking structure neighbors by homology, and we show that the HCS, LHM, and GSAS scores display considerably improved performance over the conventional measures of sequence or structural similarity. CONCLUSION: The HCS, LHM, and GSAS scores are easily computable quantities that allow users of structure-neighbor databases to more easily identify interesting structural similarities between proteins. [Abstract/Link to Full Text]