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Recent Articles in PLoS Biology

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Xiang Y, Takeo S, Florens L, Hughes SE, Huo LJ, Gilliland WD, Swanson SK, Teeter K, Schwartz JW, Washburn MP, Jaspersen SL, Hawley RS
The Inhibition of Polo Kinase by Matrimony Maintains G2 Arrest in the Meiotic Cell Cycle.
PLoS Biol. 2007 Dec 4;5(12):e323.
Many meiotic systems in female animals include a lengthy arrest in G2 that separates the end of pachytene from nuclear envelope breakdown (NEB). However, the mechanisms by which a meiotic cell can arrest for long periods of time (decades in human females) have remained a mystery. The Drosophila Matrimony (Mtrm) protein is expressed from the end of pachytene until the completion of meiosis I. Loss-of-function mtrm mutants result in precocious NEB. Coimmunoprecipitation experiments reveal that Mtrm physically interacts with Polo kinase (Polo) in vivo, and multidimensional protein identification technology mass spectrometry analysis reveals that Mtrm binds to Polo with an approximate stoichiometry of 1:1. Mutation of a Polo-Box Domain (PBD) binding site in Mtrm ablates the function of Mtrm and the physical interaction of Mtrm with Polo. The meiotic defects observed in mtrm/+ heterozygotes are fully suppressed by reducing the dose of polo(+), demonstrating that Mtrm acts as an inhibitor of Polo. Mtrm acts as a negative regulator of Polo during the later stages of G2 arrest. Indeed, both the repression of Polo expression until stage 11 and the inactivation of newly synthesized Polo by Mtrm until stage 13 play critical roles in maintaining and properly terminating G2 arrest. Our data suggest a model in which the eventual activation of Cdc25 by an excess of Polo at stage 13 triggers NEB and entry into prometaphase. [Abstract/Link to Full Text]

Palazzo AF, Springer M, Shibata Y, Lee CS, Dias AP, Rapoport TA
The Signal Sequence Coding Region Promotes Nuclear Export of mRNA.
PLoS Biol. 2007 Dec 4;5(12):e322.
In eukaryotic cells, most mRNAs are exported from the nucleus by the transcription export (TREX) complex, which is loaded onto mRNAs after their splicing and capping. We have studied in mammalian cells the nuclear export of mRNAs that code for secretory proteins, which are targeted to the endoplasmic reticulum membrane by hydrophobic signal sequences. The mRNAs were injected into the nucleus or synthesized from injected or transfected DNA, and their export was followed by fluorescent in situ hybridization. We made the surprising observation that the signal sequence coding region (SSCR) can serve as a nuclear export signal of an mRNA that lacks an intron or functional cap. Even the export of an intron-containing natural mRNA was enhanced by its SSCR. Like conventional export, the SSCR-dependent pathway required the factor TAP, but depletion of the TREX components had only moderate effects. The SSCR export signal appears to be characterized in vertebrates by a low content of adenines, as demonstrated by genome-wide sequence analysis and by the inhibitory effect of silent adenine mutations in SSCRs. The discovery of an SSCR-mediated pathway explains the previously noted amino acid bias in signal sequences and suggests a link between nuclear export and membrane targeting of mRNAs. [Abstract/Link to Full Text]

Haesler S, Rochefort C, Georgi B, Licznerski P, Osten P, Scharff C
Incomplete and Inaccurate Vocal Imitation after Knockdown of FoxP2 in Songbird Basal Ganglia Nucleus Area X.
PLoS Biol. 2007 Dec 4;5(12):e321.
The gene encoding the forkhead box transcription factor, FOXP2, is essential for developing the full articulatory power of human language. Mutations of FOXP2 cause developmental verbal dyspraxia (DVD), a speech and language disorder that compromises the fluent production of words and the correct use and comprehension of grammar. FOXP2 patients have structural and functional abnormalities in the striatum of the basal ganglia, which also express high levels of FOXP2. Since human speech and learned vocalizations in songbirds bear behavioral and neural parallels, songbirds provide a genuine model for investigating the basic principles of speech and its pathologies. In zebra finch Area X, a basal ganglia structure necessary for song learning, FoxP2 expression increases during the time when song learning occurs. Here, we used lentivirus-mediated RNA interference (RNAi) to reduce FoxP2 levels in Area X during song development. Knockdown of FoxP2 resulted in an incomplete and inaccurate imitation of tutor song. Inaccurate vocal imitation was already evident early during song ontogeny and persisted into adulthood. The acoustic structure and the duration of adult song syllables were abnormally variable, similar to word production in children with DVD. Our findings provide the first example of a functional gene analysis in songbirds and suggest that normal auditory-guided vocal motor learning requires FoxP2. [Abstract/Link to Full Text]

Goodsell DS, Johnson GT
Filling in the Gaps: Artistic License in Education and Outreach.
PLoS Biol. 2007 Dec 4;5(12):e308. [Abstract/Link to Full Text]

Anderson DH, Kickhoefer VA, Sievers SA, Rome LH, Eisenberg D
Draft crystal structure of the vault shell at 9-A resolution.
PLoS Biol. 2007 Nov 27;5(11):e318.
Vaults are the largest known cytoplasmic ribonucleoprotein structures and may function in innate immunity. The vault shell self-assembles from 96 copies of major vault protein and encapsulates two other proteins and a small RNA. We crystallized rat liver vaults and several recombinant vaults, all among the largest non-icosahedral particles to have been crystallized. The best crystals thus far were formed from empty vaults built from a cysteine-tag construct of major vault protein (termed cpMVP vaults), diffracting to about 9-A resolution. The asymmetric unit contains a half vault of molecular mass 4.65 MDa. X-ray phasing was initiated by molecular replacement, using density from cryo-electron microscopy (cryo-EM). Phases were improved by density modification, including concentric 24- and 48-fold rotational symmetry averaging. From this, the continuous cryo-EM electron density separated into domain-like blocks. A draft atomic model of cpMVP was fit to this improved density from 15 domain models. Three domains were adapted from a nuclear magnetic resonance substructure. Nine domain models originated in ab initio tertiary structure prediction. Three C-terminal domains were built by fitting poly-alanine to the electron density. Locations of loops in this model provide sites to test vault functions and to exploit vaults as nanocapsules. [Abstract/Link to Full Text]

Yang C, Czech L, Gerboth S, Kojima S, Scita G, Svitkina T
Novel roles of formin mDia2 in lamellipodia and filopodia formation in motile cells.
PLoS Biol. 2007 Nov 27;5(11):e317.
Actin polymerization-driven protrusion of the leading edge is a key element of cell motility. The important actin nucleators formins and the Arp2/3 complex are believed to have nonoverlapping functions in inducing actin filament bundles in filopodia and dendritic networks in lamellipodia, respectively. We tested this idea by investigating the role of mDia2 formin in leading-edge protrusion by loss-of-function and gain-of-function approaches. Unexpectedly, mDia2 depletion by short interfering RNA (siRNA) severely inhibited lamellipodia. Structural analysis of the actin network in the few remaining lamellipodia suggested an mDia2 role in generation of long filaments. Consistently, constitutively active mDia2 (DeltaGBD-mDia2) induced accumulation of long actin filaments in lamellipodia and increased persistence of lamellipodial protrusion. Depletion of mDia2 also inhibited filopodia, whereas expression of DeltaGBD-mDia2 promoted their formation. Correlative light and electron microscopy showed that DeltaGBD-mDia2-induced filopodia were formed from lamellipodial network through gradual convergence of long lamellipodial filaments into bundles. Efficient filopodia induction required mDia2 targeting to the membrane, likely through a scaffolding protein Abi1. Furthermore, mDia2 and Abi1 interacted through the N-terminal regulatory sequences of mDia2 and the SH3-containing Abi1 sequences. We propose that mDia2 plays an important role in formation of lamellipodia by nucleating and/or protecting from capping lamellipodial actin filaments, which subsequently exhibit high tendency to converge into filopodia. [Abstract/Link to Full Text]

Miall RC, Christensen LO, Cain O, Stanley J
Disruption of state estimation in the human lateral cerebellum.
PLoS Biol. 2007 Nov 27;5(11):e316.
The cerebellum has been proposed to be a crucial component in the state estimation process that combines information from motor efferent and sensory afferent signals to produce a representation of the current state of the motor system. Such a state estimate of the moving human arm would be expected to be used when the arm is rapidly and skillfully reaching to a target. We now report the effects of transcranial magnetic stimulation (TMS) over the ipsilateral cerebellum as healthy humans were made to interrupt a slow voluntary movement to rapidly reach towards a visually defined target. Errors in the initial direction and in the final finger position of this reach-to-target movement were significantly higher for cerebellar stimulation than they were in control conditions. The average directional errors in the cerebellar TMS condition were consistent with the reaching movements being planned and initiated from an estimated hand position that was 138 ms out of date. We suggest that these results demonstrate that the cerebellum is responsible for estimating the hand position over this time interval and that TMS disrupts this state estimate. [Abstract/Link to Full Text]

Picot M, Cusumano P, Klarsfeld A, Ueda R, Rouyer F
Light activates output from evening neurons and inhibits output from morning neurons in the Drosophila circadian clock.
PLoS Biol. 2007 Nov 27;5(11):e315.
Animal circadian clocks are based on multiple oscillators whose interactions allow the daily control of complex behaviors. The Drosophila brain contains a circadian clock that controls rest-activity rhythms and relies upon different groups of PERIOD (PER)-expressing neurons. Two distinct oscillators have been functionally characterized under light-dark cycles. Lateral neurons (LNs) that express the pigment-dispersing factor (PDF) drive morning activity, whereas PDF-negative LNs are required for the evening activity. In constant darkness, several lines of evidence indicate that the LN morning oscillator (LN-MO) drives the activity rhythms, whereas the LN evening oscillator (LN-EO) does not. Since mutants devoid of functional CRYPTOCHROME (CRY), as opposed to wild-type flies, are rhythmic in constant light, we analyzed transgenic flies expressing PER or CRY in the LN-MO or LN-EO. We show that, under constant light conditions and reduced CRY function, the LN evening oscillator drives robust activity rhythms, whereas the LN morning oscillator does not. Remarkably, light acts by inhibiting the LN-MO behavioral output and activating the LN-EO behavioral output. Finally, we show that PDF signaling is not required for robust activity rhythms in constant light as opposed to its requirement in constant darkness, further supporting the minor contribution of the morning cells to the behavior in the presence of light. We therefore propose that day-night cycles alternatively activate behavioral outputs of the Drosophila evening and morning lateral neurons. [Abstract/Link to Full Text]

Yee NS, Gong W, Huang Y, Lorent K, Dolan AC, Maraia RJ, Pack M
Mutation of RNA Pol III subunit rpc2/polr3b Leads to Deficiency of Subunit Rpc11 and disrupts zebrafish digestive development.
PLoS Biol. 2007 Nov 27;5(11):e312.
The role of RNA polymerase III (Pol III) in developing vertebrates has not been examined. Here, we identify a causative mutation of the second largest Pol III subunit, polr3b, that disrupts digestive organ development in zebrafish slim jim (slj) mutants. The slj mutation is a splice-site substitution that causes deletion of a conserved tract of 41 amino acids in the Polr3b protein. Structural considerations predict that the slj Pol3rb deletion might impair its interaction with Polr3k, the ortholog of an essential yeast Pol III subunit, Rpc11, which promotes RNA cleavage and Pol III recycling. We engineered Schizosaccharomyces pombe to carry an Rpc2 deletion comparable to the slj mutation and found that the Pol III recovered from this rpc2-delta yeast had markedly reduced levels of Rpc11p. Remarkably, overexpression of cDNA encoding the zebrafish rpc11 ortholog, polr3k, rescued the exocrine defects in slj mutants, indicating that the slj phenotype is due to deficiency of Rpc11. These data show that functional interactions between Pol III subunits have been conserved during eukaryotic evolution and support the utility of zebrafish as a model vertebrate for analysis of Pol III function. [Abstract/Link to Full Text]

Faas GC, Schwaller B, Vergara JL, Mody I
Resolving the fast kinetics of cooperative binding: Ca2+ buffering by calretinin.
PLoS Biol. 2007 Nov 27;5(11):e311.
Cooperativity is one of the most important properties of molecular interactions in biological systems. It is the ability to influence ligand binding at one site of a macromolecule by previous ligand binding at another site of the same molecule. As a consequence, the affinity of the macromolecule for the ligand is either decreased (negative cooperativity) or increased (positive cooperativity). Over the last 100 years, O2 binding to hemoglobin has served as the paradigm for cooperative ligand binding and allosteric modulation, and four practical models were developed to quantitatively describe the mechanism: the Hill, the Adair-Klotz, the Monod-Wyman-Changeux, and the Koshland-Némethy-Filmer models. The predictions of these models apply under static conditions when the binding reactions are at equilibrium. However, in a physiological setting, e.g., inside a cell, the timing and dynamics of the binding events are essential. Hence, it is necessary to determine the dynamic properties of cooperative binding to fully understand the physiological implications of cooperativity. To date, the Monod-Wyman-Changeux model was applied to determine the kinetics of cooperative binding to biologically active molecules. In this model, cooperativity is established by postulating two allosteric isoforms with different binding properties. However, these studies were limited to special cases, where transition rates between allosteric isoforms are much slower than the binding rates or where binding and unbinding rates could be measured independently. For all other cases, the complex mathematical description precludes straightforward interpretations. Here, we report on calculating for the first time the fast dynamics of a cooperative binding process, the binding of Ca2+ to calretinin. Calretinin is a Ca2+-binding protein with four cooperative binding sites and one independent binding site. The Ca2+ binding to calretinin was assessed by measuring the decay of free Ca2+ using a fast fluorescent Ca2+ indicator following rapid (<50-mus rise time) Ca2+ concentration jumps induced by uncaging Ca2+ from DM-nitrophen. To unravel the kinetics of cooperative binding, we devised several approaches based on known cooperative binding models, resulting in a novel and relatively simple model. This model revealed unexpected and highly specific nonlinear properties of cellular Ca2+ regulation by calretinin. The association rate of Ca2+ with calretinin speeds up as the free Ca2+ concentration increases from cytoplasmic resting conditions ( approximately 100 nM) to approximately 1 muM. As a consequence, the Ca2+ buffering speed of calretinin highly depends on the prevailing Ca2+ concentration prior to a perturbation. In addition to providing a novel mode of action of cellular Ca2+ buffering, our model extends the analysis of cooperativity beyond the static steady-state condition, providing a powerful tool for the investigation of the dynamics and functional significance of cooperative binding in general. [Abstract/Link to Full Text]

Cho H, Jönsson H, Campbell K, Melke P, Williams JW, Jedynak B, Stevens AM, Groisman A, Levchenko A
Self-organization in high-density bacterial colonies: efficient crowd control.
PLoS Biol. 2007 Oct 30;5(11):e302.
Colonies of bacterial cells can display complex collective dynamics, frequently culminating in the formation of biofilms and other ordered super-structures. Recent studies suggest that to cope with local environmental challenges, bacterial cells can actively seek out small chambers or cavities and assemble there, engaging in quorum sensing behavior. By using a novel microfluidic device, we showed that within chambers of distinct shapes and sizes allowing continuous cell escape, bacterial colonies can gradually self-organize. The directions of orientation of cells, their growth, and collective motion are mutually correlated and dictated by the chamber walls and locations of chamber exits. The ultimate highly organized steady state is conducive to a more-organized escape of cells from the chambers and increased access of nutrients into and evacuation of waste out of the colonies. Using a computational model, we suggest that the lengths of the cells might be optimized to maximize self-organization while minimizing the potential for stampede-like exit blockage. The self-organization described here may be crucial for the early stage of the organization of high-density bacterial colonies populating small, physically confined growth niches. It suggests that this phenomenon can play a critical role in bacterial biofilm initiation and development of other complex multicellular bacterial super-structures, including those implicated in infectious diseases. [Abstract/Link to Full Text]

Kosak ST, Scalzo D, Alworth SV, Li F, Palmer S, Enver T, Lee JS, Groudine M
Coordinate gene regulation during hematopoiesis is related to genomic organization.
PLoS Biol. 2007 Nov 20;5(11):e309.
Gene loci are found in nuclear subcompartments that are related to their expression status. For instance, silent genes are often localized to heterochromatin and the nuclear periphery, whereas active genes tend to be found in the nuclear center. Evidence also suggests that chromosomes may be specifically positioned within the nucleus; however, the nature of this organization and how it is achieved are not yet fully understood. To examine whether gene regulation is related to a discernible pattern of genomic organization, we analyzed the linear arrangement of co-regulated genes along chromosomes and determined the organization of chromosomes during the differentiation of a hematopoietic progenitor to erythroid and neutrophil cell types. Our analysis reveals that there is a significant tendency for co-regulated genes to be proximal, which is related to the association of homologous chromosomes and the spatial juxtaposition of lineage-specific gene domains. We suggest that proximity in the form of chromosomal gene distribution and homolog association may be the basis for organizing the genome for coordinate gene regulation during cellular differentiation. [Abstract/Link to Full Text]

White RJ, Nie Q, Lander AD, Schilling TF
Complex regulation of cyp26a1 creates a robust retinoic acid gradient in the zebrafish embryo.
PLoS Biol. 2007 Nov 20;5(11):e304.
Positional identities along the anterior-posterior axis of the vertebrate nervous system are assigned during gastrulation by multiple posteriorizing signals, including retinoic acid (RA), fibroblast growth factors (Fgfs), and Wnts. Experimental evidence has suggested that RA, which is produced in paraxial mesoderm posterior to the hindbrain by aldehyde dehydrogenase 1a2 (aldh1a2/raldh2), forms a posterior-to-anterior gradient across the hindbrain field, and provides the positional information that specifies the locations and fates of rhombomeres. Recently, alternative models have been proposed in which RA plays only a permissive role, signaling wherever it is not degraded. Here we use a combination of experimental and modeling tools to address the role of RA in providing long-range positional cues in the zebrafish hindbrain. Using cell transplantation and implantation of RA-coated beads into RA-deficient zebrafish embryos, we demonstrate that RA can directly convey graded positional information over long distances. We also show that expression of Cyp26a1, the major RA-degrading enzyme during gastrulation, is under complex feedback and feedforward control by RA and Fgf signaling. The predicted consequence of such control is that RA gradients will be both robust to fluctuations in RA synthesis and adaptive to changes in embryo length during gastrulation. Such control also provides an explanation for the fact that loss of an endogenous RA gradient can be compensated for by RA that is provided in a spatially uniform manner. [Abstract/Link to Full Text]

Monroe D
Looking for chinks in the armor of bacterial biofilms.
PLoS Biol. 2007 Nov 13;5(11):e307. [Abstract/Link to Full Text]

von Heimendahl M, Itskov PM, Arabzadeh E, Diamond ME
Neuronal activity in rat barrel cortex underlying texture discrimination.
PLoS Biol. 2007 Nov 13;5(11):e305.
Rats and mice palpate objects with their whiskers to generate tactile sensations. This form of active sensing endows the animals with the capacity for fast and accurate texture discrimination. The present work is aimed at understanding the nature of the underlying cortical signals. We recorded neuronal activity from barrel cortex while rats used their whiskers to discriminate between rough and smooth textures. On whisker contact with either texture, firing rate increased by a factor of two to ten. Average firing rate was significantly higher for rough than for smooth textures, and we therefore propose firing rate as the fundamental coding mechanism. The rat, however, cannot take an average across trials, but must make an immediate decision using the signals generated on each trial. To estimate single-trial signals, we calculated the mutual information between stimulus and firing rate in the time window leading to the rat's observed choice. Activity during the last 75 ms before choice transmitted the most informative signal; in this window, neuronal clusters carried, on average, 0.03 bits of information about the stimulus on trials in which the rat's behavioral response was correct. To understand how cortical activity guides behavior, we examined responses in incorrect trials and found that, in contrast to correct trials, neuronal firing rate was higher for smooth than for rough textures. Analysis of high-speed films suggested that the inappropriate signal on incorrect trials was due, at least in part, to nonoptimal whisker contact. In conclusion, these data suggest that barrel cortex firing rate on each trial leads directly to the animal's judgment of texture. [Abstract/Link to Full Text]

Snyder JB, Nelson ME, Burdick JW, Maciver MA
Omnidirectional sensory and motor volumes in electric fish.
PLoS Biol. 2007 Nov 13;5(11):e301.
Active sensing organisms, such as bats, dolphins, and weakly electric fish, generate a 3-D space for active sensation by emitting self-generated energy into the environment. For a weakly electric fish, we demonstrate that the electrosensory space for prey detection has an unusual, omnidirectional shape. We compare this sensory volume with the animal's motor volume--the volume swept out by the body over selected time intervals and over the time it takes to come to a stop from typical hunting velocities. We find that the motor volume has a similar omnidirectional shape, which can be attributed to the fish's backward-swimming capabilities and body dynamics. We assessed the electrosensory space for prey detection by analyzing simulated changes in spiking activity of primary electrosensory afferents during empirically measured and synthetic prey capture trials. The animal's motor volume was reconstructed from video recordings of body motion during prey capture behavior. Our results suggest that in weakly electric fish, there is a close connection between the shape of the sensory and motor volumes. We consider three general spatial relationships between 3-D sensory and motor volumes in active and passive-sensing animals, and we examine hypotheses about these relationships in the context of the volumes we quantify for weakly electric fish. We propose that the ratio of the sensory volume to the motor volume provides insight into behavioral control strategies across all animals. [Abstract/Link to Full Text]

Kelsch W, Mosley CP, Lin CW, Lois C
Distinct mammalian precursors are committed to generate neurons with defined dendritic projection patterns.
PLoS Biol. 2007 Nov 13;5(11):e300.
The mechanisms that regulate how dendrites target different neurons to establish connections with specific cell types remain largely unknown. In particular, the formation of cell-type-specific connectivity during postnatal neurogenesis could be either determined by the local environment of the mature neuronal circuit or by cell-autonomous properties of the immature neurons, already determined by their precursors. Using retroviral fate mapping, we studied the lamina-specific dendritic targeting of one neuronal type as defined by its morphology and intrinsic somatic electrical properties in neonatal and adult neurogenesis. Fate mapping revealed the existence of two separate populations of neuronal precursors that gave rise to the same neuronal type with two distinct patterns of dendritic targeting-innervating either a deep or superficial lamina, where they connect to different types of principal neurons. Furthermore, heterochronic and heterotopic transplantation demonstrated that these precursors were largely restricted to generate neurons with a predetermined pattern of dendritic targeting that was independent of the host environment. Our results demonstrate that, at least in the neonatal and adult mammalian brain, the pattern of dendritic targeting of a given neuron is a cell-autonomous property of their precursors. [Abstract/Link to Full Text]

Watanabe A, Toyota T, Owada Y, Hayashi T, Iwayama Y, Matsumata M, Ishitsuka Y, Nakaya A, Maekawa M, Ohnishi T, Arai R, Sakurai K, Yamada K, Kondo H, Hashimoto K, Osumi N, Yoshikawa T
Fabp7 maps to a quantitative trait locus for a schizophrenia endophenotype.
PLoS Biol. 2007 Nov 13;5(11):e297.
Deficits in prepulse inhibition (PPI) are a biological marker for schizophrenia. To unravel the mechanisms that control PPI, we performed quantitative trait loci (QTL) analysis on 1,010 F2 mice derived by crossing C57BL/6 (B6) animals that show high PPI with C3H/He (C3) animals that show low PPI. We detected six major loci for PPI, six for the acoustic startle response, and four for latency to response peak, some of which were sex-dependent. A promising candidate on the Chromosome 10-QTL was Fabp7 (fatty acid binding protein 7, brain), a gene with functional links to the N-methyl-D-aspartic acid (NMDA) receptor and expression in astrocytes. Fabp7-deficient mice showed decreased PPI and a shortened startle response latency, typical of the QTL's proposed effects. A quantitative complementation test supported Fabp7 as a potential PPI-QTL gene, particularly in male mice. Disruption of Fabp7 attenuated neurogenesis in vivo. Human FABP7 showed altered expression in schizophrenic brains and genetic association with schizophrenia, which were both evident in males when samples were divided by sex. These results suggest that FABP7 plays a novel and crucial role, linking the NMDA, neurodevelopmental, and glial theories of schizophrenia pathology and the PPI endophenotype, with larger or overt effects in males. We also discuss the results from the perspective of fetal programming. [Abstract/Link to Full Text]

Begun DJ, Holloway AK, Stevens K, Hillier LW, Poh YP, Hahn MW, Nista PM, Jones CD, Kern AD, Dewey CN, Pachter L, Myers E, Langley CH
Population genomics: whole-genome analysis of polymorphism and divergence in Drosophila simulans.
PLoS Biol. 2007 Nov 6;5(11):e310.
The population genetic perspective is that the processes shaping genomic variation can be revealed only through simultaneous investigation of sequence polymorphism and divergence within and between closely related species. Here we present a population genetic analysis of Drosophila simulans based on whole-genome shotgun sequencing of multiple inbred lines and comparison of the resulting data to genome assemblies of the closely related species, D. melanogaster and D. yakuba. We discovered previously unknown, large-scale fluctuations of polymorphism and divergence along chromosome arms, and significantly less polymorphism and faster divergence on the X chromosome. We generated a comprehensive list of functional elements in the D. simulans genome influenced by adaptive evolution. Finally, we characterized genomic patterns of base composition for coding and noncoding sequence. These results suggest several new hypotheses regarding the genetic and biological mechanisms controlling polymorphism and divergence across the Drosophila genome, and provide a rich resource for the investigation of adaptive evolution and functional variation in D. simulans. [Abstract/Link to Full Text]

Ferree PM, Barbash DA
Distorted sex ratios: a window into RNAi-mediated silencing.
PLoS Biol. 2007 Nov 6;5(11):e303. [Abstract/Link to Full Text]

McMahill MS, Sham CW, Bishop DK
Synthesis-dependent strand annealing in meiosis.
PLoS Biol. 2007 Nov 6;5(11):e299.
Recent studies led to the proposal that meiotic gene conversion can result after transient engagement of the donor chromatid and subsequent DNA synthesis-dependent strand annealing (SDSA). Double Holliday junction (dHJ) intermediates were previously proposed to form both reciprocal crossover recombinants (COs) and noncrossover recombinants (NCOs); however, dHJs are now thought to give rise mainly to COs, with SDSA forming most or all NCOs. To test this model in Saccharomyces cerevisiae, we constructed a random spore system in which it is possible to identify a subset of NCO recombinants that can readily be accounted for by SDSA, but not by dHJ-mediated recombination. The diagnostic class of recombinants is one in which two markers on opposite sides of a double-strand break site are converted, without conversion of an intervening heterologous insertion located on the donor chromatid. This diagnostic class represents 26% of selected NCO recombinants. Tetrad analysis using the same markers provided additional evidence that SDSA is a major pathway for NCO gene conversion in meiosis. [Abstract/Link to Full Text]

Tao Y, Araripe L, Kingan SB, Ke Y, Xiao H, Hartl DL
A sex-ratio meiotic drive system in Drosophila simulans. II: an X-linked distorter.
PLoS Biol. 2007 Nov 6;5(11):e293.
The evolution of heteromorphic sex chromosomes creates a genetic condition favoring the invasion of sex-ratio meiotic drive elements, resulting in the biased transmission of one sex chromosome over the other, in violation of Mendel's first law. The molecular mechanisms of sex-ratio meiotic drive may therefore help us to understand the evolutionary forces shaping the meiotic behavior of the sex chromosomes. Here we characterize a sex-ratio distorter on the X chromosome (Dox) in Drosophila simulans by genetic and molecular means. Intriguingly, Dox has very limited coding capacity. It evolved from another X-linked gene, which also evolved de nova. Through retrotransposition, Dox also gave rise to an autosomal suppressor, not much yang (Nmy). An RNA interference mechanism seems to be involved in the suppression of the Dox distorter by the Nmy suppressor. Double mutant males of the genotype dox; nmy are normal for both sex-ratio and spermatogenesis. We postulate that recurrent bouts of sex-ratio meiotic drive and its subsequent suppression might underlie several common features observed in the heterogametic sex, including meiotic sex chromosome inactivation and achiasmy. [Abstract/Link to Full Text]

Tao Y, Masly JP, Araripe L, Ke Y, Hartl DL
A sex-ratio meiotic drive system in Drosophila simulans. I: an autosomal suppressor.
PLoS Biol. 2007 Nov 6;5(11):e292.
Sex ratio distortion (sex-ratio for short) has been reported in numerous species such as Drosophila, where distortion can readily be detected in experimental crosses, but the molecular mechanisms remain elusive. Here we characterize an autosomal sex-ratio suppressor from D. simulans that we designate as not much yang (nmy, polytene chromosome position 87F3). Nmy suppresses an X-linked sex-ratio distorter, contains a pair of near-perfect inverted repeats of 345 bp, and evidently originated through retrotransposition from the distorter itself. The suppression is likely mediated by sequence homology between the suppressor and distorter. The strength of sex-ratio is greatly enhanced by lower temperature. This temperature sensitivity was used to assign the sex-ratio etiology to the maturation process of the Y-bearing sperm, a hypothesis corroborated by both light microscope observations and ultrastructural studies. It has long been suggested that an X-linked sex-ratio distorter can evolve by exploiting loopholes in the meiotic machinery for its own transmission advantage, which may be offset by other changes in the genome that control the selfish distorter. Data obtained in this study help to understand this evolutionary mechanism in molecular detail and provide insight regarding its evolutionary impact on genomic architecture and speciation. [Abstract/Link to Full Text]

Diester I, Nieder A
Semantic associations between signs and numerical categories in the prefrontal cortex.
PLoS Biol. 2007 Oct 30;5(11):e294.
The utilization of symbols such as words and numbers as mental tools endows humans with unrivalled cognitive flexibility. In the number domain, a fundamental first step for the acquisition of numerical symbols is the semantic association of signs with cardinalities. We explored the primitives of such a semantic mapping process by recording single-cell activity in the monkey prefrontal and parietal cortices, brain structures critically involved in numerical cognition. Monkeys were trained to associate visual shapes with varying numbers of items in a matching task. After this long-term learning process, we found that the responses of many prefrontal neurons to the visual shapes reflected the associated numerical value in a behaviorally relevant way. In contrast, such association neurons were rarely found in the parietal lobe. These findings suggest a cardinal role of the prefrontal cortex in establishing semantic associations between signs and abstract categories, a cognitive precursor that may ultimately give rise to symbolic thinking in linguistic humans. [Abstract/Link to Full Text]

Luheshi LM, Tartaglia GG, Brorsson AC, Pawar AP, Watson IE, Chiti F, Vendruscolo M, Lomas DA, Dobson CM, Crowther DC
Systematic in vivo analysis of the intrinsic determinants of amyloid Beta pathogenicity.
PLoS Biol. 2007 Oct 30;5(11):e290.
Protein aggregation into amyloid fibrils and protofibrillar aggregates is associated with a number of the most common neurodegenerative diseases. We have established, using a computational approach, that knowledge of the primary sequences of proteins is sufficient to predict their in vitro aggregation propensities. Here we demonstrate, using rational mutagenesis of the Abeta42 peptide based on such computational predictions of aggregation propensity, the existence of a strong correlation between the propensity of Abeta42 to form protofibrils and its effect on neuronal dysfunction and degeneration in a Drosophila model of Alzheimer disease. Our findings provide a quantitative description of the molecular basis for the pathogenicity of Abeta and link directly and systematically the intrinsic properties of biomolecules, predicted in silico and confirmed in vitro, to pathogenic events taking place in a living organism. [Abstract/Link to Full Text]

Menashe I, Abaffy T, Hasin Y, Goshen S, Yahalom V, Luetje CW, Lancet D
Genetic elucidation of human hyperosmia to isovaleric acid.
PLoS Biol. 2007 Oct 30;5(11):e284.
The genetic basis of odorant-specific variations in human olfactory thresholds, and in particular of enhanced odorant sensitivity (hyperosmia), remains largely unknown. Olfactory receptor (OR) segregating pseudogenes, displaying both functional and nonfunctional alleles in humans, are excellent candidates to underlie these differences in olfactory sensitivity. To explore this hypothesis, we examined the association between olfactory detection threshold phenotypes of four odorants and segregating pseudogene genotypes of 43 ORs genome-wide. A strong association signal was observed between the single nucleotide polymorphism variants in OR11H7P and sensitivity to the odorant isovaleric acid. This association was largely due to the low frequency of homozygous pseudogenized genotype in individuals with specific hyperosmia to this odorant, implying a possible functional role of OR11H7P in isovaleric acid detection. This predicted receptor-ligand functional relationship was further verified using the Xenopus oocyte expression system, whereby the intact allele of OR11H7P exhibited a response to isovaleric acid. Notably, we also uncovered another mechanism affecting general olfactory acuity that manifested as a significant inter-odorant threshold concordance, resulting in an overrepresentation of individuals who were hyperosmic to several odorants. An involvement of polymorphisms in other downstream transduction genes is one possible explanation for this observation. Thus, human hyperosmia to isovaleric acid is a complex trait, contributed to by both receptor and other mechanisms in the olfactory signaling pathway. [Abstract/Link to Full Text]

Gross L
Poverty, human development, and basic biology.
PLoS Biol. 2007 Oct 23;5(11):e295. [Abstract/Link to Full Text]

Ancrenaz M, Dabek L, O'Neil S
The costs of exclusion: recognizing a role for local communities in biodiversity conservation.
PLoS Biol. 2007 Oct 23;5(11):e289. [Abstract/Link to Full Text]

Overath T, Cusack R, Kumar S, von Kriegstein K, Warren JD, Grube M, Carlyon RP, Griffiths TD
An information theoretic characterisation of auditory encoding.
PLoS Biol. 2007 Oct 23;5(11):e288.
The entropy metric derived from information theory provides a means to quantify the amount of information transmitted in acoustic streams like speech or music. By systematically varying the entropy of pitch sequences, we sought brain areas where neural activity and energetic demands increase as a function of entropy. Such a relationship is predicted to occur in an efficient encoding mechanism that uses less computational resource when less information is present in the signal: we specifically tested the hypothesis that such a relationship is present in the planum temporale (PT). In two convergent functional MRI studies, we demonstrated this relationship in PT for encoding, while furthermore showing that a distributed fronto-parietal network for retrieval of acoustic information is independent of entropy. The results establish PT as an efficient neural engine that demands less computational resource to encode redundant signals than those with high information content. [Abstract/Link to Full Text]

Sterpenich V, Albouy G, Boly M, Vandewalle G, Darsaud A, Balteau E, Dang-Vu TT, Desseilles M, D'Argembeau A, Gais S, Rauchs G, Schabus M, Degueldre C, Luxen A, Collette F, Maquet P
Sleep-related hippocampo-cortical interplay during emotional memory recollection.
PLoS Biol. 2007 Oct 23;5(11):e282.
Emotional events are usually better remembered than neutral ones. This effect is mediated in part by a modulation of the hippocampus by the amygdala. Sleep plays a role in the consolidation of declarative memory. We examined the impact of sleep and lack of sleep on the consolidation of emotional (negative and positive) memories at the macroscopic systems level. Using functional MRI (fMRI), we compared the neural correlates of successful recollection by humans of emotional and neutral stimuli, 72 h after encoding, with or without total sleep deprivation during the first post-encoding night. In contrast to recollection of neutral and positive stimuli, which was deteriorated by sleep deprivation, similar recollection levels were achieved for negative stimuli in both groups. Successful recollection of emotional stimuli elicited larger responses in the hippocampus and various cortical areas, including the medial prefrontal cortex, in the sleep group than in the sleep deprived group. This effect was consistent across subjects for negative items but depended linearly on individual memory performance for positive items. In addition, the hippocampus and medial prefrontal cortex were functionally more connected during recollection of either negative or positive than neutral items, and more so in sleeping than in sleep-deprived subjects. In the sleep-deprived group, recollection of negative items elicited larger responses in the amygdala and an occipital area than in the sleep group. In contrast, no such difference in brain responses between groups was associated with recollection of positive stimuli. The results suggest that the emotional significance of memories influences their sleep-dependent systems-level consolidation. The recruitment of hippocampo-neocortical networks during recollection is enhanced after sleep and is hindered by sleep deprivation. After sleep deprivation, recollection of negative, potentially dangerous, memories recruits an alternate amygdalo-cortical network, which would keep track of emotional information despite sleep deprivation. [Abstract/Link to Full Text]

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Recent Articles in BMC Biology

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Kabacoff C, Xiong Y, Musib R, Reichl EM, Kim J, Iglesias PA, Robinson DN
Dynacortin facilitates polarization of chemotaxing cells.
BMC Biol. 2007 Nov 26;5(1):53.
ABSTRACT: BACKGROUND: Cell shape changes during cytokinesis and chemotaxis require regulation of the actin cytoskeletal network. Dynacortin, an actin crosslinking protein, localizes to the cell cortex and contributes to cortical resistance, thereby helping to define the cell shape changes of cytokinesis. Dynacortin also becomes highly enriched in cortical protrusions, which are sites of new actin assembly. RESULTS: We studied the effect of dynacortin on cell motility during chemotaxis and on actin dynamics in vivo and in vitro. Dynacortin enriches with the actin, particularly at the leading edge of chemotaxing cells. Cells devoid of dynacortin do not become as polarized as wild type control cells but move with similar velocities as wild type cells. In particular, they send out multiple pseudopods that radiate at a broader distribution of angles relative to the chemoattractant gradient. Wild type cells typically only send out one pseudopod at a time that does not diverge much from 0 on average relative to the gradient. Though dynacortin-deficient cells show normal bulk (whole-cell) actin assembly upon chemoattractant stimulation, dynacortin can promote actin assembly in vitro. By fluorescence spectroscopy, co-sedimentation and transmission electron microscopy, dynacortin acts as an actin scaffolder in which it assembles actin monomers into polymers with a stoichiometry of 1 Dyn2:1 actin under salt conditions that disfavor polymer assembly. CONCLUSION: Dynacortin contributes to cell polarization during chemotaxis. By crosslinking and possibly stabilizing actin polymers, dynacortin also contributes to cortical viscoelasticity, which may be critical for establishing cell polarity. Though not essential for directional sensing or motility, dynacortin is required to establish cell polarity, the third core feature of chemotaxis. [Abstract/Link to Full Text]

Okamoto H, Stracke H, Ross B, Kakigi R, Pantev C
Left hemispheric dominance during auditory processing in noisy environment.
BMC Biol. 2007 Nov 15;5(1):52.
ABSTRACT: BACKGROUND: In daily life, we are exposed to different sound inputs simultaneously. During neural encoding in the auditory pathway, neural activities elicited by these different sounds interact with each other. In the present study, we investigated neural interactions elicited by masker and amplitude-modulated test stimulus in primary and non-primary human auditory cortex during ipsi-lateral and contra-lateral masking by means of magnetoencephalography (MEG). RESULTS: We observed significant decrements of auditory evoked responses and a significant inter-hemispheric difference for the N1m response during both ipsi- and contra-lateral masking. CONCLUSIONS: The decrements of auditory evoked neural activities during simultaneous masking can be explained by neural interactions evoked by masker and test stimulus in peripheral and central auditory systems. The inter-hemispheric differences of N1m decrements during ipsi- and contra-lateral masking reflect a basic hemispheric specialization contributing to the processing of complex auditory stimuli like speech signals in noisy environments. [Abstract/Link to Full Text]

Salzburger W, Braasch I, Meyer A
Adaptive sequence evolution in a color gene involved in the formation of the characteristic egg-dummies of male haplochromine cichlid fishes.
BMC Biol. 2007 Nov 15;5(1):51.
ABSTRACT: BACKGROUND: The exceptionally diverse species flocks of cichlid fishes in East Africa are prime examples of parallel adaptive radiations. About 80% of East Africa's more than 1,800 endemic cichlid species, and all species of the flocks of Lakes Victoria and Malawi, belong to a particularly rapidly evolving lineage, the haplochromines. One characteristic feature of the haplochromines is their possession of egg-dummies on the males' anal fins. These egg-spots mimic real eggs and play an important role in the mating system of these maternal mouthbrooding fish. RESULTS: Here we show that the egg-spots of haplochromines are made up of yellow pigment cells, xanthophores, and that a gene coding for a type III receptor tyrosine kinase, the colony-stimulating factor 1 receptor a (csf1ra), is expressed in egg-spot tissue. Molecular evolutionary analyses reveal that the extracellular ligand-binding and receptor-interacting domain of csf1ra underwent adaptive sequence evolution in the ancestral lineage of the haplochromines coinciding with the emergence of egg-dummies. We also find that csf1ra is expressed in the egg-dummies of a distantly related cichlid species, the ectodine cichlid Ophthalmotilapia ventralis, in which markings with similar functions evolved on the pectoral fin in convergence to those of the haplochromines. CONCLUSION: We conclude that modifications of existing signal transduction mechanisms might have evolved in the haplochromine lineage in association with the origination of anal fin egg-dummies. That positive selection has acted during the evolution of a color gene that seems to be involved in the morphogenesis of a sexually selected trait, the egg-dummies, highlights the importance of further investigations of the comparative genomic basis of the phenotypic diversification of cichlid fishes. [Abstract/Link to Full Text]

Le Rouzic A, Siegel PB, Carlborg O
Phenotypic evolution from genetic polymorphisms in a radial network architecture.
BMC Biol. 2007 Nov 14;5(1):50.
ABSTRACT: BACKGROUND: The genetic architecture of a quantitative trait influences the phenotypic response to natural or artificial selection. One of the main objectives of genetic mapping studies is to identify the genetic factors underlying complex traits and understand how they contribute to phenotypic expression. Presently, we are good at identifying and locating individual loci with large effects, but there is a void in describing more complex genetic architectures. Although large networks of connected genes have been reported, there is an almost complete lack of information on how polymorphisms in these networks contribute to phenotypic variation and change. To date, most of our understanding comes from theoretical, model-based studies, and it remains difficult to assess how realistic the conclusions from this work are as they lack empirical support. RESULTS: A previous study provided evidence that nearly half of the difference in eight-week body weight between two divergently selected lines of chickens was a result of four loci organized in a 'radial' network (one central locus interacting with three 'radial' loci that, in turn, only interacted with the central locus). Here, we study the relationship between phenotypic change and genetic polymorphism in this empirically detected network. We use a model-free approach to study, through individual-based simulations, the dynamic properties of this polymorphic and epistatic genetic architecture. The study provides new insights to how epistasis can modify the selection response, buffer and reveal effects of major loci leading to a progressive release of genetic variation. We also illustrate the difficulty of predicting genetic architecture from observed selection response, and discuss mechanisms that might lead to misleading conclusions on underlying genetic architectures from quantitative trait locus (QTL) experiments in selected populations. CONCLUSION: Considering both molecular (QTL) and phenotypic (selection response) data, as suggested in this work, provides additional insights into the genetic mechanisms involved in the response to selection. Such dissection of genetic architectures and in-depth studies of their ability to contribute to short- or long-term selection response represents an important step towards a better understanding of the genetic bases of complex traits and, consequently, of the evolutionary properties of populations. [Abstract/Link to Full Text]

Nyman T, Bokma F, Kopelke JP
Reciprocal diversification in a complex plant-herbivore-parasitoid food web.
BMC Biol. 2007 Nov 1;5(1):49.
ABSTRACT: BACKGROUND: Plants, plant-feeding insects, and insect parasitoids form some of the most complex and species-rich food webs. According to the classic escape-and-radiate (EAR) hypothesis, these hyperdiverse communities result from coevolutionary arms races consisting of successive cycles of enemy escape, radiation, and colonization by new enemy lineages. It has also been suggested that "enemy-free space" provided by novel host plants could promote host shifts by herbivores, and that parasitoids could similarly drive diversification of gall form in insects that induce galls on plants. Because these central coevolutionary hypotheses have never been tested in a phylogenetic framework, we combined phylogenetic information on willow-galling sawflies with data on their host plants, gall types, and enemy communities. RESULTS: We found that evolutionary shifts in host plant use and habitat have led to dramatic prunings of parasitoid communities, and that changes in gall phenotype can provide "enemy-free morphospace" for millions of years even in the absence of host plant shifts. Some parasites have nevertheless managed to colonize recently-evolved gall types, and this has apparently led to adaptive speciation in several enemy groups. However, having fewer enemies does not in itself increase speciation probabilities in individual sawfly lineages, partly because the high diversity of the enemy community facilitates compensatory attack by remaining parasite taxa. CONCLUSIONS: Taken together, our results indicate that niche-dependent parasitism is a major force promoting ecological divergence in herbivorous insects, and that prey divergence can cause speciation in parasite lineages. However, the results also show that the EAR hypothesis is too simplistic for species-rich food webs: instead, diversification seems to be spurred by a continuous stepwise process, in which ecological and phenotypic shifts in prey lineages are followed by a lagged evolutionary response by some of the associated enemies. [Abstract/Link to Full Text]

Feldhaar H, Straka J, Krischke M, Berthold K, Stoll S, Mueller MJ, Gross R
Nutritional Upgrading for Omnivorous Carpenter Ants by the Endosymbiont Blochmannia.
BMC Biol. 2007 Oct 30;5(1):48.
ABSTRACT: BACKGROUND: Carpenter ants (genus Camponotus) are considered to be omnivores. Nonetheless, the genome sequence of Blochmannia floridanus, the obligate intracellular endosymbiont of Camponotus floridanus, suggests a function in nutritional upgrading of host resources by the bacterium. Thus, the strongly reduced genome of the endosymbiont retains genes for all subunits of a functional urease, as well as those for biosynthetic pathways for all but one (arginine) of the amino acids essential to the host. RESULTS: Nutritional upgrading by Blochmannia was tested in 90-day feeding experiments with brood-raising in worker-groups on chemically defined diets with and without essential amino acids and treated or not with antibiotics. Control groups were fed with cockroaches, honey water and Bhatkar agar. Worker-groups were provided with brood collected from the queenright mother-colonies (45 eggs and 45 first instar larvae each). Brood production did not differ significantly between groups of symbiotic workers on diets with and without essential amino acids. However, aposymbiotic worker groups raised significantly less brood on a diet lacking essential amino acids. Reduced brood production by aposymbiotic workers was compensated when those groups were provided with essential amino acids in their diet. Decrease of endosymbionts due to treatment with antibiotic was monitored by qRT-PCR and FISH after the 90-day experimental period. Urease function was confirmed by feeding experiments using 15N-labelled urea. GC-MS analysis of 15N-enrichment of free amino acids in workers revealed significant labelling of the non-essential amino acids alanine, glycine, aspartic acid, and glutamic acid, as well as of the essential amino acids methionine and phenylalanine. CONCLUSIONS: Our results show that endosymbiotic Blochmannia nutritionally upgrade the diet of C. floridanus hosts to provide essential amino acids, and that it may also play a role in nitrogen recycling via its functional urease. Blochmannia may confer a significant fitness advantage via nutritional upgrading by enhancing competitive ability of Camponotus with other ant species lacking such an endosymbiont. Domestication of the endosymbiont may have facilitated the evolutionary success the genus of Camponotus. [Abstract/Link to Full Text]

Holland PW, Booth HA, Bruford EA
Classification and nomenclature of all human homeobox genes.
BMC Biol. 2007 Oct 26;5(1):47.
ABSTRACT: BACKGROUND: The homeobox genes are a large and diverse group of genes, many of which play important roles in the embryonic development of animals. Increasingly, homeobox genes are being compared between genomes in an attempt to understand the evolution of animal development. Despite their importance, the full diversity of human homeobox genes has not previously been described. RESULTS: We have identified all homeobox genes and pseudogenes in the euchromatic regions of the human genome, finding many unannotated, incorrectly annotated, unnamed, misnamed or misclassified genes and pseudogenes. We describe 300 human homeobox loci, which we divide into 235 probable functional genes and 65 probable pseudogenes. These totals include three genes with partial homeoboxes and thirteen pseudogenes that lack homeoboxes but are clearly derived from homeobox genes. These figures exclude the repetitive DUX1 to DUX5 homeobox sequences of which we identified 35 probable pseudogenes, with many more expected in heterochromatic regions. Nomenclature is established for approximately 40 formerly unnamed loci, reflecting their evolutionary relationships to other loci in human and other species, and nomenclature revisions are proposed for around 30 other loci. We use a classification that recognizes eleven homeobox gene 'classes' subdivided into 102 homeobox gene 'families'. CONCLUSIONS: We have conducted a comprehensive survey of homeobox genes and pseudogenes in the human genome, described many new loci, and revised the classification and nomenclature of homeobox genes. The classification scheme may be widely applicable to homeobox genes in other animal genomes and will facilitate comparative genomics of this important gene superclass. [Abstract/Link to Full Text]

Noack S, Wahl A, Qeli E, Wiechert W
Visualizing Regulatory Interactions in Metabolic Networks.
BMC Biol. 2007 Oct 16;5(1):46.
ABSTRACT: BACKGROUND: Direct visualization of data sets in the context of biochemical network drawings is one of the most appealing approaches in the field of data evaluation within systems biology. One important type of information that is very helpful in interpreting and understanding metabolic networks has been overlooked so far. Here we focus on the representation of this type of information given by the strength of regulatory interactions between metabolite pools and reaction steps. RESULTS: The visualization of such interactions in a given metabolic network is based on a novel concept defining the regulatory strength (RS) of effectors regulating certain reaction steps. It is applicable to any mechanistic reaction kinetic formula. The RS values are measures for the strength of an up- or down-regulation of a reaction step compared with the completely non-inhibited or non-activated state, respectively. One numerical RS value is associated to any effector edge contained in the network. The RS is approximately interpretable on a percentage scale where 100% means the maximal possible inhibition or activation, respectively, and 0% means the absence of a regulatory interaction. If many effectors influence a certain reaction step, the respective percentages indicate the proportion in which the different effectors contribute to the total regulation of the reaction step. The benefits of the proposed method are demonstrated with a complex example system of a dynamic E. coli network. CONCLUSIONS: The presented visualization approach is suitable for an intuitive interpretation of simulation data of metabolic networks under dynamic as well as steady-state conditions. Huge amounts of simulation data can be analyzed in a quick and comprehensive way. An extended time-resolved graphical network presentation provides a series of information about regulatory interaction within the biological system under investigation. [Abstract/Link to Full Text]

Kuballa AV, Merritt DJ, Elizur A
Gene Expression Profiling of Cuticular Proteins across the Moult Cycle of the Crab Portunus pelagicus.
BMC Biol. 2007 Oct 10;5(1):45.
ABSTRACT: BACKGROUND: Crustaceans represent an attractive model to study biomineralization and cuticle matrix formation as these events are precisely timed to occur at certain stages of the moult cycle. Moulting, the process by which crustaceans shed their exoskeleton, involves the partial breakdown of the old exoskeleton and the synthesis of a new cuticle. This cuticle is subdivided into layers some of which become calcified and some which remain uncalcified. The cuticle matrix consists of many different proteins which confer the physical properties, such as pliability, of the exoskeleton. RESULTS: We have used a custom cDNA microarray chip, developed for the blue swimmer crab Portunus pelagicus, to generate expression profiles of genes involved in exoskeletal formation across the moult cycle. Twenty-one distinct moult cycle related differentially expressed transcripts representing crustacean cuticular proteins were isolated. Thirteen contain copies of the cuticle_1 domain, previously isolated from calcified regions of the crustacean exoskeleton. Four transcripts contain a chitin_bind_4 domain (RR consensus sequence), associated with both the calcified and un-calcified cuticle of crustaceans. Four transcripts contain an unannotated domain (PfamB_109992) previously isolated from C. pagurus. Additionally cryptocyanin, a hemolymph protein, involved in cuticle synthesis and structural integrity, also displays differential expression related to the moult cycle. Moult stage-specific expression analysis of these transcripts revealed that differential gene expression occurs both among transcripts containing the same domain and among transcripts containing different domains. CONCLUSIONS: The large variety of genes associated with cuticle formation, and their differential expression across the crustacean moult cycle, point to the complexity of the processes associated with cuticle formation and hardening which involve many components and require strict regulatory mechanisms. This study provides a molecular entry path into the investigation of the gene networks associated with cuticle formation. [Abstract/Link to Full Text]

Kerrien S, Orchard S, Montecchi-Palazzi L, Aranda B, Quinn AF, Vinod N, Bader GD, Xenarios I, Wojcik J, Sherman D, Tyers M, Salama JJ, Moore S, Ceol A, Chatr-Aryamontri A, Oesterheld M, Stumpflen V, Salwinski L, Nerothin J, Cerami E, Cusick ME, Vidal M, Gilson M, Armstrong J, Woollard P, Hogue C, Eisenberg D, Cesareni G, Apweiler R, Hermjakob H
Broadening the Horizon - Level 2.5 of the HUPO-PSI Format for Molecular Interactions.
BMC Biol. 2007 Oct 9;5(1):44.
ABSTRACT: BACKGROUND: Information about molecular interaction is a key resource in modern biomedical research. Publicly available data have previously been provided in a broad array of diverse formats, making access to this very difficult. The publication and wide implementation of the Human Proteome Organisation Proteomics Standards Initiative Molecular Interactions (HUPO PSI-MI) format in 2004 was a major step towards the establishment of a single, unified format by which molecular interactions should be presented, but focused purely on protein-protein interactions. RESULTS: The HUPO-PSI has further developed the PSI-MI XML schema to enable the description of interactions between a wider range of molecular types, for example nucleic acids, chemical entities, and molecular complexes. Extensive details about each supported molecular interaction can now be captured, including the biological role of each molecule within that interaction, detailed description of interacting domains, and the kinetic parameters of the interaction. The format is supported by data management and analysis tools and has been adopted by major interaction data providers. Additionally, a simpler, tab-delimited format MITAB2.5 has been developed for the benefit of users who require only minimal information in an easy to access configuration. CONCLUSIONS: The PSI-MI XML2.5 and MITAB2.5 formats have been jointly developed by interaction data producers and providers from both the academic and commercial sector, and are already widely implemented and well supported by an active development community. PSI-MI XML2.5 enables the description of highly detailed molecular interaction data and facilitates data exchange between databases and users without loss of information. MITAB2.5 is a simpler format appropriate for fast Perl parsing or loading into Microsoft Excel. [Abstract/Link to Full Text]

Shulman-Peleg A, Shatsky M, Nussinov R, Wolfson HJ
Spatial chemical conservation of hot spot interactions in protein-protein complexes.
BMC Biol. 2007 Oct 9;5(1):43.
ABSTRACT: BACKGROUND: Conservation of the spatial binding organizations at the level of physico-chemical interactions is important for the formation and stability of protein-protein complexes as well as protein and drug design. Due to the lack of computational tools for recognition of spatial patterns of interactions shared by a set of protein-protein complexes, the conservation of such interactions was not addressed previously. RESULTS: Here, we performed extensive spatial comparisons of physico-chemical interactions common to different types of protein-protein complexes. We observed that 80% of these interactions correspond to known hot spots. Moreover, we show that spatially conserved interactions allow prediction of hot spots with a success rate higher than obtained by methods based on energy calculations, sequence or backbone similarity. Detection of spatially conserved interaction patterns is performed by our novel MAPPIS algorithm. MAPPIS performs multiple alignments of the physico-chemical interactions and the binding properties in three dimensional space. It is independent of the overall similarity in the protein sequences, folds or amino acid identities. We present examples of interactions shared between complexes of colicins with immunity proteins, serine proteases with inhibitors and T-cell receptors with superantigens. We unravel previously overlooked similarities, such as the interactions shared by the structurally different RNase-inhibitor families. CONCLUSIONS: The key contribution of MAPPIS is in discovering the 3D patterns of physico-chemical interactions. The detected patterns describe the conserved binding organizations which involve energetically important hot spot residues and are crucial for the protein-protein associations. AVAILABILITY: http://bioinfo3d.cs.tau.ac.il/mappis/ [Abstract/Link to Full Text]

Douglas CL, Vyazovskiy VV, Southard TL, Chiu SY, Messing A, Tononi G, Cirelli C
Sleep in Kcna2 knockout mice.
BMC Biol. 2007 Oct 9;5(1):42.
ABSTRACT: BACKGROUND: Shaker codes for a Drosophila voltage-dependent potassium channel. Flies carrying Shaker null or hypomorphic mutations sleep 3-4 hours/day instead of 8-14 hours/day like their wild-type siblings. Shaker-like channels are conserved across species but it is unknown whether they affect sleep in mammals. To address this issue, we studied sleep in Kcna2 knockout (KO) mice. Kcna2 codes for Kv1.2, the alpha subunit of a Shaker-like voltage-dependent potassium channel with high expression in the mammalian thalamocortical system. RESULTS: 24-hour EEG, EMG, and video recordings were used to measure sleep and waking in Kcna2 KO, heterozygous (HZ) and wild-type (WT) pups (P17) and HZ and WT adult mice (P67). Sleep stages were scored visually based on 4-sec epochs. EEG power spectra (0-20Hz) were calculated on consecutive 4-sec epochs. KO pups die by P28 due to generalized seizures. At P17 seizures are either absent or very rare in KO pups (<1% of the 24-hour recording time), and abnormal EEG activity is only present during the seizure. KO pups have significantly less NREM sleep (-23%) and significantly more waking (+21%) than HZ and WT siblings with no change in REM sleep time. The decrease in NREM sleep is due to an increase in the number of waking episodes, with no change in number or duration of sleep episodes. Sleep patterns, daily amounts of sleep and waking, and the response to 6 hours of sleep deprivation are similar in HZ and WT adult mice. CONCLUSIONS: Kv1.2, a mammalian homologue of Shaker, regulates neuronal excitability and affects NREM sleep. [Abstract/Link to Full Text]

Jackson CJ, Norman JE, Schnare MN, Gray MW, Keeling PJ, Waller RF
Broad genomic and transcriptional analysis reveals a highly derived genome in dinoflagellate mitochondria.
BMC Biol. 2007 Sep 27;5(1):41.
ABSTRACT: BACKGROUND: Dinoflagellates comprise an ecologically significant and diverse eukaryotic phylum that is sister to the phylum containing apicomplexan endoparasites. The mitochondrial genome of apicomplexans is uniquely reduced in gene content and size, encoding only three proteins and two ribosomal RNAs (rRNAs) within a highly compacted 6 kb DNA. Dinoflagellate mitochondrial genomes have been comparatively poorly studied: limited available data suggest some similarities with apicomplexan mitochondrial genomes but an even more radical type of genomic organization. Here, we investigate structure, content and expression of dinoflagellate mitochondrial genomes. RESULTS: From two dinoflagellates, Crypthecodinium cohnii and Karlodinium micrum, we generated over 42 kb of mitochondrial genomic data that indicate a reduced gene content paralleling that of mitochondrial genomes in apicomplexans, i.e., only three protein-encoding genes and at least eight conserved components of the highly fragmented large and small subunit rRNAs. Unlike in apicomplexans, dinoflagellate mitochondrial genes occur in multiple copies, often as gene fragments, and in numerous genomic contexts. Analysis of cDNAs suggests several novel aspects of dinoflagellate mitochondrial gene expression. Polycistronic transcripts were found, standard start codons are absent, and oligoadenylation occurs upstream of stop codons, resulting in the absence of termination codons. Transcripts of at least one gene, cox3, are apparently trans-spliced to generate full-length mRNAs. RNA substitutional editing, a process previously identified for mRNAs in dinoflagellate mitochondria, is also implicated in rRNA expression. CONCLUSIONS: The dinoflagellate mitochondrial genome shares the same gene complement and fragmentation of rRNA genes with its apicomplexan counterpart. However, it also exhibits several unique characteristics. Most notable are the expansion of gene copy numbers and their arrangements within the genome, RNA editing, loss of stop codons, and use of trans-splicing. [Abstract/Link to Full Text]

Ellegren H, Hultin-Rosenberg L, Brunström B, Dencker L, Kultima K, Scholz B
Faced with inequality: chicken do not have a general dosage compensation of sex-linked genes.
BMC Biol. 2007;540.
BACKGROUND: The contrasting dose of sex chromosomes in males and females potentially introduces a large-scale imbalance in levels of gene expression between sexes, and between sex chromosomes and autosomes. In many organisms, dosage compensation has thus evolved to equalize sex-linked gene expression in males and females. In mammals this is achieved by X chromosome inactivation and in flies and worms by up- or down-regulation of X-linked expression, respectively. While otherwise widespread in systems with heteromorphic sex chromosomes, the case of dosage compensation in birds (males ZZ, females ZW) remains an unsolved enigma. RESULTS: Here, we use a microarray approach to show that male chicken embryos generally express higher levels of Z-linked genes than female birds, both in soma and in gonads. The distribution of male-to-female fold-change values for Z chromosome genes is wide and has a mean of 1.4-1.6, which is consistent with absence of dosage compensation and sex-specific feedback regulation of gene expression at individual loci. Intriguingly, without global dosage compensation, the female chicken has significantly lower expression levels of Z-linked compared to autosomal genes, which is not the case in male birds. CONCLUSION: The pronounced sex difference in gene expression is likely to contribute to sexual dimorphism among birds, and potentially has implication to avian sex determination. Importantly, this report, together with a recent study of sex-biased expression in somatic tissue of chicken, demonstrates the first example of an organism with a lack of global dosage compensation, providing an unexpected case of a viable system with large-scale imbalance in gene expression between sexes. [Abstract/Link to Full Text]

Walker T, Klasson L, Sebaihia M, Sanders MJ, Thomson NR, Parkhill J, Sinkins SP
Ankyrin repeat domain-encoding genes in the wPip strain of Wolbachia from the Culex pipiens group.
BMC Biol. 2007;539.
BACKGROUND: Wolbachia are obligate endosymbiotic bacteria maternally transmitted through the egg cytoplasm that are responsible for several reproductive disorders in their insect hosts, such as cytoplasmic incompatibility (CI) in infected mosquitoes. Species in the Culex pipiens complex display an unusually high number of Wolbachia-induced crossing types, and based on present data, only the wPip strain is present. RESULTS: The sequencing of the wPip strain of Wolbachia revealed the presence of 60 ankyrin repeat domain (ANK) encoding genes and expression studies of these genes were carried out in adult mosquitoes. One of these ANK genes, pk2, is shown to be part of an operon of three prophage-associated genes with sex-specific expression, and is present in two identical copies in the genome. Another homolog of pk2 is also present that is differentially expressed in different Cx. pipiens group strains. A further two ANK genes showed sex-specific regulation in wPip-infected Cx. pipiens group adults. CONCLUSION: The high number, variability and differential expression of ANK genes in wPip suggest an important role in Wolbachia biology, and the gene family provides both markers and promising candidates for the study of reproductive manipulation. [Abstract/Link to Full Text]

Suter B, Pogoutse O, Guo X, Krogan NJ, Lewis P, Greenblatt JF, Rine J, Emili A
Association with the Origin Recognition Complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p.
BMC Biol. 2007 Sep 19;5(1):38.
ABSTRACT: BACKGROUND: Histone modifications have been implicated in the regulation of transcription and, more recently, in DNA replication and repair. In yeast, a major conserved histone acetyltransferase, Hat1p, preferentially acetylates lysine residues 5 and 12 on histone H4. RESULTS: Here, we report that a nuclear sub-complex consisting of Hat1p and its partner Hat2p interacts physically and functionally with the origin recognition complex (ORC). While mutational inactivation of HAT1 alone does not compromise origin firing or initiation of DNA replication, a deletion in HAT1 (or HAT2) exacerbates the growth defects of conditional orc-ts mutants. Thus, the ORC-associated Hat1p-dependent histone acetyltransferase activity suggests a novel linkage between histone modification and DNA replication. Additional genetic and biochemical evidence points to the existence of partly overlapping histone H3 acetyltransferase activities in addition to Hat1p/Hat2p for proper DNA replication efficiency. Furthermore, we demonstrated a dynamic association of Hat1p with chromatin during S-phase that suggests a role of this enzyme at the replication fork. CONCLUSION: We have found an intriguing new association of the Hat1p-dependent histone acetyltransferase besides its previously known role in nuclear chromatin assembly (Hat1p/Hat2p-Hif1p). The participation of a distinct Hat1p/Hat2p sub-complex suggests a linkage of histone H4 modification with ORC-dependent DNA replication. [Abstract/Link to Full Text]

Kessler D, Papatheodorou P, Stratmann T, Dian EA, Hartmann-Fatu C, Rassow J, Bayer P, Mueller JW
The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae.
BMC Biol. 2007;537.
BACKGROUND: The parvulin-type peptidyl prolyl cis/trans isomerase Par14 is highly conserved in all metazoans. The recently identified parvulin Par17 contains an additional N-terminal domain whose occurrence and function was the focus of the present study. RESULTS: Based on the observation that the human genome encodes Par17, but bovine and rodent genomes do not, Par17 exon sequences from 10 different primate species were cloned and sequenced. Par17 is encoded in the genomes of Hominidae species including humans, but is absent from other mammalian species. In contrast to Par14, endogenous Par17 was found in mitochondrial and membrane fractions of human cell lysates. Fluorescence of EGFP fusions of Par17, but not Par14, co-localized with mitochondrial staining. Par14 and Par17 associated with isolated human, rat and yeast mitochondria at low salt concentrations, but only the Par17 mitochondrial association was resistant to higher salt concentrations. Par17 was imported into mitochondria in a time and membrane potential-dependent manner, where it reached the mitochondrial matrix. Moreover, Par17 was shown to bind to double-stranded DNA under physiological salt conditions. CONCLUSION: Taken together, the DNA binding parvulin Par17 is targeted to the mitochondrial matrix by the most recently evolved mitochondrial prepeptide known to date, thus adding a novel protein constituent to the mitochondrial proteome of Hominidae. [Abstract/Link to Full Text]

Munfus DL, Haga CL, Burrows PD, Cooper MD
A conserved gene family encodes transmembrane proteins with fibronectin, immunoglobulin and leucine-rich repeat domains (FIGLER).
BMC Biol. 2007;536.
BACKGROUND: In mouse the cytokine interleukin-7 (IL-7) is required for generation of B lymphocytes, but human IL-7 does not appear to have this function. A bioinformatics approach was therefore used to identify IL-7 receptor related genes in the hope of identifying the elusive human cytokine. RESULTS: Our database search identified a family of nine gene candidates, which we have provisionally named fibronectin immunoglobulin leucine-rich repeat (FIGLER). The FIGLER 1-9 genes are predicted to encode type I transmembrane glycoproteins with 6-12 leucine-rich repeats (LRR), a C2 type Ig domain, a fibronectin type III domain, a hydrophobic transmembrane domain, and a cytoplasmic domain containing one to four tyrosine residues. Members of this multichromosomal gene family possess 20-47% overall amino acid identity and are differentially expressed in cell lines and primary hematopoietic lineage cells. Genes for FIGLER homologs were identified in macaque, orangutan, chimpanzee, mouse, rat, dog, chicken, toad, and puffer fish databases. The non-human FIGLER homologs share 38-99% overall amino acid identity with their human counterpart. CONCLUSION: The extracellular domain structure and absence of recognizable cytoplasmic signaling motifs in members of the highly conserved FIGLER gene family suggest a trophic or cell adhesion function for these molecules. [Abstract/Link to Full Text]

Bennett JS, Jolley KA, Sparling PF, Saunders NJ, Hart CA, Feavers IM, Maiden MC
Species status of Neisseria gonorrhoeae: evolutionary and epidemiological inferences from multilocus sequence typing.
BMC Biol. 2007;535.
BACKGROUND: Various typing methods have been developed for Neisseria gonorrhoeae, but none provide the combination of discrimination, reproducibility, portability, and genetic inference that allows the analysis of all aspects of the epidemiology of this pathogen from a single data set. Multilocus sequence typing (MLST) has been used successfully to characterize the related organisms Neisseria meningitidis and Neisseria lactamica. Here, the same seven locus Neisseria scheme was used to characterize a diverse collection of N. gonorrhoeae isolates to investigate whether this method would allow differentiation among isolates, and to distinguish these three species. RESULTS: A total of 149 gonococcal isolates were typed and submitted to the Neisseria MLST database. Although relatively few (27) polymorphisms were detected among the seven MLST loci, a total of 66 unique allele combinations (sequence types, STs), were observed, a number comparable to that seen among isolate collections of the more diverse meningococcus. Patterns of genetic variation were consistent with high levels of recombination generating this diversity. There was no evidence for geographical structuring among the isolates examined, with isolates collected in Liverpool, UK, showing levels of diversity similar to a global collection of isolates. There was, however, evidence that populations of N. meningitidis, N. gonorrhoeae and N. lactamica were distinct, with little support for frequent genetic recombination among these species, with the sequences from the gdh locus alone grouping the species into distinct clusters. CONCLUSION: The seven loci Neisseria MLST scheme was readily adapted to N. gonorrhoeae isolates, providing a highly discriminatory typing method. In addition, these data permitted phylogenetic and population genetic inferences to be made, including direct comparisons with N. meningitidis and N. lactamica. Examination of these data demonstrated that alleles were rarely shared among the three species. Analysis of variation at a single locus, gdh, provided a rapid means of identifying misclassified isolates and determining whether mixed cultures were present. [Abstract/Link to Full Text]

Bryson-Richardson RJ, Berger S, Schilling TF, Hall TE, Cole NJ, Gibson AJ, Sharpe J, Currie PD
FishNet: an online database of zebrafish anatomy.
BMC Biol. 2007;534.
BACKGROUND: Over the last two decades, zebrafish have been established as a genetically versatile model system for investigating many different aspects of vertebrate developmental biology. With the credentials of zebrafish as a developmental model now well recognized, the emerging new opportunity is the wider application of zebrafish biology to aspects of human disease modelling. This rapidly increasing use of zebrafish as a model for human disease has necessarily generated interest in the anatomy of later developmental phases such as the larval, juvenile, and adult stages, during which many of the key aspects of organ morphogenesis and maturation take place. Anatomical resources and references that encompass these stages are non-existent in zebrafish and there is therefore an urgent need to understand how different organ systems and anatomical structures develop throughout the life of the fish. RESULTS: To overcome this deficit we have utilized the technique of optical projection tomography to produce three-dimensional (3D) models of larval fish. In order to view and display these models we have created FishNet http://www.fishnet.org.au, an interactive reference of zebrafish anatomy spanning the range of zebrafish development from 24 h until adulthood. CONCLUSION: FishNet contains more than 36,000 images of larval zebrafish, with more than 1,500 of these being annotated. The 3D models can be manipulated on screen or virtually sectioned. This resource represents the first complete embryo to adult atlas for any species in 3D. [Abstract/Link to Full Text]

Dawe HR, Shaw MK, Farr H, Gull K
The hydrocephalus inducing gene product, Hydin, positions axonemal central pair microtubules.
BMC Biol. 2007;533.
BACKGROUND: Impairment of cilia and flagella function underlies a growing number of human genetic diseases. Mutations in hydin in hy3 mice cause lethal communicating hydrocephalus with early onset. Hydin was recently identified as an axonemal protein; however, its function is as yet unknown. RESULTS: Here we use RNAi in Trypanosoma brucei to address this issue and demonstrate that loss of Hydin causes slow growth and a loss of cell motility. We show that two separate defects in newly-formed flagellar central pair microtubules underlie the loss of cell motility. At early time-points after RNAi induction, the central pair becomes mispositioned, while at later time points the central pair is lost. While the basal body is unaffected, both defects originate at the basal plate, reflecting a role for TbHydin throughout the length of the central pair. CONCLUSION: Our data provide the first evidence of Hydin's role within the trypanosome axoneme, and reveal central pair anomalies and thus impairment of ependymal ciliary motility as the likely cause of the hydrocephalus observed in the hy3 mouse. [Abstract/Link to Full Text]

Hayden CA, Jorgensen RA
Identification of novel conserved peptide uORF homology groups in Arabidopsis and rice reveals ancient eukaryotic origin of select groups and preferential association with transcription factor-encoding genes.
BMC Biol. 2007;532.
BACKGROUND: Upstream open reading frames (uORFs) can mediate translational control over the largest, or major ORF (mORF) in response to starvation, polyamine concentrations, and sucrose concentrations. One plant uORF with conserved peptide sequences has been shown to exert this control in an amino acid sequence-dependent manner but generally it is not clear what kinds of genes are regulated, or how extensively this mechanism is invoked in a given genome. RESULTS: By comparing full-length cDNA sequences from Arabidopsis and rice we identified 26 distinct homology groups of conserved peptide uORFs, only three of which have been reported previously. Pairwise Ka/Ks analysis showed that purifying selection had acted on nearly all conserved peptide uORFs and their associated mORFs. Functions of predicted mORF proteins could be inferred for 16 homology groups and many of these proteins appear to have a regulatory function, including 6 transcription factors, 5 signal transduction factors, 3 developmental signal molecules, a homolog of translation initiation factor eIF5, and a RING finger protein. Transcription factors are clearly overrepresented in this data set when compared to the frequency calculated for the entire genome (p = 1.2 x 10(-7)). Duplicate gene pairs arising from a whole genome duplication (ohnologs) with a conserved uORF are much more likely to have been retained in Arabidopsis (Arabidopsis thaliana) than are ohnologs of other genes (39% vs 14% of ancestral genes, p = 5 x 10(-3)). Two uORF groups were found in animals, indicating an ancient origin of these putative regulatory elements. CONCLUSION: Conservation of uORF amino acid sequence, association with homologous mORFs over long evolutionary time periods, preferential retention after whole genome duplications, and preferential association with mORFs coding for transcription factors suggest that the conserved peptide uORFs identified in this study are strong candidates for translational controllers of regulatory genes. [Abstract/Link to Full Text]

Hellsten U, Khokha MK, Grammer TC, Harland RM, Richardson P, Rokhsar DS
Accelerated gene evolution and subfunctionalization in the pseudotetraploid frog Xenopus laevis.
BMC Biol. 2007;531.
BACKGROUND: Ancient whole genome duplications have been implicated in the vertebrate and teleost radiations, and in the emergence of diverse angiosperm lineages, but the evolutionary response to such a perturbation is still poorly understood. The African clawed frog Xenopus laevis experienced a relatively recent tetraploidization ~40 million years ago. Analysis of the considerable amount of EST sequence available for this species together with the genome sequence of the related diploid Xenopus tropicalis provides a unique opportunity to study the genomic response to whole genome duplication. RESULTS: We identified 2218 gene triplets in which a single gene in X. tropicalis corresponds to precisely two co-orthologous genes in X. laevis--the largest such collection published from any duplication event in animals. Analysis of these triplets reveals accelerated evolution or relaxation of constraint in the peptides of the X. laevis pairs compared with the orthologous sequences in X. tropicalis and other vertebrates. In contrast, single-copy X. laevis genes do not show this acceleration. Duplicated genes can differ substantially in expression levels and patterns. We find no significant difference in gene content in the duplicated set, versus the single-copy set based on molecular and biological function ontologies. CONCLUSION: These results support a scenario in which duplicate genes are retained through a process of subfunctionalization and/or relaxation of constraint on both copies of an ancestral gene. [Abstract/Link to Full Text]

Fontaine MC, Baird SJ, Piry S, Ray N, Tolley KA, Duke S, Birkun A, Ferreira M, Jauniaux T, Llavona A, Oztürk B, A Oztürk A, Ridoux V, Rogan E, Sequeira M, Siebert U, Vikingsson GA, Bouquegneau JM, Michaux JR
Rise of oceanographic barriers in continuous populations of a cetacean: the genetic structure of harbour porpoises in Old World waters.
BMC Biol. 2007;530.
BACKGROUND: Understanding the role of seascape in shaping genetic and demographic population structure is highly challenging for marine pelagic species such as cetaceans for which there is generally little evidence of what could effectively restrict their dispersal. In the present work, we applied a combination of recent individual-based landscape genetic approaches to investigate the population genetic structure of a highly mobile extensive range cetacean, the harbour porpoise in the eastern North Atlantic, with regards to oceanographic characteristics that could constrain its dispersal. RESULTS: Analyses of 10 microsatellite loci for 752 individuals revealed that most of the sampled range in the eastern North Atlantic behaves as a 'continuous' population that widely extends over thousands of kilometres with significant isolation by distance (IBD). However, strong barriers to gene flow were detected in the south-eastern part of the range. These barriers coincided with profound changes in environmental characteristics and isolated, on a relatively small scale, porpoises from Iberian waters and on a larger scale porpoises from the Black Sea. CONCLUSION: The presence of these barriers to gene flow that coincide with profound changes in oceanographic features, together with the spatial variation in IBD strength, provide for the first time strong evidence that physical processes have a major impact on the demographic and genetic structure of a cetacean. This genetic pattern further suggests habitat-related fragmentation of the porpoise range that is likely to intensify with predicted surface ocean warming. [Abstract/Link to Full Text]

Bloom JD, Lu Z, Chen D, Raval A, Venturelli OS, Arnold FH
Evolution favors protein mutational robustness in sufficiently large populations.
BMC Biol. 2007;529.
BACKGROUND: An important question is whether evolution favors properties such as mutational robustness or evolvability that do not directly benefit any individual but can influence the course of future evolution. Functionally similar proteins can differ substantially in their robustness to mutations and capacity to evolve new functions, but it has remained unclear whether any of these differences might be due to evolutionary selection for these properties. RESULTS: Here, we use laboratory experiments to demonstrate that evolution favors protein mutational robustness if the evolving population is sufficiently large. We neutrally evolve cytochrome P450 proteins under identical selection pressures and mutation rates in populations of different sizes, and show that proteins from the larger and thus more polymorphic population tend towards higher mutational robustness. Proteins from the larger population also evolve greater stability, a biophysical property that is known to enhance both mutational robustness and evolvability. The excess mutational robustness and stability is well described by mathematical theory, and can be quantitatively related to the way that the proteins occupy their neutral network. CONCLUSION: Our work is the first experimental demonstration of the general tendency of evolution to favor mutational robustness and protein stability in highly polymorphic populations. We suggest that this phenomenon could contribute to the mutational robustness and evolvability of viruses and bacteria that exist in large populations. [Abstract/Link to Full Text]

Nozaki H, Takano H, Misumi O, Terasawa K, Matsuzaki M, Maruyama S, Nishida K, Yagisawa F, Yoshida Y, Fujiwara T, Takio S, Tamura K, Chung SJ, Nakamura S, Kuroiwa H, Tanaka K, Sato N, Kuroiwa T
A 100%-complete sequence reveals unusually simple genomic features in the hot-spring red alga Cyanidioschyzon merolae.
BMC Biol. 2007;528.
BACKGROUND: All previously reported eukaryotic nuclear genome sequences have been incomplete, especially in highly repeated units and chromosomal ends. Because repetitive DNA is important for many aspects of biology, complete chromosomal structures are fundamental for understanding eukaryotic cells. Our earlier, nearly complete genome sequence of the hot-spring red alga Cyanidioschyzon merolae revealed several unique features, including just three ribosomal DNA copies, very few introns, and a small total number of genes. However, because the exact structures of certain functionally important repeated elements remained ambiguous, that sequence was not complete. Obviously, those ambiguities needed to be resolved before the unique features of the C. merolae genome could be summarized, and the ambiguities could only be resolved by completing the sequence. Therefore, we aimed to complete all previous gaps and sequence all remaining chromosomal ends, and now report the first nuclear-genome sequence for any eukaryote that is 100% complete. RESULTS: Our present complete sequence consists of 16546747 nucleotides covering 100% of the 20 linear chromosomes from telomere to telomere, representing the simple and unique chromosomal structures of the eukaryotic cell. We have unambiguously established that the C. merolae genome contains the smallest known histone-gene cluster, a unique telomeric repeat for all chromosomal ends, and an extremely low number of transposons. CONCLUSION: By virtue of these attributes and others that we had discovered previously, C. merolae appears to have the simplest nuclear genome of the non-symbiotic eukaryotes. These unusually simple genomic features in the 100% complete genome sequence of C. merolae are extremely useful for further studies of eukaryotic cells. [Abstract/Link to Full Text]

Rogers MB, Patron NJ, Keeling PJ
Horizontal transfer of a eukaryotic plastid-targeted protein gene to cyanobacteria.
BMC Biol. 2007;526.
BACKGROUND: Horizontal or lateral transfer of genetic material between distantly related prokaryotes has been shown to play a major role in the evolution of bacterial and archaeal genomes, but exchange of genes between prokaryotes and eukaryotes is not as well understood. In particular, gene flow from eukaryotes to prokaryotes is rarely documented with strong support, which is unusual since prokaryotic genomes appear to readily accept foreign genes. RESULTS: Here, we show that abundant marine cyanobacteria in the related genera Synechococcus and Prochlorococcus acquired a key Calvin cycle/glycolytic enzyme from a eukaryote. Two non-homologous forms of fructose bisphosphate aldolase (FBA) are characteristic of eukaryotes and prokaryotes respectively. However, a eukaryotic gene has been inserted immediately upstream of the ancestral prokaryotic gene in several strains (ecotypes) of Synechococcus and Prochlorococcus. In one lineage this new gene has replaced the ancestral gene altogether. The eukaryotic gene is most closely related to the plastid-targeted FBA from red algae. This eukaryotic-type FBA once replaced the plastid/cyanobacterial type in photosynthetic eukaryotes, hinting at a possible functional advantage in Calvin cycle reactions. The strains that now possess this eukaryotic FBA are scattered across the tree of Synechococcus and Prochlorococcus, perhaps because the gene has been transferred multiple times among cyanobacteria, or more likely because it has been selectively retained only in certain lineages. CONCLUSION: A gene for plastid-targeted FBA has been transferred from red algae to cyanobacteria, where it has inserted itself beside its non-homologous, functional analogue. Its current distribution in Prochlorococcus and Synechococcus is punctate, suggesting a complex history since its introduction to this group. [Abstract/Link to Full Text]

Steigele S, Huber W, Stocsits C, Stadler PF, Nieselt K
Comparative analysis of structured RNAs in S. cerevisiae indicates a multitude of different functions.
BMC Biol. 2007;525.
BACKGROUND: Non-coding RNAs (ncRNAs) are an emerging focus for both computational analysis and experimental research, resulting in a growing number of novel, non-protein coding transcripts with often unknown functions. Whole genome screens in higher eukaryotes, for example, provided evidence for a surprisingly large number of ncRNAs. To supplement these searches, we performed a computational analysis of seven yeast species and searched for new ncRNAs and RNA motifs. RESULTS: A comparative analysis of the genomes of seven yeast species yielded roughly 2800 genomic loci that showed the hallmarks of evolutionary conserved RNA secondary structures. A total of 74% of these regions overlapped with annotated non-coding or coding genes in yeast. Coding sequences that carry predicted structured RNA elements belong to a limited number of groups with common functions, suggesting that these RNA elements are involved in post-transcriptional regulation and/or cellular localization. About 700 conserved RNA structures were found outside annotated coding sequences and known ncRNA genes. Many of these predicted elements overlapped with UTR regions of particular classes of protein coding genes. In addition, a number of RNA elements overlapped with previously characterized antisense transcripts. Transcription of about 120 predicted elements located in promoter regions and other, previously un-annotated, intergenic regions was supported by tiling array experiments, ESTs, or SAGE data. CONCLUSION: Our computational predictions strongly suggest that yeasts harbor a substantial pool of several hundred novel ncRNAs. In addition, we describe a large number of RNA structures in coding sequences and also within antisense transcripts that were previously characterized using tiling arrays. [Abstract/Link to Full Text]

Hajibabaei M, Singer GA, Clare EL, Hebert PD
Design and applicability of DNA arrays and DNA barcodes in biodiversity monitoring.
BMC Biol. 2007;524.
BACKGROUND: The rapid and accurate identification of species is a critical component of large-scale biodiversity monitoring programs. DNA arrays (micro and macro) and DNA barcodes are two molecular approaches that have recently garnered much attention. Here, we compare these two platforms for identification of an important group, the mammals. RESULTS: Our analyses, based on the two commonly used mitochondrial genes cytochrome c oxidase I (the standard DNA barcode for animal species) and cytochrome b (a common species-level marker), suggest that both arrays and barcodes are capable of discriminating mammalian species with high accuracy. We used three different datasets of mammalian species, comprising different sampling strategies. For DNA arrays we designed three probes for each species to address intraspecific variation. As for DNA barcoding, our analyses show that both cytochrome c oxidase I and cytochrome b genes, and even smaller fragments of them (mini-barcodes) can successfully discriminate species in a wide variety of specimens. CONCLUSION: This study showed that DNA arrays and DNA barcodes are valuable molecular methods for biodiversity monitoring programs. Both approaches were capable of discriminating among mammalian species in our test assemblages. However, because designing DNA arrays require advance knowledge of target sequences, the use of this approach could be limited in large scale monitoring programs where unknown haplotypes might be encountered. DNA barcodes, by contrast, are sequencing-based and therefore could provide more flexibility in large-scale studies. [Abstract/Link to Full Text]

Hoffman EA, Goodisman MA
Gene expression and the evolution of phenotypic diversity in social wasps.
BMC Biol. 2007;523.
BACKGROUND: Organisms are capable of developing different phenotypes by altering the genes they express. This phenotypic plasticity provides a means for species to respond effectively to environmental conditions. One of the most dramatic examples of phenotypic plasticity occurs in the highly social hymenopteran insects (ants, social bees, and social wasps), where distinct castes and sexes all arise from the same genes. To elucidate how variation in patterns of gene expression affects phenotypic variation, we conducted a study to simultaneously address the influence of developmental stage, sex, and caste on patterns of gene expression in Vespula wasps. Furthermore, we compared the patterns found in this species to those found in other taxa in order to investigate how variation in gene expression leads to phenotypic evolution. RESULTS: We constructed 11 different cDNA libraries derived from various developmental stages and castes of Vespula squamosa. Comparisons of overall expression patterns indicated that gene-expression differences distinguishing developmental stages were greater than expression differences differentiating sex or caste. Furthermore, we determined that certain sets of genes showed similar patterns of expression in the same phenotypic forms of different species. Specifically, larvae upregulated genes related to metabolism and genes possessing structural activity. Surprisingly, our data indicated that at least a few specific gene functions and at least one specific gene family are important components of caste differentiation across social insect taxa. CONCLUSION: Despite research on various aspects of development originating from model systems, growth in understanding how development is related to phenotypic diversity relies on a growing literature of contrasting studies in non-model systems. In this study, we found that comparisons of patterns of gene expression with model systems highlighted areas of conserved and convergent developmental evolution across diverse taxa. Indeed, conserved biological functions across species implicated key functions related to how phenotypes are built. Finally, overall differences between social insect taxa suggest that the independent evolution of caste arose via distinct developmental trajectories. [Abstract/Link to Full Text]

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Recent Articles in Journal of Biology

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Hansen MM, Hemmer-Hansen J
Landscape genetics goes to sea.
J Biol. 2007 Nov 16;6(3):6.
ABSTRACT: A recent study revealing geographical and environmental barriers to gene flow in the harbour porpoise shows the great potential of 'landscape genetics' when applied to marine organisms. [Abstract/Link to Full Text]

Byrne AB, Weirauch MT, Wong V, Koeva M, Dixon SJ, Stuart JM, Roy PJ
A global analysis of genetic interactions in Caenorhabditis elegans.
J Biol. 2007 Sep 26;6(3):8.
ABSTRACT: BACKGROUND: Understanding gene function and genetic relationships is fundamental to our efforts to better understand biological systems. Previous studies systematically describing genetic interactions on a global scale have either focused on core biological processes in protozoans or surveyed catastrophic interactions in metazoans. Here, we describe a reliable high-throughput approach capable of revealing both weak and strong genetic interactions in the nematode Caenorhabditis elegans. RESULTS: We investigated interactions between 11 'query' mutants in conserved signal transduction pathways and hundreds of 'target' genes compromised by RNA interference (RNAi). Mutant-RNAi combinations that grew more slowly than controls were identified, and genetic interactions inferred through an unbiased global analysis of the interaction matrix. A network of 1,246 interactions was uncovered, establishing the largest metazoan genetic-interaction network to date. We refer to this approach as systematic genetic interaction analysis (SGI). To investigate how genetic interactions connect genes on a global scale, we superimposed the SGI network on existing networks of physical, genetic, phenotypic and coexpression interactions. We identified 56 putative functional modules within the superimposed network, one of which regulates fat accumulation and is coordinated by interactions with bar-1(ga80), which encodes a homolog of beta-catenin. We also discovered that SGI interactions link distinct subnetworks on a global scale. Finally, we showed that the properties of genetic networks are conserved between C. elegans and Saccharomyces cerevisiae, but that the connectivity of interactions within the current networks is not. CONCLUSIONS: Synthetic genetic interactions may reveal redundancy among functional modules on a global scale, which is a previously unappreciated level of organization within metazoan systems. Although the buffering between functional modules may differ between species, studying these differences may provide insight into the evolution of divergent form and function. [Abstract/Link to Full Text]


Journal of Biology celebrates its fifth anniversary.
J Biol. 2007 Jun 29;6(3):5. [Abstract/Link to Full Text]

Ramanathan A, Schreiber SL
Multilevel regulation of growth rate in yeast revealed using systems biology.
J Biol. 2007;6(2):3.
ABSTRACT : The effect of changing growth rates on the transcriptome, proteome and metabolome has been systematically studied. Measurements made under varying nutrient conditions, corresponding to biochemical pathways that correlate primarily with growth rate, reveal a central role for mitochondrial metabolism and the TOR (target of rapamycin) signaling pathway. [Abstract/Link to Full Text]

Castrillo JI, Zeef LA, Hoyle DC, Zhang N, Hayes A, Gardner DC, Cornell MJ, Petty J, Hakes L, Wardleworth L, Rash B, Brown M, Dunn WB, Broadhurst D, O'donoghue K, Hester SS, Dunkley TP, Hart SR, Swainston N, Li P, Gaskell SJ, Paton NW, Lilley KS, Kell DB, Oliver SG
Growth control of the eukaryote cell: a systems biology study in yeast.
J Biol. 2007;6(2):4.
ABSTRACT : BACKGROUND : Cell growth underlies many key cellular and developmental processes, yet a limited number of studies have been carried out on cell-growth regulation. Comprehensive studies at the transcriptional, proteomic and metabolic levels under defined controlled conditions are currently lacking. RESULTS : Metabolic control analysis is being exploited in a systems biology study of the eukaryotic cell. Using chemostat culture, we have measured the impact of changes in flux (growth rate) on the transcriptome, proteome, endometabolome and exometabolome of the yeast Saccharomyces cerevisiae. Each functional genomic level shows clear growth-rate-associated trends and discriminates between carbon-sufficient and carbon-limited conditions. Genes consistently and significantly upregulated with increasing growth rate are frequently essential and encode evolutionarily conserved proteins of known function that participate in many protein-protein interactions. In contrast, more unknown, and fewer essential, genes are downregulated with increasing growth rate; their protein products rarely interact with one another. A large proportion of yeast genes under positive growth-rate control share orthologs with other eukaryotes, including humans. Significantly, transcription of genes encoding components of the TOR complex (a major controller of eukaryotic cell growth) is not subject to growth-rate regulation. Moreover, integrative studies reveal the extent and importance of post-transcriptional control, patterns of control of metabolic fluxes at the level of enzyme synthesis, and the relevance of specific enzymatic reactions in the control of metabolic fluxes during cell growth. CONCLUSION : This work constitutes a first comprehensive systems biology study on growth-rate control in the eukaryotic cell. The results have direct implications for advanced studies on cell growth, in vivo regulation of metabolic fluxes for comprehensive metabolic engineering, and for the design of genome-scale systems biology models of the eukaryotic cell. [Abstract/Link to Full Text]

Graves JA, Disteche CM
Does gene dosage really matter?
J Biol. 2007;6(1):1.
ABSTRACT : Mechanisms to compensate for dosage differences of genes on sex chromosomes are widespread in animals and have been thought to be critical for viability. However, in birds, compensation is inefficient, implying that for many genes dosage compensation is not critical, and for some genes, dosage differences have even been selected for. [Abstract/Link to Full Text]

Itoh Y, Melamed E, Yang X, Kampf K, Wang S, Yehya N, Van Nas A, Replogle K, Band MR, Clayton DF, Schadt EE, Lusis AJ, Arnold AP
Dosage compensation is less effective in birds than in mammals.
J Biol. 2007;6(1):2.
ABSTRACT : BACKGROUND : In animals with heteromorphic sex chromosomes, dosage compensation of sex-chromosome genes is thought to be critical for species survival. Diverse molecular mechanisms have evolved to effectively balance the expressed dose of X-linked genes between XX and XY animals, and to balance expression of X and autosomal genes. Dosage compensation is not understood in birds, in which females (ZW) and males (ZZ) differ in the number of Z chromosomes. RESULTS : Using microarray analysis, we compared the male:female ratio of expression of sets of Z-linked and autosomal genes in two bird species, zebra finch and chicken, and in two mammalian species, mouse and human. Male:female ratios of expression were significantly higher for Z genes than for autosomal genes in several finch and chicken tissues. In contrast, in mouse and human the male:female ratio of expression of X-linked genes is quite similar to that of autosomal genes, indicating effective dosage compensation even in humans, in which a significant percentage of genes escape X-inactivation. CONCLUSION : Birds represent an unprecedented case in which genes on one sex chromosome are expressed on average at constitutively higher levels in one sex compared with the other. Sex-chromosome dosage compensation is surprisingly ineffective in birds, suggesting that some genomes can do without effective sex-specific sex-chromosome dosage compensation mechanisms. [Abstract/Link to Full Text]

Weitzman JB
Imaging with isotopes: high resolution and quantitation.
J Biol. 2006;5(6):17. [Abstract/Link to Full Text]

Duffner PK
The long term effects of chemotherapy on the central nervous system.
J Biol. 2006;5(7):21.
Cranial radiotherapy is known to have adverse effects on intelligence. A new study shows that chemotherapy is also toxic to the central nervous system, especially to neural progenitor cells and oligodendrocytes. By identifying the cell populations at risk, these results may help explain the neurological problems previously seen after chemotherapy. [Abstract/Link to Full Text]

Dietrich J, Han R, Yang Y, Mayer-Pröschel M, Noble M
CNS progenitor cells and oligodendrocytes are targets of chemotherapeutic agents in vitro and in vivo.
J Biol. 2006;5(7):22.
BACKGROUND: Chemotherapy in cancer patients can be associated with serious short- and long-term adverse neurological effects, such as leukoencephalopathy and cognitive impairment, even when therapy is delivered systemically. The underlying cellular basis for these adverse effects is poorly understood. RESULTS: We found that three mainstream chemotherapeutic agents--carmustine (BCNU), cisplatin, and cytosine arabinoside (cytarabine), representing two DNA cross-linking agents and an antimetabolite, respectively--applied at clinically relevant exposure levels to cultured cells are more toxic for the progenitor cells of the CNS and for nondividing oligodendrocytes than they are for multiple cancer cell lines. Enhancement of cell death and suppression of cell division were seen in vitro and in vivo. When administered systemically in mice, these chemotherapeutic agents were associated with increased cell death and decreased cell division in the subventricular zone, in the dentate gyrus of the hippocampus and in the corpus callosum of the CNS. In some cases, cell division was reduced, and cell death increased, for weeks after drug administration ended. CONCLUSION: Identifying neural populations at risk during any cancer treatment is of great importance in developing means of reducing neurotoxicity and preserving quality of life in long-term survivors. Thus, as well as providing possible explanations for the adverse neurological effects of systemic chemotherapy, the strong correlations between our in vitro and in vivo analyses indicate that the same approaches we used to identify the reported toxicities can also provide rapid in vitro screens for analyzing new therapies and discovering means of achieving selective protection or targeted killing. [Abstract/Link to Full Text]

Williams P
Biological imaging using secondary ions.
J Biol. 2006;5(6):18.
Biological materials are morphologically and chemically complex. A quantitative imaging tool is now available that can produce chemical, and even metabolic, information from morphological features as small as a few nanometers. [Abstract/Link to Full Text]

Lechene C, Hillion F, McMahon G, Benson D, Kleinfeld AM, Kampf JP, Distel D, Luyten Y, Bonventre J, Hentschel D, Park KM, Ito S, Schwartz M, Benichou G, Slodzian G
High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry.
J Biol. 2006;5(6):20.
BACKGROUND: Secondary-ion mass spectrometry (SIMS) is an important tool for investigating isotopic composition in the chemical and materials sciences, but its use in biology has been limited by technical considerations. Multi-isotope imaging mass spectrometry (MIMS), which combines a new generation of SIMS instrument with sophisticated ion optics, labeling with stable isotopes, and quantitative image-analysis software, was developed to study biological materials. RESULTS: The new instrument allows the production of mass images of high lateral resolution (down to 33 nm), as well as the counting or imaging of several isotopes simultaneously. As MIMS can distinguish between ions of very similar mass, such as 12C15N- and 13C14N-, it enables the precise and reproducible measurement of isotope ratios, and thus of the levels of enrichment in specific isotopic labels, within volumes of less than a cubic micrometer. The sensitivity of MIMS is at least 1,000 times that of 14C autoradiography. The depth resolution can be smaller than 1 nm because only a few atomic layers are needed to create an atomic mass image. We illustrate the use of MIMS to image unlabeled mammalian cultured cells and tissue sections; to analyze fatty-acid transport in adipocyte lipid droplets using 13C-oleic acid; to examine nitrogen fixation in bacteria using 15N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic kidney cells using 15N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus using 15N-uridine and 81Br of bromodeoxyuridine or 14C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes 12C, 16O, 14N and 31P; and to track a few 15N-labeled donor spleen cells in the lymph nodes of the host mouse. CONCLUSION: MIMS makes it possible for the first time to both image and quantify molecules labeled with stable or radioactive isotopes within subcellular compartments. [Abstract/Link to Full Text]

Parker J, Johnston LA
The proximate determinants of insect size.
J Biol. 2006;5(5):15.
One of the least understood aspects of animal development--the determination of body size--is currently the subject of intense scrutiny. A new study employs a modeling approach to expose the factors that matter in the control of insect size. [Abstract/Link to Full Text]

Nijhout HF, Davidowitz G, Roff DA
A quantitative analysis of the mechanism that controls body size in Manduca sexta.
J Biol. 2006;5(5):16.
BACKGROUND: Body size is controlled by mechanisms that terminate growth when the individual reaches a species-specific size. In insects, it is a pulse of ecdysone at the end of larval life that causes the larva to stop feeding and growing and initiate metamorphosis. Body size is a quantitative trait, so it is important that the problem of control of body size be analyzed quantitatively. The processes that control the timing of ecdysone secretion in larvae of the moth Manduca sexta are sufficiently well understood that they can be described in a rigorous manner. RESULTS: We develop a quantitative description of the empirical data on body size determination that accurately predicts body size for diverse genetic strains. We show that body size is fully determined by three fundamental parameters: the growth rate, the critical weight (which signals the initiation of juvenile hormone breakdown), and the interval between the critical weight and the secretion of ecdysone. All three parameters are easily measured and differ between genetic strains and environmental conditions. The mathematical description we develop can be used to explain how variables such as growth rate, nutrition, and temperature affect body size. CONCLUSION: Our analysis shows that there is no single locus of control of body size, but that body size is a system property that depends on interactions among the underlying determinants of the three fundamental parameters. A deeper mechanistic understanding of body size will be obtained by research aimed at uncovering the molecular mechanisms that give these three parameters their particular quantitative values. [Abstract/Link to Full Text]

Lloyd AC
Distinct functions for ERKs?
J Biol. 2006;5(5):13.
The Ras/Raf/MEK/ERK signaling pathway is one of the best understood signal routes in cells. Recent studies add complexity to this cascade by indicating that the two ERK kinases, ERK1 (p44ERK1) and ERK2 (p42ERK2), may have distinct functions. [Abstract/Link to Full Text]

Pelech S
Dimerization in protein kinase signaling.
J Biol. 2006;5(5):12.
The closely related mitogen-activated protein kinases ERK1 and ERK2 have now been shown to have opposing roles in Ras-mediated cell proliferation. I propose that dimerization of these highly related protein kinases could underlie these surprising observations and that this could be a common paradigm for widespread regulation of protein phosphorylation by kinase-substrate interactions. [Abstract/Link to Full Text]

Vantaggiato C, Formentini I, Bondanza A, Bonini C, Naldini L, Brambilla R
ERK1 and ERK2 mitogen-activated protein kinases affect Ras-dependent cell signaling differentially.
J Biol. 2006;5(5):14.
BACKGROUND: The mitogen-activated protein (MAP) kinases p44ERK1 and p42ERK2 are crucial components of the regulatory machinery underlying normal and malignant cell proliferation. A currently accepted model maintains that ERK1 and ERK2 are regulated similarly and contribute to intracellular signaling by phosphorylating a largely common subset of substrates, both in the cytosol and in the nucleus. RESULTS: Here, we show that ablation of ERK1 in mouse embryo fibroblasts and NIH 3T3 cells by gene targeting and RNA interference results in an enhancement of ERK2-dependent signaling and in a significant growth advantage. By contrast, knockdown of ERK2 almost completely abolishes normal and Ras-dependent cell proliferation. Ectopic expression of ERK1 but not of ERK2 in NIH 3T3 cells inhibits oncogenic Ras-mediated proliferation and colony formation. These phenotypes are independent of the kinase activity of ERK1, as expression of a catalytically inactive form of ERK1 is equally effective. Finally, ectopic expression of ERK1 but not ERK2 is sufficient to attenuate Ras-dependent tumor formation in nude mice. CONCLUSION: These results reveal an unexpected interplay between ERK1 and ERK2 in transducing Ras-dependent cell signaling and proliferation. Whereas ERK2 seems to have a positive role in controlling normal and Ras-dependent cell proliferation, ERK1 probably affects the overall signaling output of the cell by antagonizing ERK2 activity. [Abstract/Link to Full Text]

Mellor J, DeLisi C
The interaction map of yeast: terra incognita?
J Biol. 2006;5(4):10.
A systematic curation of the literature on Saccharomyces cerevisiae has yielded a comprehensive collection of experimentally observed interactions. This new resource augments current views of the topological structure of yeast's physical and genetic networks, but also reveals that existing studies cover only a fraction of the cell. [Abstract/Link to Full Text]

Reguly T, Breitkreutz A, Boucher L, Breitkreutz BJ, Hon GC, Myers CL, Parsons A, Friesen H, Oughtred R, Tong A, Stark C, Ho Y, Botstein D, Andrews B, Boone C, Troyanskya OG, Ideker T, Dolinski K, Batada NN, Tyers M
Comprehensive curation and analysis of global interaction networks in Saccharomyces cerevisiae.
J Biol. 2006;5(4):11.
BACKGROUND: The study of complex biological networks and prediction of gene function has been enabled by high-throughput (HTP) methods for detection of genetic and protein interactions. Sparse coverage in HTP datasets may, however, distort network properties and confound predictions. Although a vast number of well substantiated interactions are recorded in the scientific literature, these data have not yet been distilled into networks that enable system-level inference. RESULTS: We describe here a comprehensive database of genetic and protein interactions, and associated experimental evidence, for the budding yeast Saccharomyces cerevisiae, as manually curated from over 31,793 abstracts and online publications. This literature-curated (LC) dataset contains 33,311 interactions, on the order of all extant HTP datasets combined. Surprisingly, HTP protein-interaction datasets currently achieve only around 14% coverage of the interactions in the literature. The LC network nevertheless shares attributes with HTP networks, including scale-free connectivity and correlations between interactions, abundance, localization, and expression. We find that essential genes or proteins are enriched for interactions with other essential genes or proteins, suggesting that the global network may be functionally unified. This interconnectivity is supported by a substantial overlap of protein and genetic interactions in the LC dataset. We show that the LC dataset considerably improves the predictive power of network-analysis approaches. The full LC dataset is available at the BioGRID (http://www.thebiogrid.org) and SGD (http://www.yeastgenome.org/) databases. CONCLUSION: Comprehensive datasets of biological interactions derived from the primary literature provide critical benchmarks for HTP methods, augment functional prediction, and reveal system-level attributes of biological networks. [Abstract/Link to Full Text]

Luo S, Nonet ML
Regulators of kinesin involved in polarized trafficking and axon outgrowth.
J Biol. 2006;5(4):8.
Proteins such as UNC-76 that associate with kinesin motors are important in directing neurite extension. A small Caenorhabditis elegans coiled-coil protein, UNC-69, has now been shown to interact with UNC-76 and to be involved in axonal (but not dendritic) transport and outgrowth, as well as synapse formation. [Abstract/Link to Full Text]

Su CW, Tharin S, Jin Y, Wightman B, Spector M, Meili D, Tsung N, Rhiner C, Bourikas D, Stoeckli E, Garriga G, Horvitz HR, Hengartner MO
The short coiled-coil domain-containing protein UNC-69 cooperates with UNC-76 to regulate axonal outgrowth and normal presynaptic organization in Caenorhabditis elegans.
J Biol. 2006;5(4):9.
BACKGROUND: The nematode Caenorhabditis elegans has been used extensively to identify the genetic requirements for proper nervous system development and function. Key to this process is the direction of vesicles to the growing axons and dendrites, which is required for growth-cone extension and synapse formation in the developing neurons. The contribution and mechanism of membrane traffic in neuronal development are not fully understood, however. RESULTS: We show that the C. elegans gene unc-69 is required for axon outgrowth, guidance, fasciculation and normal presynaptic organization. We identify UNC-69 as an evolutionarily conserved 108-amino-acid protein with a short coiled-coil domain. UNC-69 interacts physically with UNC-76, mutations in which produce similar defects to loss of unc-69 function. In addition, a weak reduction-of-function allele, unc-69(ju69), preferentially causes mislocalization of the synaptic vesicle marker synaptobrevin. UNC-69 and UNC-76 colocalize as puncta in neuronal processes and cooperate to regulate axon extension and synapse formation. The chicken UNC-69 homolog is highly expressed in the developing central nervous system, and its inactivation by RNA interference leads to axon guidance defects. CONCLUSION: We have identified a novel protein complex, composed of UNC-69 and UNC-76, which promotes axonal growth and normal presynaptic organization in C. elegans. As both proteins are conserved through evolution, we suggest that the mammalian homologs of UNC-69 and UNC-76 (SCOCO and FEZ, respectively) may function similarly. [Abstract/Link to Full Text]

Miller RH
Building bridges with astrocytes for spinal cord repair.
J Biol. 2006;5(3):6.
Simultaneous suppression of glial scarring and a general enhancement of axonal outgrowth has now been accomplished in an adult rat model of spinal cord transection. Transplantation of a novel astrocyte cell type derived from glial-restricted precursors in vitro raise the eventual possibility of cellular therapy for spinal cord injury. [Abstract/Link to Full Text]

Davies JE, Huang C, Proschel C, Noble M, Mayer-Proschel M, Davies SJ
Astrocytes derived from glial-restricted precursors promote spinal cord repair.
J Biol. 2006;5(3):7.
BACKGROUND: Transplantation of embryonic stem or neural progenitor cells is an attractive strategy for repair of the injured central nervous system. Transplantation of these cells alone to acute spinal cord injuries has not, however, resulted in robust axon regeneration beyond the sites of injury. This may be due to progenitors differentiating to cell types that support axon growth poorly and/or their inability to modify the inhibitory environment of adult central nervous system (CNS) injuries. We reasoned therefore that pre-differentiation of embryonic neural precursors to astrocytes, which are thought to support axon growth in the injured immature CNS, would be more beneficial for CNS repair. RESULTS: Transplantation of astrocytes derived from embryonic glial-restricted precursors (GRPs) promoted robust axon growth and restoration of locomotor function after acute transection injuries of the adult rat spinal cord. Transplantation of GRP-derived astrocytes (GDAs) into dorsal column injuries promoted growth of over 60% of ascending dorsal column axons into the centers of the lesions, with 66% of these axons extending beyond the injury sites. Grid-walk analysis of GDA-transplanted rats with rubrospinal tract injuries revealed significant improvements in locomotor function. GDA transplantation also induced a striking realignment of injured tissue, suppressed initial scarring and rescued axotomized CNS neurons with cut axons from atrophy. In sharp contrast, undifferentiated GRPs failed to suppress scar formation or support axon growth and locomotor recovery. CONCLUSION: Pre-differentiation of glial precursors into GDAs before transplantation into spinal cord injuries leads to significantly improved outcomes over precursor cell transplantation, providing both a novel strategy and a highly effective new cell type for repairing CNS injuries. [Abstract/Link to Full Text]

Urban BC, Todryk S
Malaria pigment paralyzes dendritic cells.
J Biol. 2006 Apr 12;5(2):4.
ABSTRACT : The capacity of malarial infection to suppress the patient's immune responses both to the parasite and to other antigens has long puzzled researchers. A prime suspect, the parasite-produced pigment hemozoin, has now been clearly shown to mediate immunosuppression by inhibiting dendritic cell activity. [Abstract/Link to Full Text]

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]

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Recent Articles in The Journal of Experimental Biology

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Wiltschko W, Freire R, Munro U, Ritz T, Rogers L, Thalau P, Wiltschko R
The magnetic compass of domestic chickens, Gallus gallus.
J Exp Biol. 2007 Jul;210(Pt 13):2300-10.
By directional training, young domestic chickens have been shown to use a magnetic compass; the same method has now been used to analyse the functional characteristics and the physical principles underlying the chickens' magnetic compass. Tests in magnetic fields with different intensities revealed a functional window around the intensity of the local geomagnetic field, with this window extending further towards lower than higher intensities. Testing chickens under monochromatic 465 nm blue and 645 nm red light suggested a wavelength dependence, with orientation possible under blue but not under red light. Exposing chickens to an oscillating field of 1.566 MHz led to disorientation, identifying an underlying radical pair mechanism. Local anesthesia of the upper beak, where iron-rich structures have been described as potential magnetoreceptors, did not affect the performance, suggesting that these receptors are not involved in compass orientation. These findings show obvious parallels to the magnetic compass described for European robins, indicating that chickens and small passerines use the same type of magnetic compass mechanism. This suggests that the avian magnetic compass may have evolved in the common ancestor of all present-day birds to facilitate orientation within the home range. [Abstract/Link to Full Text]

Hama N, Tsuchida Y, Takahata M
Behavioral context-dependent modulation of descending statocyst pathways during free walking, as revealed by optical telemetry in crayfish.
J Exp Biol. 2007 Jun;210(Pt 12):2199-211.
Crustacean posture control is based on a complex interaction between the statocyst input and other sensory inputs as well as the animal's behavioral context. We examined the effects of behavioral condition on the activity of descending statocyst pathways using an optical telemetry system that allowed underwater recording of neuronal signals from freely behaving crayfish. A functionally identified statocyst-driven interneuron that directionally responded to body tilting without a footboard and to tilting of the footboard was found to show complicated responses depending upon the ongoing behavior of the animal when it freely walked around in water on the aquarium floor. The spike firing frequency of the interneuron increased significantly during walking. When the animal stood or walked on the tilted floor, the interneuron activity represented the tilt angle and direction if the abdomen was actively flexed, but not if it was extended. Two other statocyst-driven descending interneurons were found to be affected differently by the animal's behavioral condition: the spike activity of one interneuron increased during walking, but its directional response on the tilted floor was completely absent during abdominal posture movements, whereas that of another interneuron was enhanced during abdominal extension only, representing the tilt angle and direction. The results obtained in this study provide the first experimental demonstration that crustacean postural control under natural conditions is dependent on very fine aspects of the animal's locomotor behavioral context, suggesting far more complex control mechanisms than those expected from the experimental data obtained in isolated and fixed animals. [Abstract/Link to Full Text]

Guadagnoli JA, Tobita K, Reiber CL
Assessment of the pressure-volume relationship of the single ventricle of the grass shrimp, Palaemonetes pugio.
J Exp Biol. 2007 Jun;210(Pt 12):2192-8.
The ventricular pressure-volume (PV) relationship has been used extensively to study the mechanics and energetics in multi-chambered hearts of closed circulatory system vertebrates. In the current study we applied the use of PV loops in the assessment of cardiac mechanics and energetics in the single ventricle of a decapod crustacean possessing an open circulatory system. Anatomical differences between multi-and single-chambered hearts include multiple ostia entering and valved multiple arterial systems exiting the ventricle, and the neurogenic origin of the heartbeat in decapod crustaceans. However, the microscopic architecture and excitation-contraction coupling events are similar in both systems. Ventricular pressure and area were obtained independently and integrated into pressure-area loops. Area was then converted to volume to generate PV loops. Based on the PV loops generated in this study, the ventricle of Palaemonetes pugio processes the same primary phases of the cardiac cycle as ventricles from the multi-chambered hearts of vertebrates: (1) isovolumic contraction, (2) ventricular emptying, (3) isovolumic relaxation and (4) ventricular filling. The area enclosed by the PV loop provides a measure of stroke work and when multiplied by heart rate provides an assessment of cardiac work. This initial examination of PV loops from a single-ventricle decapod crustacean demonstrates the utility of this technique to further elucidate the cardiac mechanics and energetics of this system, and in particular during times of physiological stress. [Abstract/Link to Full Text]

Wu G, Yang Y, Zeng L
Kinematics, hydrodynamics and energetic advantages of burst-and-coast swimming of koi carps (Cyprinus carpio koi).
J Exp Biol. 2007 Jun;210(Pt 12):2181-91.
Koi carps frequently swim in burst-and-coast style, which consists of a burst phase and a coast phase. We quantify the swimming kinematics and the flow patterns generated by the carps in burst-and-coast swimming. In the burst phase, the carps burst in two modes: in the first, the tail beats for at least one cycle (multiple tail-beat mode); in the second, the tail beats for only a half-cycle (half tail-beat mode). The carp generates a vortex ring in each half-cycle beat. The vortex rings generated during bursting in multiple tail-beat mode form a linked chain, but only one vortex ring is generated in half tail-beat mode. The wake morphologies, such as momentum angle and jet angle, also show much difference between the two modes. In the burst phase, the kinematic data and the impulse obtained from the wake are linked to obtain the drag coefficient (C(d,burst) approximately 0.242). In the coast phase, drag coefficient (C(d,coast) approximately 0.060) is estimated from swimming speed deceleration. Our estimation suggests that nearly 45% of energy is saved when burst-and-coast swimming is used by the koi carps compared with steady swimming at the same mean speed. [Abstract/Link to Full Text]

Rottenberg H
Exceptional longevity in songbirds is associated with high rates of evolution of cytochrome b, suggesting selection for reduced generation of free radicals.
J Exp Biol. 2007 Jun;210(Pt 12):2170-80.
In animals, longevity (maximal lifespan) is inversely related to mass-specific basal metabolic rates. However, contrary to expectation, in several mammalian taxa, exceptional longevity is associated with high basal metabolic rate, and also fast evolution of mtDNA-coded proteins. The association of these traits was suggested to result from adaptive selection of mutations in mtDNA-coded proteins, which accelerates basal respiration, thus inhibiting the generation of reactive oxygen species that constrain longevity. In birds, all the genera with high rate of cytochrome b evolution are songbirds (oscines). Within the songbirds group, both longevity residuals and lifetime expenditure of energy are positively correlated with the rate of cytochrome b evolution. Moreover, within the large songbirds family Fringillidae (true finches) mass-specific basal metabolic rates, longevity, longevity residuals and lifetime expenditure of energy are all positively correlated with the rate of evolution of cytochrome b. In Serinus, a genus of finches (canaries) that exhibits the highest rate of cytochrome b evolution, and the highest values of exceptional longevity and lifetime expenditure of energy in all birds, many of the substitutions in cytochrome b are clustered around Qi, a ubiquinone binding site adjacent to the mitochondrial matrix, apparently selected to increase the rate of ubiquinone reduction. We therefore suggest that, in songbirds, the accelerated evolution of cytochrome b involved selection of mutations that reduce the generation of reactive oxygen species, thus contributing to the evolution of exceptional longevity, and possibly also exceptional long-term memory, which is necessary for learning songs. [Abstract/Link to Full Text]

Ruther J, Stahl LM, Steiner S, Garbe LA, Tolasch T
A male sex pheromone in a parasitic wasp and control of the behavioral response by the female's mating status.
J Exp Biol. 2007 Jun;210(Pt 12):2163-9.
Male insects may increase their chance of successful reproduction by releasing pheromones that attract females or elicit sexual acceptance. In parasitic wasps, male pheromones have been suggested for a few species but no chemicals have been identified so far. Here we report the first identification of a male sex pheromone in parasitic Hymenoptera. In abdomens of male jewel wasps, Nasonia vitripennis Walker, we found a mixture of (4R,5R)- and (4R,5S)-5-hydroxy-4-decanolide (HDL), which was released intermittently and attracted virgin females, but no males, in an olfactometer bioassay. However, only a few minutes after copulation mated females avoided the male-derived pheromone. Neither preference nor avoidance was shown by mated females after 24 h and even after they had been allowed to oviposit for 6 days. Nasonia vitripennis females normally mate only once. Thus, their variable response to the sex attractant depending on the mating status makes sense from an evolutionary perspective. Firstly, it increases the chance of virgins to be inseminated. Secondly, by terminating the response or even avoiding the male pheromone, mated females decrease the probability of encountering males and being disturbed by their courtship activities when searching for new oviposition sites. [Abstract/Link to Full Text]

Welch KC, Suarez RK
Oxidation rate and turnover of ingested sugar in hovering Anna's (Calypte anna) and rufous (Selasphorus rufus) hummingbirds.
J Exp Biol. 2007 Jun;210(Pt 12):2154-62.
Hummingbirds obtain most of their dietary calories from floral nectar ingested during hovering flight. Despite the importance of dietary sugar to hummingbird metabolism, the turnover of newly ingested carbon in the pool of actively metabolized substrates has not been adequately characterized in hovering hummingbirds. By combining respirometry with stable carbon isotope analysis of respired breath, we show that in rufous (Selasphorus rufus) and Anna's (Calypte anna) hummingbirds at high foraging frequencies, utilization of newly ingested sugars increased over a period of 30-45 min until it accounted for virtually 100% of the fuel oxidized. This newly ingested sugar disappears from the actively metabolized pool of substrates over a similar time course. These results demonstrate that turnover of carbon in the pool of actively metabolized substrates is rapid; carbon from ingested sucrose is available for oxidation for 30-45 min before being cleared. By monitoring expired CO2 for the appearance and disappearance of the signature characteristic of newly ingested sugar and then calculating energy budgets using video recordings of hummingbird activity, we estimated the proportion of recently ingested sugar used to fuel ongoing metabolism as well as the proportion devoted to energy storage. Consistent differences between species in the percentage of ingested cane sugar oxidized during the 2 h experimental periods suggest that individuals of each species adopted energy intake patterns appropriate to their needs. This approach provides a means by which to examine the partitioning of dietary carbon intake between energy expenditure and storage using non-invasive, field-compatible techniques. [Abstract/Link to Full Text]

Welch KC, Altshuler DL, Suarez RK
Oxygen consumption rates in hovering hummingbirds reflect substrate-dependent differences in P/O ratios: carbohydrate as a 'premium fuel'.
J Exp Biol. 2007 Jun;210(Pt 12):2146-53.
The stoichiometric relationship of ATP production to oxygen consumption, i.e. the P/O ratio, varies depending on the nature of the metabolic substrate used. The latest estimates reveal a P/O ratio approximately 15% higher when glucose is oxidized compared with fatty acid oxidation. Because the energy required to produce aerodynamic lift for hovering is independent of the metabolic fuel oxidized, we hypothesized that the rate of oxygen consumption, VO2, should decline as the respiratory quotient, RQ (VCO2/VO2), increases from 0.71 to 1.0 as hummingbirds transition from a fasted to a fed state. Here, we show that hovering VO2 values in rufous (Selasphorus rufus) and Anna's hummingbirds (Calypte anna) are significantly greater when fats are metabolized (RQ=0.71) than when carbohydrates are used (RQ=1.0). Because hummingbirds gained mass during our experiments, making mass a confounding variable, we estimated VO2 per unit mechanical power output. Expressed in this way, the difference in VO2 when hummingbirds display an RQ=0.71 (fasted) and an RQ=1.0 (fed) is between 16 and 18%, depending on whether zero or perfect elastic energy storage is assumed. These values closely match theoretical expectations, indicating that a combination of mechanical power estimates and ;indirect calorimetry', i.e. the measurement of rates of gas exchange, enables precise estimates of ATP turnover and metabolic flux rates in vivo. The requirement for less oxygen when oxidizing carbohydrate suggests that carbohydrate oxidation may facilitate hovering flight in hummingbirds at high altitude. [Abstract/Link to Full Text]

Werneke SW, Swann C, Farquharson LA, Hamilton KS, Smith AM
The role of metals in molluscan adhesive gels.
J Exp Biol. 2007 Jun;210(Pt 12):2137-45.
Several gastropod molluscs produce glues that are interesting because they are dilute gels and yet they produce strong adhesion. Specific glue proteins have been identified that play a central role in this adhesion, possibly by crosslinking other polymers in the gel. This study investigates the role of metals in the action of these glue proteins. Atomic absorption spectrometry showed that glue from the slug Arion subfuscus contains substantial quantities of zinc (46+/-7 p.p.m. and 189+/-80 p.p.m. in two different sets of experiments) and also iron, copper and manganese (2-7 p.p.m.). Iron-specific staining demonstrates that iron is bound specifically to the 15 kDa glue protein. Several approaches were used to show that these metals have important functional effects. Adding iron or copper to dissolved glue causes the proteins to precipitate rapidly, although zinc has no effect. Removing iron and related transition metals with a chelator during secretion of the glue causes a sixfold increase in the solubility of the glue. Once the glue has set, however, removing these metals has no effect. Finally, the gel-stiffening activity of the glue proteins was measured in the presence and absence of the chelator. The chelator eliminated the gel-stiffening effect of the proteins, suggesting that transition metals were necessary for the proteins to act on the gel. Thus, the glue contains transition metals and these metals play an essential role in glue function. [Abstract/Link to Full Text]

Graham P, Durier V, Collett T
The co-activation of snapshot memories in wood ants.
J Exp Biol. 2007 Jun;210(Pt 12):2128-36.
Insects can guide themselves along a familiar route to a familiar place by retrieving and using visual snapshots that they have stored both along the route and at their destination and moving so that their current views match the target snapshots. To learn more about the matching process, we have investigated the interaction of snapshots by engineering a situation in which ants simultaneously retrieve two sets of memories. Ants were trained from a fixed start position to feed in one site, after which the feeder was switched to a new one. It could take up to 30 trials after the switch before the ants headed directly to the new food site. We suppose that during this transition phase ants retrieve memories appropriate for both sites. We compared the ants' behaviour for two different sized separations between feeder sites. When the sites are relatively close together, the initial headings of the ants' paths rotated gradually from aiming directly at the first food site to aiming at the second food site, suggesting that ants' paths are controlled by the weighted average of two simultaneously activated snapshot attractors. By contrast, when the food sites were further apart, initial headings switched abruptly between the two sites - ants either headed for food site 1 or for food site 2. We show that these differences in transition behaviour can be simulated by the co-activation of snapshot attractors of restricted spatial extent, such that features encoded in a snapshot are only recognised if they occur within a limited retinal distance of the stored position of the feature. [Abstract/Link to Full Text]

Khokhlova IS, Hovhanyan A, Krasnov BR, Degen AA
Reproductive success in two species of desert fleas: density dependence and host effect.
J Exp Biol. 2007 Jun;210(Pt 12):2121-7.
We tested the hypothesis that a negative fitness-density relationship exists in haematophagous ectoparasites. We studied the effect of flea density on the number of blood meals necessary for starting oviposition and egg production in Xenopsylla conformis and Xenopsylla ramesis when exploiting two rodent hosts, Meriones crassus and Gerbillus dasyurus. The number of blood meals taken by a flea prior to first oviposition was similar in both flea species but was dependent on flea density and differed significantly between hosts. When parasitizing G. dasyurus, females of both flea species required a similar number of blood meals to start oviposition, independent of density. By contrast, fleas on M. crassus at higher densities needed less blood meals than at lower densities. Egg production of female fleas differed significantly between flea and host species and was affected by flea density. X. ramesis produced more eggs than X. conformis. When parasitizing G. dasyurus, density did not affect the number of eggs produced by X. conformis, however, when on M. crassus, this flea produced significantly less eggs at the highest density. The number of eggs produced by X. ramesis at high densities was significantly lower than at low densities when it parasitized either host species. Results of this study demonstrated that reproductive success of fleas was density dependent and, in general, decreased with an increase in density. However, the effect of density on reproductive performance was manifested differently on different host species. [Abstract/Link to Full Text]

Tse WK, Au DW, Wong CK
Effect of osmotic shrinkage and hormones on the expression of Na+/H+ exchanger-1, Na+/K+/2Cl- cotransporter and Na+/K+ -ATPase in gill pavement cells of freshwater adapted Japanese eel, Anguilla japonica.
J Exp Biol. 2007 Jun;210(Pt 12):2113-20.
It is well-known that gill epithelial cells are important in fish osmoregulation. However, studies on the effect of osmotic stress on the direct cellular responses of the gill epithelial cells are limited. In this paper, we aimed to determine the effects of osmotic hypertonicity, hormones and cellular signaling molecules on the expression of ion transporters in the cultured primary freshwater pavement cells (PVCs), prepared from freshwater-adapted eels (Anguilla japonica). Our data demonstrated that the hypertonic (500 mOsmol l(-1)) treatment of the isolated PVCs induced cell shrinkage, followed by regulatory volume increase (RVI). Application of blockers (i.e. ouabain, bumetanide and EIPA) demonstrated that Na+/K+ -ATPase, Na+/K+/2Cl- cotransporter (NKCC) and Na+/H+ exchanger-1 (NHE-1) were involved in RVI. Western blot analysis of the hypertonic-treated cells revealed a significant induction of NHE-1, NKCC and, alpha and beta subunits of Na+/K+ -ATPase. In nonshrunken cultured PVCs, we found that dexamethasone and dibutyryl cAMP treatments significantly stimulated the expression levels of the three ion transporters. Both prolactin and insulin-like growth factor-1, can only induce the expression of NKCC. The effect of thyroid hormone (T3) and dibutyryl cGMP was negligible. In this study, the induction of ion transporter expression was found to be post-transcriptionally regulated as no significant change in mRNA levels was detected. This observation implies that the regulation is rapid and is probably induced via nongenomic actions. [Abstract/Link to Full Text]

Vasconcelos RO, Amorim MC, Ladich F
Effects of ship noise on the detectability of communication signals in the Lusitanian toadfish.
J Exp Biol. 2007 Jun;210(Pt 12):2104-12.
Underwater noise pollution is an increasing environmental problem which might affect communication, behaviour, fitness and consequently species' survival. The most common anthropogenic noises in aquatic habitats derive from shipping. In the present study we investigated the implications of noise pollution from a ship on the sound detectability, namely of conspecific vocalizations in the Lusitanian toadfish, Halobatrachus didactylus. Ambient and ferry-boat noises were recorded in the Tagus River estuary (Portugal), as well as toadfish sounds, and their sound pressure levels determined. Hearing sensitivities were measured under quiet lab conditions and in the presence of these masking noises at levels encountered in the field, using the auditory evoked potentials (AEP) recording technique. The Lusitanian toadfish is a hearing generalist, with best hearing sensitivity at low frequencies between 50 and 200 Hz (below 100 dB re. 1 microPa). Under ambient noise conditions, hearing was only slightly masked at lower frequencies. In the presence of ship noise, auditory thresholds increased considerably, by up to 36 dB, at most frequencies tested. This is mainly because the main energies of ferry-boat noise were within the most sensitive hearing range of this species. Comparisons between masked audiograms and sound spectra of the toadfish's mating and agonistic vocalizations revealed that ship noise decreased the ability to detect conspecific acoustic signals. This study provides the first evidence that fishes' auditory sensitivity can be impaired by ship noise and that acoustic communication, which is essential during agonistic encounters and mate attraction, might be restricted in coastal environments altered by human activity. [Abstract/Link to Full Text]

Seternes T, Tonheim TC, Lřvoll M, Břgwald J, Dalmo RA
Specific endocytosis and degradation of naked DNA in the endocardial cells of cod (Gadus morhua L.).
J Exp Biol. 2007 Jun;210(Pt 12):2091-103.
DNA vaccines are administered in the form of plasmid DNA (pDNA) carrying a strong promoter and the gene of interest. In this study we investigated the tissue distribution, cellular uptake and the fate of intravenously (i.v.) and intramuscularly (i.m.) injected pDNA in Atlantic cod (Gadus morhua L.). The anatomical distribution of pDNA was determined using both morphological and radiotracing methods. Cellular uptake and receptor specificity were studied in cultures of cod atrial endocardial endothelial cells (aEEC) and head kidney leukocytes. The short-term fate of the endocytosed pDNA in vivo and in vitro was investigated by Southern blot. Expression of the pDNA (R70pRomiLuc)-derived gene was investigated in cod tissues and cultures of cod aEEC by means of real-time RT-PCR and luciferase activity assay. 125I-labelled pDNA was rapidly eliminated from the blood by the aEEC of the cod heart atrium and ventricle. Co-injection of trace amounts of 125I-labelled pDNA with excess amounts of non-labelled pDNA or formaldehyde-treated albumin (FSA), a ligand for the cod EEC scavenger receptor, significantly inhibited the accumulation of the radiotracer in the heart. The organ to blood ratio of radioactivity after inhibition of the cod EEC scavenger receptor demonstrated that the radioactivity not taken up by the EEC remained in the blood. Fluorescence microscopy of tissue sections from cod injected with fluorescein-labelled pDNA confirmed intracellular uptake of pDNA by the endocardial cells of the atrium and ventricle. In purified cultures of cod aEEC the fluorescein-labelled pDNA was taken up in structures reminiscent of endosomal/lysosomal vesicles. Uptake of 125I-labelled pDNA in cultures of cod aEEC was specific. Incubation of cultures with 125I-labelled pDNA together with excess amounts of FSA and fucoidan, which are molecules also known to bind to the scavenger receptors, reduced the uptake of the pDNA by at least 70%. Mannan, a ligand for the mannose receptor, did not inhibit the uptake of 125I-labelled pDNA. Despite, low uptake of 125I-fluorescein-pDNA in the kidney of the cod, the uptake of pDNA in cultured cod head kidney leukocytes was significant. Southern blot analysis of cod tissues after injection of pDNA and culture of aEEC given 10 microg pDNA per 10(6) cells demonstrated the presence of degradation products in tissues and in the cell cultures. Real-time RT-PCR studies showed expression of luciferase mRNA only at the injection site 168 h after injection. Neither expression of luciferase mRNA nor luciferase activity was present in cod aEEC incubated for 48 h with 10 microg pDNA. These results suggest that the EEC are very important for removal of blood borne pDNA in cod and that the uptake by these cells was mediated in a scavenger-receptor-like manner. Uptake of pDNA by head kidney leukocytes was only observed in vitro. The endocytosed DNA was subjected to intracellular degradation and was not expressed by the cod EEC. Despite the low amount of radioactivity found in the head kidney after i.v. injection of 125I-labelled pDNA, the head kidney leukocytes seem to have a high capacity for uptake of 125I-labelled pDNA in vitro. [Abstract/Link to Full Text]

Wittenberg JB, Wittenberg BA
Myoglobin-enhanced oxygen delivery to isolated cardiac mitochondria.
J Exp Biol. 2007 Jun;210(Pt 12):2082-90.
The heart, red skeletal muscles and the nitrogen-fixing legume root nodule function in steady states of high oxygen influx, partial oxygenation of cytoplasmic myoglobin or leghemoglobin and correspondingly low oxygen partial pressure. Here, we ask: what conditions are required at the surface of actively respiring, state III, tightly coupled mitochondria to enhance oxygen flow to cytochrome oxidase? Pigeon heart mitochondria were isolated with minimal damage to the outer mitochondrial membrane and were incubated at low oxygen pressures, where respiration is oxygen limited, with solutions of each of six monomeric hemoglobins with widely divergent kinetics and equilibria in their reactions with oxygen: Busycon myoglobin, horse myoglobin, Lucina hemoglobins I and II, soybean leghemoglobin c and Gasterophilus hemoglobin. Each augments oxygen uptake. The declining fractional saturation of each hemoglobin with oxygen was monitored spectrophotometrically as mitochondrial respiration depleted the oxygen; the oxygen partial pressure at half-maximal rate of oxygen uptake was similar for each hemoglobin, supporting the conclusion that the hemoglobins did not interact with the mitochondrial surface in oxygen delivery. The oxygen pressure required to support state III mitochondrial oxygen uptake, 0.005 kPa (0.04 torr), is small compared with that obtained in the sarcoplasm and at the mitochondrial surface of the working heart, 0.32 kPa (2.4 torr). We conclude that, in normal steady states of contraction of the myoglobin-containing heart, oxygen utilization by mitochondrial cytochrome oxidase is not limited by oxygen availability. [Abstract/Link to Full Text]

Jayasundara N, Towle DW, Weihrauch D, Spanings-Pierrot C
Gill-specific transcriptional regulation of Na+/K+ -ATPase alpha-subunit in the euryhaline shore crab Pachygrapsus marmoratus: sequence variants and promoter structure.
J Exp Biol. 2007 Jun;210(Pt 12):2070-81.
The sodium pump (Na+/K+ -ATPase) has been implicated in osmoregulatory ion transport in many aquatic animals. In the euryhaline hyper-hypoosmoregulating shore crab Pachygrapsus marmoratus, induction of Na+/K+ -ATPase alpha-subunit mRNA varies between gills in response to osmotic stress. Following transfer of crabs from normal seawater (36 per thousand salinity) to diluted seawater (10 per thousand), a condition in which gills exhibit net ion uptake, alpha-subunit mRNA expression is upregulated in all tested gills, albeit with differing time courses. By contrast, following transfer from seawater to hypertonic (45 per thousand) seawater, a condition in which the animal is excreting ions, alpha-subunit mRNA is induced primarily in gill no. 7 (nine in total), suggesting that this gill may be associated specifically with ion excretion in P. marmoratus. Full-length sequencing of alpha-subunit cDNA revealed the existence of two isoforms differing only in the inclusion of an 81-nucleotide segment within the N-terminal open reading frame of the long (D) form in comparison to the short (C) form. The 81-nucleotide segment encodes a 14-3-3 protein binding site that may facilitate movement of the alpha-subunit protein between intracellular compartments and the plasma membrane. mRNA expression of the two forms followed similar patterns upon salinity transfer. Genomic DNA sequencing of the putative promoter region of the alpha-subunit gene demonstrated a spectrum of predicted transcription factor binding sites that are likely associated with the complex expression pattern observed among gills following osmotic stress. [Abstract/Link to Full Text]

Vincent SE, Moon BR, Herrel A, Kley NJ
Are ontogenetic shifts in diet linked to shifts in feeding mechanics? Scaling of the feeding apparatus in the banded watersnake Nerodia fasciata.
J Exp Biol. 2007 Jun;210(Pt 12):2057-69.
The effects of size on animal behaviour, ecology, and physiology are widespread. Theoretical models have been developed to predict how animal form, function, and performance should change with increasing size. Yet, numerous animals undergo dramatic shifts in ecology (e.g. habitat use, diet) that may directly influence the functioning and presumably the scaling of the musculoskeletal system. For example, previous studies have shown that banded watersnakes (Nerodia fasciata) switch from fish prey as juveniles to frog prey as adults, and that fish and frogs represent functionally distinct prey types to watersnakes. We therefore tested whether this ontogenetic shift in diet was coupled to changes in the scaling patterns of the cranial musculoskeletal system in an ontogenetic size series (70-600 mm snout-vent length) of banded watersnakes. We found that all cranial bones and gape size exhibited significant negative allometry, whereas the muscle physiological cross-sectional area (pCSAs) scaled either isometrically or with positive allometry against snout-vent length. By contrast, we found that gape size, most cranial bones, and muscle pCSAs exhibited highly significant positive allometry against head length. Furthermore, the mechanical advantage of the jaw-closing lever system remained constant over ontogeny. Overall, these cranial allometries should enable watersnakes to meet the functional requirements of switching from fusiform fish to bulky frog prey. However, recent studies have reported highly similar allometries in a wide diversity of vertebrate taxa, suggesting that positive allometry within the cranial musculoskeletal system may actually be a general characteristic of vertebrates. [Abstract/Link to Full Text]

Belzile O, Gulemetova R, Kinkead R
Effects of medullary Raphé stimulation on fictive lung ventilation during development in Rana catesbeiana.
J Exp Biol. 2007 Jun;210(Pt 12):2046-56.
To better understand serotonergic modulation of air breathing during bullfrog development, we measured changes in fictive lung ventilation frequency associated with focal stimulation of the rostral region of the medullary Raphé neurons. Electrical (3 to 33 Hz) and chemical (glutamate microinjections; 0.5 mol l(-1), 0.3-10 nl) activation of Raphé neurons was performed in brainstem preparations from three developmental stages (pre- and metamorphic tadpoles and adult frogs). Fictive lung ventilation was recorded extracelluarly from the Vth and Xth cranial nerves. Electrical stimulation of Raphé neurons caused a frequency-dependent increase in lung burst frequency in pre-metamorphic tadpoles only. In metamorphic tadpoles, an increase in fictive lung ventilation was observed at 20 Hz only. Electrical stimulation had no effect in preparations from adult frogs. Glutamate microinjections elicited similar responses as a lung burst frequency increase was observed in the pre-metamorphic group only. Regardless of the stimulation technique used, the increase in fictive lung ventilation was attenuated by the selective 5-HT3 antagonist tropisetron (5-20 micromol l(-1)). Results from immunohistochemical analysis of the Raphé region stimulated do not correlate with functional data as the number of 5-HT immunoreactive neurons within this region increases during development. We conclude that, in this preparation, stimulation of lung ventilation by the medullary Raphé is restricted to early (pre-metamorphic) stages. [Abstract/Link to Full Text]

Starck JM, Cruz-Neto AP, Abe AS
Physiological and morphological responses to feeding in broad-nosed caiman (Caiman latirostris).
J Exp Biol. 2007 Jun;210(Pt 12):2033-45.
Broad nosed caiman are ectotherm sauropsids that naturally experience long fasting intervals. We have studied the postprandial responses by measuring oxygen consumption using respirometry, the size changes of the duodenum, the distal small intestine, and the liver, using repeated non-invasive ultrasonography, and by investigating structural changes on the level of tissues and cells by using light- and electron microscopy. The caimans showed the same rapid and reversible changes of organ size and identical histological features, down to the ultrastructure level, as previously described for other ectothermic sauropsids. We found a configuration change of the mucosa epithelium from pseudostratified during fasting to single layered during digestion, in association with hypertrophy of enterocytes by loading them with lipid droplets. Similar patterns were also found for the hepatocytes of the liver. By placing the results of our study in comparative relationship and by utilizing the phylogenetic bracket of crocodiles, birds and squamates, we suggest that the observed features are plesiomorphic characters of sauropsids. By extending the comparison to anurans, we suggest that morphological and physiological adjustments to feeding and fasting described here may have been a character of early tetrapods. In conclusion, we suggest that the ability to tolerate long fasting intervals and then swallow a single large meal as described for many sit-an-wait foraging sauropsids is a functional feature that was already present in ancestral tetrapods. [Abstract/Link to Full Text]

Collett TS, Graham P, Harris RA
Novel landmark-guided routes in ants.
J Exp Biol. 2007 Jun;210(Pt 12):2025-32.
We review studies in which ants familiar with fixed routes between their nest and a feeding site are displaced from one of these destinations to an unfamiliar site away from the route. Ants can reach their goal from such novel release sites guided by distant landmarks. We suggest that an ant's ability to take such novel landmark-guided routes after displacement is a by-product of the robustness of normal route-following and is unlikely to reflect the ant's use of a map-like knowledge of its surroundings. [Abstract/Link to Full Text]

Weibel ER
In memoriam Knut Schmidt-Nielsen. 24 September 1915 - 25 January 2007.
J Exp Biol. 2007 Apr;210(Pt 8):1299-301. [Abstract/Link to Full Text]

Vaanholt LM, De Jong B, Garland T, Daan S, Visser GH
Behavioural and physiological responses to increased foraging effort in male mice.
J Exp Biol. 2007 Jun;210(Pt 11):2013-24.
Free-living animals must forage for food and hence may face energetic constraints imposed by their natural environmental conditions (e.g. ambient temperature, food availability). Simulating the variation in such constraints, we have experimentally manipulated the rate of work (wheel running) mice must do to obtain their food, and studied the ensuing behavioural and physiological responses. This was done with a line of mice selectively bred for high spontaneous wheel running and a randomly bred control line that vary in the amount of baseline wheel-running activity. We first determined the maximum workload for each individual. The maximum workload animals could engage in was around 23 km d(-1) in both control and activity-selected mice, and was not associated with baseline wheel-running activity. We then kept mice at 90% of their individual maximum and measured several physiological and behavioural traits. At this high workload, mice increased wheel-running activity from an average of 10 to 20 km d(-1), and decreased food intake and body mass by approximately 20%. Mass-specific resting metabolic rate strongly decreased from 1.43 to 0.98 kJ g(-1) d(-1), whereas daily energy expenditure slightly increased from 2.09 to 2.25 kJ g(-1) d(-1). Costs of running decreased from 2.3 to 1.6 kJ km(-1) between baseline and workload conditions. At high workloads, animals were in a negative energy balance, resulting in a sharp reduction in fat mass as well as a slight decrease in dry lean mass. In addition, corticosterone levels increased, and body temperature was extremely low in some animals at high workloads. When challenged to work for food, mice thus show significant physiological and behavioural adjustments. [Abstract/Link to Full Text]

de Heij ME, van der Graaf AJ, Hafner D, Tinbergen JM
Metabolic rate of nocturnal incubation in female great tits, Parus major, in relation to clutch size measured in a natural environment.
J Exp Biol. 2007 Jun;210(Pt 11):2006-12.
To study the energetic costs of incubation in relation to clutch size, clutch sizes were manipulated and the metabolic rate of female great tits, Parus major (Linnaeus), during nocturnal incubation (MR(inc)) was measured using mobile oxygen analysers. Individuals were measured on consecutive nights while incubating their own or manipulated clutches. The experiment was performed under field conditions in order to place possible effects of clutch size manipulation within the context of other factors explaining variation in MR(inc). Females spent more energy when incubating enlarged clutches as compared with controls (6-10% more energy for three additional eggs) but did not spend significantly less energy when incubating reduced clutches. MR(inc) was strongly negatively related to ambient temperature. The effect of clutch enlargement is consistent with previous studies whereas the absence of an effect of clutch reduction is not. The small effect of clutch enlargement on MR(inc) highlights the need for further studies to include measurements of daily energy expenditure in order to judge how important energy expenditure can be in explaining fitness consequences of incubating experimentally enlarged clutches. [Abstract/Link to Full Text]

Nespolo RF, Franco M
Whole-animal metabolic rate is a repeatable trait: a meta-analysis.
J Exp Biol. 2007 Jun;210(Pt 11):2000-5.
Repeatability studies are gaining considerable interest among physiological ecologists, particularly in traits affected by high environmental/residual variance, such as whole-animal metabolic rate (MR). The original definition of repeatability, known as the intraclass correlation coefficient, is computed from the components of variance obtained in a one-way ANOVA on several individuals from which two or more measurements are performed. An alternative estimation of repeatability, popular among physiological ecologists, is the Pearson product-moment correlation between two consecutive measurements. However, despite the more than 30 studies reporting repeatability of MR, so far there is not a definite synthesis indicating: (1) whether repeatability changes in different types of animals; (2) whether some kinds of metabolism are more repeatable than others; and most important, (3) whether metabolic rate is significantly repeatable. We performed a meta-analysis to address these questions, as well as to explore the historical trend in repeatability studies. Our results show that metabolic rate is significantly repeatable and its effect size is not statistically affected by any of the mentioned factors (i.e. repeatability of MR does not change in different species, type of metabolism, time between measurements, and number of individuals). The cumulative meta-analysis revealed that repeatability studies in MR have already reached an asymptotical effect size with no further change either in its magnitude and/or variance (i.e. additional studies will not contribute significantly to the estimator). There was no evidence of strong publication bias. [Abstract/Link to Full Text]

Matsumura K, Matsunaga S, Fusetani N
Phosphatidylcholine profile-mediated group recognition in catfish.
J Exp Biol. 2007 Jun;210(Pt 11):1992-9.
Animal groups are integrated by emission of discrete signals from members, so-called social signals, which have evolved for each species. Among communication signals, chemical signals play an important role for recognition of group membership. The catfish Plotosus lineatus forms a dense school immediately after hatching, and school recognition is under the control of chemical signals emitted by the school members. The key substance(s) governing this recognition are deduced to be a mixture of phosphatidylcholines (PC). To substantiate this hypothesis that a mixture of PC molecular species functions as recognition of school-specific odor, we examined the ability of P. lineatus to discriminate between familiar and unfamiliar PCs. P. lineatus responded only to PCs from a familiar school, and not to those from unfamiliar schools. PC molecular species were then analyzed by quantitative high performance liquid chromatography, which resulted in not only a complex mixture of PC molecular species, but also school-specific PC profiles. Furthermore, multivariate analysis of the quantified PC peaks revealed the presence of various PC profiles. Finally, we showed that the modification of PC profiles disrupts the recognition of school odor in P. lineatus. Therefore, we conclude that the recognition of school odor in P. lineatus is governed by school-specific PC profiles. [Abstract/Link to Full Text]

Santini MS, Ronderos JR
Allatotropin-like peptide released by Malpighian tubules induces hindgut activity associated with diuresis in the Chagas disease vector Triatoma infestans (Klug).
J Exp Biol. 2007 Jun;210(Pt 11):1986-91.
Haematophagous insects incorporate a large amount of blood with each meal, producing a big quantity of urine in a few hours to eliminate the excess water and Na(+). Malpighian tubules (MTs) have traditionally been seen as a system that responds to neuroendocrine stimulus. In a related paper, we demonstrated that MTs of Triatoma infestans produce an autonomous endocrine secretion of an allatotropin-like (AT-like) peptide. In the present study, we report a myostimulatory activity of AT at the level of the hindgut (HG), associated with endocrine mechanisms regulating post-prandial diuresis. Allatotropin induced an increase in frequency and intensity of peristaltic contractions at the level of the HG. The release of the HG content in MTs-HG in vitro preparations undergoing an osmotic shock occurred at different times, depending on the number of MTs present, and there was no release in treatments without MTs. The application of an AT-antiserum to MTs-HG preparations undergoing osmotic shock produced a delay or a long-term blockade of diuresis, depending on the antiserum dilution applied. Similar results were obtained when AT-antiserum was applied in vivo prior to blood intake, decreasing the volume of urine eliminated during the first 2 h. Our results allow us to assign a specific endocrine function to the AT-like peptide released by MTs that is linked to the elimination of urine after blood meals. [Abstract/Link to Full Text]

Bystriansky JS, Frick NT, Ballantyne JS
Intermediary metabolism of Arctic char Salvelinus alpinus during short-term salinity exposure.
J Exp Biol. 2007 Jun;210(Pt 11):1971-85.
The migration of Arctic char Salvelinus alpinus from freshwater to seawater requires a substantial reorganization of the osmoregulatory tissues to regulate plasma ion levels. These modifications have an inherent metabolic cost, which must be met through the upregulation of intermediary metabolism. Arctic char intermediary metabolism was monitored during the initial 96 h of seawater acclimation through measurement of key enzymes in gill, liver, red and white muscle as well as tissue and blood free amino acid (FAA) levels, and plasma glucose and non-esterified fatty acid content. In general, seawater exposure stimulated large changes in amino acid metabolism, but no change in lipid or carbohydrate metabolism. White muscle FAA content increased significantly following seawater exposure, with levels of essential FAAs doubling after 96 h. Similar increases were seen in the plasma, suggesting a rapid mobilization of FAAs to the circulation. These changes were accompanied by significant increases in the activities of enzymes involved in amino acid metabolism in the gill, liver, red and white muscle, suggesting seawater-acclimated fish have an enhanced capacity for energy production from amino acids. Increased energy requirements were evident in the gill of seawater-acclimated char, as citrate synthase activity increased significantly. The results of this study suggest a rapid upregulation of amino acid metabolism may be critical for the successful acclimation of Arctic char to seawater. [Abstract/Link to Full Text]

Yeates LC, Williams TM, Fink TL
Diving and foraging energetics of the smallest marine mammal, the sea otter (Enhydra lutris).
J Exp Biol. 2007 Jun;210(Pt 11):1960-70.
As the smallest and one of the most recently evolved marine mammals, sea otters face physiological challenges rarely encountered by larger, more derived aquatic species. To examine the effect of these challenges on foraging costs and resultant daily energy budgets, we measured the energetics of resting, grooming, diving and foraging for adult, male sea otters. The energy expended for these different behaviors as determined from open flow respirometry was then standardized across activity budgets measured for wild sea otters to estimate field metabolic rates (FMR). We found that the metabolic rate of captive otters performing single dives ranging in duration from 40 to 192 s was 17.6+/-0.5 ml O(2) kg(-1) min(-1) and only 1.3 times resting rates. This rate increased significantly if the animals foraged during submergence. The cost of a foraging dive for sea otters was nearly twice that predicted for phocid seals, which was attributed in part to elevated locomotor costs associated with buoyancy and swimming style. Our behavioral studies indicate that wild sea otters spend the greatest proportion of the day feeding and resting, with the largest daily energy expenditure (6.1+/-1.1 MJ day(-1)) associated with foraging. The resulting mean FMR for wild sea otters based on the energy expended for all behaviors was 15.7+/-2.7 MJ day(-1) and matched predicted FMR values based upon a regression of known FMR values for other marine mammals across a range of body sizes. This was achieved by counterbalancing elevated foraging costs with prolonged periods of rest on the water surface. [Abstract/Link to Full Text]

Gilmour KM, Euverman RM, Esbaugh AJ, Kenney L, Chew SF, Ip YK, Perry SF
Mechanisms of acid-base regulation in the African lungfish Protopterus annectens.
J Exp Biol. 2007 Jun;210(Pt 11):1944-59.
African lungfish Protopterus annectens utilized both respiratory and metabolic compensation to restore arterial pH to control levels following the imposition of a metabolic acidosis or alkalosis. Acid infusion (3 mmol kg(-1) NH(4)Cl) to lower arterial pH by 0.24 units increased both pulmonary (by 1.8-fold) and branchial (by 1.7-fold) ventilation frequencies significantly, contributing to 4.8-fold and 1.9-fold increases in, respectively, aerial and aquatic CO(2) excretion. This respiratory compensation appeared to be the main mechanism behind the restoration of arterial pH, because even though net acid excretion (J(net)H(+)) increased following acid infusion in 7 of 11 fish, the mean increase in net acid excretion, 184.5+/-118.5 micromol H(+) kg(-1) h(-1) (mean +/- s.e.m., N=11), was not significantly different from zero. Base infusion (3 mmol kg(-1) NaHCO(3)) to increase arterial pH by 0.29 units halved branchial ventilation frequency, although pulmonary ventilation frequency was unaffected. Correspondingly, aquatic CO(2) excretion also fell significantly (by 3.7-fold) while aerial CO(2) excretion was unaffected. Metabolic compensation consisting of negative net acid excretion (net base excretion) accompanied this respiratory compensation, with J(net)H(+) decreasing from 88.5+/-75.6 to -337.9+/-199.4 micromol H(+) kg(-1) h(-1) (N=8). Partitioning of net acid excretion into renal and extra-renal (assumed to be branchial and/or cutaneous) components revealed that under control conditions, net acid excretion occurred primarily by extra-renal routes. Finally, several genes that are involved in the exchange of acid-base equivalents between the animal and its environment (carbonic anhydrase, V-type H(+)-ATPase and Na(+)/HCO (-)(3) cotransporter) were cloned, and their branchial and renal mRNA expressions were examined prior to and following acid or base infusion. In no case was mRNA expression significantly altered by metabolic acid-base disturbance. These findings suggest that lungfish, like tetrapods, alter ventilation to compensate for metabolic acid-base disturbances, a mechanism that is not employed by water-breathing fish. Like fish and amphibians, however, extra-renal routes play a key role in metabolic compensation. [Abstract/Link to Full Text]

Lewis JM, Costa I, Val AL, Almeida-Val VM, Gamperl AK, Driedzic WR
Responses to hypoxia and recovery: repayment of oxygen debt is not associated with compensatory protein synthesis in the Amazonian cichlid, Astronotus ocellatus.
J Exp Biol. 2007 Jun;210(Pt 11):1935-43.
Oxygen consumption, as an indicator of routine metabolic rate (RoMR), and tissue-specific changes in protein synthesis, as measured by (3)H-labelled phenylalanine incorporation rates, were determined in Astronotus ocellatus to investigate the cellular mechanisms behind hypoxia-induced metabolic depression and recovery. RoMR was significantly depressed, by approximately 50%, when dissolved oxygen levels reached 10% saturation (0.67+/-0.01 mg l(-1) at 28+/-1 degrees C). This depression in RoMR was accompanied by a 50-60% decrease in liver, heart and gill protein synthesis, but only a 30% decrease in brain protein synthesis. During recovery from hypoxia, an overshoot in RoMR to 270% of the normoxic rate was observed, indicating the accumulation of an oxygen debt during hypoxia. This conclusion was consistent with significant increase in plasma lactate levels during the hypoxic exposure, and the fact that lactate levels rapidly returned to pre-hypoxic levels. In contrast, a hyperactivation of protein synthesis did not occur, suggesting the overshoot in oxygen consumption during recovery is attributed to an increase in cellular processes other than protein synthesis. [Abstract/Link to Full Text]

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Recent Articles in Biological Procedures Online

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Murphy M, Greferath U, Wilson YM
A method for detecting functional activity related expression in gross brain regions, specific brain nuclei and individual neuronal cell bodies and their projections.
Biol Proced Online. 2007;91-8.
We have developed a system to visualize functionally activated neurons and their projections in the brain. This system utilizes a transgenic mouse, fos-tau-lacZ (FTL), which expresses the marker gene, lacZ, in neurons and their processes after activation by many different stimuli. This system allows the imaging of activation from the level of the entire brain surface, through to individual neurons and their projections. The use of this system involves detection of neuronal activation by histochemical or immunohistochemical detection of beta-galactosidase (betagal), the product of the lacZ gene. Furthermore, the underlying brain state of the FTL mice determines the basal levels of expression of betagal. Here we describe in detail our protocols for detection of FTL expression in these mice and discuss the main variables which need to be considered in the use of these mice for the detection and mapping of functionally activated neurons, circuits and regions in the brain. [Abstract/Link to Full Text]

Harrison JJ, Ceri H, Yerly J, Stremick CA, Hu Y, Martinuzzi R, Turner RJ
The use of microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms cultivated in the Calgary Biofilm Device.
Biol Proced Online. 2006;8194-215.
Microbes frequently live within multicellular, solid surface-attached assemblages termed biofilms. These microbial communities have architectural features that contribute to population heterogeneity and consequently to emergent cell functions. Therefore, three-dimensional (3D) features of biofilm structure are important for understanding the physiology and ecology of these microbial systems. This paper details several protocols for scanning electron microscopy and confocal laser scanning microscopy (CLSM) of biofilms grown on polystyrene pegs in the Calgary Biofilm Device (CBD). Furthermore, a procedure is described for image processing of CLSM data stacks using amira, a virtual reality tool, to create surface and/or volume rendered 3D visualizations of biofilm microorganisms. The combination of microscopy with microbial cultivation in the CBD - an apparatus that was designed for high-throughput susceptibility testing - allows for structure-function analysis of biofilms under multivariate growth and exposure conditions. [Abstract/Link to Full Text]

Morey JS, Ryan JC, Van Dolah FM
Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR.
Biol Proced Online. 2006;8175-93.
Quantitative real-time PCR (qPCR) is a commonly used validation tool for confirming gene expression results obtained from microarray analysis; however, microarray and qPCR data often result in disagreement. The current study assesses factors contributing to the correlation between these methods in five separate experiments employing two-color 60-mer oligonucleotide microarrays and qPCR using SYBR green. Overall, significant correlation was observed between microarray and qPCR results (rho=0.708, p<0.0001, n=277) using these platforms. The contribution of factors including up- vs. down-regulation, spot intensity, rho-value, fold-change, cycle threshold (C(t)), array averaging, tissue type, and tissue preparation was assessed. Filtering of microarray data for measures of quality (fold-change and rho-value) proves to be the most critical factor, with significant correlations of rho>0.80 consistently observed when quality scores are applied. [Abstract/Link to Full Text]

Lin M, Zhao W, Wu R
A statistical framework for genetic association studies of power curves in bird flight.
Biol Proced Online. 2006;8164-74.
How the power required for bird flight varies as a function of forward speed can be used to predict the flight style and behavioral strategy of a bird for feeding and migration. A U-shaped curve was observed between the power and flight velocity in many birds, which is consistent to the theoretical prediction by aerodynamic models. In this article, we present a general genetic model for fine mapping of quantitative trait loci (QTL) responsible for power curves in a sample of birds drawn from a natural population. This model is developed within the maximum likelihood context, implemented with the EM algorithm for estimating the population genetic parameters of QTL and the simplex algorithm for estimating the QTL genotype-specific parameters of power curves. Using Monte Carlo simulation derived from empirical observations of power curves in the European starling (Sturnus vulgaris), we demonstrate how the underlying QTL for power curves can be detected from molecular markers and how the QTL detected affect the most appropriate flight speeds used to design an optimal migration strategy. The results from our model can be directly integrated into a conceptual framework for understanding flight origin and evolution. [Abstract/Link to Full Text]

Gallup JM, Ackermann MR
Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions.
Biol Proced Online. 2006;887-152.
The purpose of this manuscript is to discuss fluorogenic real-time quantitative polymerase chain reaction (qPCR) inhibition and to introduce/define a novel Microsoft Excel-based file system which provides a way to detect and avoid inhibition, and enables investigators to consistently design dynamically-sound, truly LOG-linear qPCR reactions very quickly. The qPCR problems this invention solves are universal to all qPCR reactions, and it performs all necessary qPCR set-up calculations in about 52 seconds (using a pentium 4 processor) for up to seven qPCR targets and seventy-two samples at a time - calculations that commonly take capable investigators days to finish. We have named this custom Excel-based file system "FocusField2-6GallupqPCRSet-upTool-001" (FF2-6-001 qPCR set-up tool), and are in the process of transforming it into professional qPCR set-up software to be made available in 2007. The current prototype is already fully functional. [Abstract/Link to Full Text]

Badtke MP, Cao F, Tavis JE
Combining genetic and biochemical approaches to identify functional molecular contact points.
Biol Proced Online. 2006;877-86.
Protein-protein interactions are required for many viral and cellular functions and are potential targets for novel therapies. Here we detail a series of genetic and biochemical techniques used in combination to find an essential molecular contact point on the duck hepatitis B virus polymerase. These techniques include differential immunoprecipitation, mutagenesis and peptide competition. The strength of these techniques is their ability to identify contact points on intact proteins or protein complexes employing functional assays. This approach can be used to aid identification of putative binding sites on proteins and protein complexes which are resistant to characterization by other methods. [Abstract/Link to Full Text]

Ray S, Das SK
Chromatin immunoprecipitation assay detects ERalpha recruitment to gene specific promoters in uterus.
Biol Proced Online. 2006;869-76.
Chromatin immunoprecipitation (ChIP) technique allows detection of proteins that bind to chromatin. While this technique has been applied extensively in cell-based studies, its tissue-based application remains poorly explored. We are specifically interested in examining estrogen-dependent transcriptional mechanism in respect of recruitment of estrogen receptor-alpha (ERalpha), a ligand-activated transcription factor, to uterine gene promoters in mice. Recent gene-array studies, utilizing ERalpha knock-out vs. wild-type mice, have revealed that estrogen regulates numerous uterine genes temporally and most importantly via ERalpha during the phase-II response, including three well characterized genes viz., lactoferrin (Ltf), progesterone receptor (Pgr) and cyclinD1 (Ccnd1). Here, utilizing systematic ChIP studies, we demonstrate endogenous recruitment of ERalpha to above uterine gene promoters following estradiol-17beta (E(2)) injection in mice. [Abstract/Link to Full Text]

Emmerson JR, Gally DL, Roe AJ
Generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system.
Biol Proced Online. 2006;8153-62.
In this work we describe protocols for the generation of gene deletions and gene replacements using a temperature sensitive plasmid in Escherichia coli O157:H7. This technology requires flanking DNA to be cloned into a temperature sensitive vector but the resulting clone allows great flexibility for further modification of the target sequence. It is therefore highly suited to the study of genes in which several rounds of changes are envisaged. A number of examples are used to illustrate the flexibility of the system which has been used to create novel gene replacements including fusions for protein localisation work and reporters for transcriptional analyses. In this paper we describe protocols which can be used with a high degree of success when applied to E. coli O157. The deletion and replacement of the LEE4 operon of E. coli O157 is detailed to show the advantages and limitations of the technology. [Abstract/Link to Full Text]

Xia J, Kim SH, Macmillan S, Truant R
Practical three color live cell imaging by widefield microscopy.
Biol Proced Online. 2006;863-8.
Live cell fluorescence microscopy using fluorescent protein tags derived from jellyfish and coral species has been a successful tool to image proteins and dynamics in many species. Multi-colored aequorea fluorescent protein (AFP) derivatives allow investigators to observe multiple proteins simultaneously, but overlapping spectral properties sometimes require the use of sophisticated and expensive microscopes. Here, we show that the aequorea coerulescens fluorescent protein derivative, PS-CFP2 has excellent practical properties as a blue fluorophore that are distinct from green or red fluorescent proteins and can be imaged with standard filter sets on a widefield microscope. We also find that by widefield illumination in live cells, that PS-CFP2 is very photostable. When fused to proteins that form concentrated puncta in either the cytoplasm or nucleus, PSCFP2 fusions do not artifactually interact with other AFP fusion proteins, even at very high levels of over-expression. PSCFP2 is therefore a good blue fluorophore for distinct three color imaging along with eGFP and mRFP using a relatively simple and inexpensive microscope. [Abstract/Link to Full Text]

Austin BA, James CM, Härle P, Carr DJ
Direct application of plasmid DNA containing type I interferon transgenes to vaginal mucosa inhibits HSV-2 mediated mortality.
Biol Proced Online. 2006;855-62.
The application of naked DNA containing type I interferon (IFN) transgenes is a promising potential therapeutic approach for controlling chronic viral infections. Herein, we detail the application of this approach that has been extensively used to restrain ocular HSV-1 infection, for antagonizing vaginal HSV-2 infection. We show that application of IFN-alpha1, -alpha5, and -beta transgenes to vaginal mouse lumen 24 hours prior to HSV-2 infection reduces HSV-2 mediated mortality by 2.5 to 3-fold. However, other type I IFN transgenes (IFN- alpha4, -alpha5, -alpha6, and -alpha9) are non effectual against HSV-2. We further show that the efficacy of IFN-alpha1 transgene treatment is independent of CD4+ T lymphocytes. However, in mice depleted of CD8+ T lymphocytes, the ability of IFN-alpha1 transgene treatment to antagonize HSV-2 was lost. [Abstract/Link to Full Text]

Dryer RL, Covey LR
Use of chromatin immunoprecipitation (ChIP) to detect transcription factor binding to highly homologous promoters in chromatin isolated from unstimulated and activated primary human B cells.
Biol Proced Online. 2006;844-54.
The Chromatin Immunoprecipiation (ChIP) provides a powerful technique for identifying the in vivo association of transcription factors with regulatory elements. However, obtaining meaningful information for promoter interactions is extremely challenging when the promoter is a member of a class of highly homologous elements. Use of PCR primers with small numbers of mutations can limit cross-hybridization with non-targeted sequences and distinguish a pattern of binding for factors with the regulatory element of interest. In this report, we demonstrate the selective in vivo association of NF-kappaB, p300 and CREB with the human Igamma1 promoter located in the intronic region upstream of the Cgamma1 exons in the immunoglobulin heavy chain locus. These methods have the ability to extend ChIP analysis to promoters with a high degree of homology. [Abstract/Link to Full Text]

Yamada HY, Gorbsky GJ
Cell-based expression cloning for identification of polypeptides that hypersensitize mammalian cells to mitotic arrest.
Biol Proced Online. 2006;836-43.
Microtubule inhibitors such as Vinblastine and Paclitaxel are chemotherapy agents that activate the mitotic spindle checkpoint, arresting cells in mitosis and leading to cell death. The pathways that connect mitotic arrest to cell death are not well characterized. We developed a mammalian cell-based cDNA cloning method to isolate proteins and protein fragments whose expression inhibits colony formation in the presence of microtubule inhibitors. Understanding how these proteins impact cellular responses to microtubule drugs will lead to better understanding of the biochemical pathways connecting mitotic arrest and cell death in mammalian cells and may provide novel targets that can enhance microtubule inhibitor-mediated chemotherapy. [Abstract/Link to Full Text]

Raez MB, Hussain MS, Mohd-Yasin F
Techniques of EMG signal analysis: detection, processing, classification and applications.
Biol Proced Online. 2006;811-35.
Electromyography (EMG) signals can be used for clinical/biomedical applications, Evolvable Hardware Chip (EHW) development, and modern human computer interaction. EMG signals acquired from muscles require advanced methods for detection, decomposition, processing, and classification. The purpose of this paper is to illustrate the various methodologies and algorithms for EMG signal analysis to provide efficient and effective ways of understanding the signal and its nature. We further point up some of the hardware implementations using EMG focusing on applications related to prosthetic hand control, grasp recognition, and human computer interaction. A comparison study is also given to show performance of various EMG signal analysis methods. This paper provides researchers a good understanding of EMG signal and its analysis procedures. This knowledge will help them develop more powerful, flexible, and efficient applications. [Abstract/Link to Full Text]

Dussault AA, Pouliot M
Rapid and simple comparison of messenger RNA levels using real-time PCR.
Biol Proced Online. 2006;81-10.
Real-time polymerase chain reaction (PCR) constitutes a significant improvement over traditional end-point PCR, as it allows the quantification of starting amounts of nucleic acid templates, in real-time. However, quantification requires validation through numerous internal controls and standard curves. We describe in this paper a simple protocol which uses real-time PCR to compare mRNA levels of a gene of interest between different experimental conditions. Comparative real-time PCR can be a relatively low-cost method and does not require sequence-specific fluorescent reporters. Moreover, several genes from a set of experiments can be assessed in a single run. Thus, in addition to providing a comparative profile for the expression of a gene of interest, this method can also provide information regarding the relative abundance of different mRNA species. [Abstract/Link to Full Text]

Wozniak MA, Keely PJ
Use of three-dimensional collagen gels to study mechanotransduction in T47D breast epithelial cells.
Biol Proced Online. 2005;7144-61.
Several pathological and disease conditions can alter the mechanical properties of the extracellular matrix (ECM). Conversely, some diseases may arise from changes in the density or rigidity of the ECM. This necessitates the use and development of in vitro models to understand how both biophysical and biochemical signals regulate complex cellular behaviors. T47D breast epithelial cells will differentiate into duct-like tubules when cultured in a floating three-dimensional (3D) collagen gel, but not a 3D collagen gel that is left attached to the culture dish. This paper details several protocols we have developed for analyzing breast cell biology in 3D matrices, including culturing cells in 3D collagen gels, immunostaining cellular structures, and performing biochemical procedures directly from cells embedded in collagen gels. [Abstract/Link to Full Text]

Mellado-Gil JM, Aguilar-Diosdado M
Assay for high glucose-mediated islet cell sensitization to apoptosis induced by streptozotocin and cytokines.
Biol Proced Online. 2005;7162-71.
Pancreatic beta-cell apoptosis is known to participate in the beta-cell destruction process that occurs in diabetes. It has been described that high glucose level induces a hyperfunctional status which could provoke apoptosis. This phenomenon is known as glucotoxicity and has been proposed that it can play a role in type 1 diabetes mellitus pathogenesis. In this study we develop an experimental design to sensitize pancreatic islet cells by high glucose to streptozotocin (STZ) and proinflammatory cytokines [interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma]-induced apoptosis. This method is appropriate for subsequent quantification of apoptotic islet cells stained with Tdt-mediated dUTP Nick-End Labeling (TUNEL) and protein expression assays by Western Blotting (WB). [Abstract/Link to Full Text]

Encarnación S, Hernández M, Martínez-Batallar G, Contreras S, Vargas Mdel C, Mora J
Comparative proteomics using 2-D gel electrophoresis and mass spectrometry as tools to dissect stimulons and regulons in bacteria with sequenced or partially sequenced genomes.
Biol Proced Online. 2005;7117-35.
We propose two-dimensional gel electrophoresis (2-DE) and mass spectrometry to define the protein components of regulons and stimulons in bacteria, including those organisms where genome sequencing is still in progress. The basic 2-DE protocol allows high resolution and reproducibility and enables the direct comparison of hundreds or even thousands of proteins simultaneously. To identify proteins that comprise stimulons and regulons, peptide mass fingerprint (PMF) with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) analysis is the first option and, if results from this tool are insufficient, complementary data obtained with electrospray ionization tandem-MS (ESI-MS/MS) may permit successful protein identification. ESI-MS/MS and MALDI-TOF-MS provide complementary data sets, and so a more comprehensive coverage of a proteome can be obtained using both techniques with the same sample, especially when few sequenced proteins of a particular organism exist or genome sequencing is still in progress. [Abstract/Link to Full Text]

Jud C, Schmutz I, Hampp G, Oster H, Albrecht U
A guideline for analyzing circadian wheel-running behavior in rodents under different lighting conditions.
Biol Proced Online. 2005;7101-16.
Most behavioral experiments within circadian research are based on the analysis of locomotor activity. This paper introduces scientists to chronobiology by explaining the basic terminology used within the field. Furthermore, it aims to assist in designing, carrying out, and evaluating wheel-running experiments with rodents, particularly mice. Since light is an easily applicable stimulus that provokes strong effects on clock phase, the paper focuses on the application of different lighting conditions. [Abstract/Link to Full Text]

Chandra PK, Wikel SK
Analyzing ligation mixtures using a PCR based method.
Biol Proced Online. 2005;793-100.
We have developed a simple and effective method (Lig-PCR) for monitoring ligation reactions using PCR and primers that are common to many cloning vectors. Ligation mixtures can directly be used as templates and the results can be analyzed by conventional gel electrophoresis. The PCR products are representative of the recombinant molecules created during ligation and the corresponding transformants. Orientation of inserts can also be determined using an internal primer. The usefulness of this method has been demonstrated using ligation mixtures of two cDNA's derived from the salivary glands of Aedes aegypti mosquitoes. The method described here is sensitive and easy to perform compared to currently available methods. [Abstract/Link to Full Text]

Gallup JM, Kawashima K, Lucero G, Ackermann MR
New quick method for isolating RNA from laser captured cells stained by immunofluorescent immunohistochemistry; RNA suitable for direct use in fluorogenic TaqMan one-step real-time RT-PCR.
Biol Proced Online. 2005;770-92.
We describe a new approach for reliably isolating one-step real-time quantitative RT-PCR-quality RNA from laser captured cells retrieved from frozen sections previously subjected to immunofluorescent immunohistochemistry (IF-IHC) and subsequently subjected to fluorogenic one-step real-time RT-PCR analysis without the need for costly, time-consuming linear amplification. One cell's worth of RNA can now be interrogated with confidence. This approach represents an amalgam of technologies already offered commercially by Applied Biosystems, Arcturus and Invitrogen. It is the primary focus of this communication to expose the details and execution of an important new LCM RNA isolation technique, but also provide a detailed account of the IF-IHC procedure preceding RNA isolation, and provide information regarding our approach to fluorogenic one-step real-time RT-PCR in general. Experimental results shown here are meant to supplement the primary aim and are not intended to represent a complete scientific study. It is important to mention, that since LCM-RT-PCR is still far less expensive than micro-array analysis, we feel this approach to isolating RNA from LCM samples will be of continuing use to many researchers with limited budgets in the years ahead. [Abstract/Link to Full Text]

Ricke RM, Bielinsky AK
Easy detection of chromatin binding proteins by the Histone Association Assay.
Biol Proced Online. 2005;760-9.
The Histone Association Assay provides an easy approach for detecting proteins that bind chromatin in vivo. This technique is based on a chromatin immunoprecipitation protocol using histone H3-specific antibodies to precipitate bulk chromatin from crosslinked whole cell extracts. Proteins that co-precipitate with chromatin are subsequently detected by conventional SDS-PAGE and Western blot analysis. Unlike techniques that separate chromatin and non-chromatin interacting proteins by centrifugation, this method can be used to delineate whether a protein is chromatin associated regardless of its innate solubility. Moreover, the relative amount of protein bound to DNA can be ascertained under quantitative conditions. Therefore, this technique may be utilized for analyzing the chromatin association of proteins involved in diverse cellular processes. [Abstract/Link to Full Text]

Akiyama T, Miyazaki T, Bouillet P, Nakamura K, Strasser A, Tanaka S
In vitro and in vivo assays for osteoclast apoptosis.
Biol Proced Online. 2005;748-59.
Mature osteoclasts, multinucleated giant cells responsible for bone resorption, are terminally differentiated cells with a short life span. Recently, we have demonstrated that osteoclast apoptosis is regulated by ERK activity and Bcl-2 family member Bim. In this paper, we summarize the methods we used to study osteoclast apoptosis in vitro and in vivo. Using adenovirus and retrovirus vectors, we were able to introduce foreign genes into osteoclasts and examine their effects on osteoclast survival in vitro. In addition, we established the modified methods for in situ hybridization and BrdU labeling of bone sections from mice to study osteoclast survival in vivo. The detailed methods described here could be useful for studying the biological process in bone. [Abstract/Link to Full Text]

Watanabe M, Fujioka-Kaneko Y, Kobayashi H, Kiniwa M, Kuwano M, Basaki Y
Involvement of integrin-linked kinase in capillary/tube-like network formation of human vascular endothelial cells.
Biol Proced Online. 2005;741-7.
Angiogenesis is a complex process involving an ECM and vascular endothelial cells (EC), and is regulated by various angiogenic factors including VEGF. The ability to form a capillary/tube-like network is a specialized function of EC. Therefore, in vitro angiogenesis was assessed by a capillary/tube-like network formation assay. There are three angiogenic parameters: capillary length, number of capillaries, and relative capillary area per field. We evaluated capillary length per field in the assay. VEGF promoted capillary/tube-like network formation of EC in a type I collagen gel matrix in vitro. Moreover, we demonstrated the involvement of ILK in a VEGF signaling pathway mediating capillary/tube-like network formation of EC using dominant-negative, kinase deficient ILK. This is a straightforward assay to monitor responses of human vascular endothelial cells. [Abstract/Link to Full Text]

Deveau JS, Lindinger MI, Grodzinski B
An improved method for constructing and selectively silanizing double-barreled, neutral liquid-carrier, ion-selective microelectrodes.
Biol Proced Online. 2005;731-40.
We describe an improved, efficient and reliable method for the vapour-phase silanization of multi-barreled, ion-selective microelectrodes of which the silanized barrel(s) are to be filled with neutral liquid ion-exchanger (LIX). The technique employs a metal manifold to exclusively and simultaneously deliver dimethyldichlorosilane to only the ion-selective barrels of several multi-barreled microelectrodes. Compared to previously published methods the technique requires fewer procedural steps, less handling of individual microelectrodes, improved reproducibility of silanization of the selected microelectrode barrels and employs standard borosilicate tubing rather than the less-conventional theta-type glass. The electrodes remain stable for up to 3 weeks after the silanization procedure. The efficacy of a double-barreled electrode containing a proton ionophore in the ion-selective barrel is demonstrated in situ in the leaf apoplasm of pea (Pisum) and sunflower (Helianthus). Individual leaves were penetrated to depth of approximately 150 microm through the abaxial surface. Microelectrode readings remained stable after multiple impalements without the need for a stabilizing PVC matrix. [Abstract/Link to Full Text]

Corsini J, Mann E
Rapid cryopreservation of five mammalian and one mosquito cell line at -80 degrees C while attached to flasks in a serum free cryopreservative.
Biol Proced Online. 2005;726-30.
Cell culturing, and the requisite storage of cell lines at ultra-low temperatures, is used in most laboratories studying or using eukaryotic proteomics, genomics, microarray, and RNA technologies. In this study we have observed that A72(dog), CRFK(cat), NB324K(human), MCF7(human), WI38(human), and C636(mosquito) cells were effectively cryopreserved at -80 degrees C while attached to the substratum of 25 cm2 tissue culture flasks. This was accomplished using a serum free crypreservative recently developed by Corsini and co-workers. The technique allows for significant savings of time and money in laboratories that rapidly process numerous cell lines. [Abstract/Link to Full Text]

Lefrancois S, Canuel M, Zeng J, Morales CR
Inactivation of sortilin (a novel lysosomal sorting receptor) by dominant negative competition and RNA interference.
Biol Proced Online. 2005;717-25.
To assess the role of sortilin in the sorting and trafficking of sphingolipid activator proteins (SAPs) the function of sortilin was abolished by a dominant-negative mutant and by the use of RNAi. Mutant sortilin lacking the carboxyl-terminal region that contains the sorting signal abolished the trafficking of SAPs to the lysosomes. Both sortilin and SAPs were retained in the Golgi apparatus. The use of chemically synthesized siRNA effectively blocked the trafficking of SAPs to the lysosomes as well. Additionally, we created a stable COS-7 cell line transfected with the pSilencer 3.1 H1 neo vector containing a selected siRNA template oligonucleotide (small hairpin interference RNA) where the levels of sortilin were greatly suppressed. The elimination of sortilin by this method will permit to determine whether or not sortilin is involved in a general mechanism of lysosomal sorting that involves the trafficking of various soluble lysosomal proteins other than SAPs. [Abstract/Link to Full Text]

Corcoran KE, Patel PS, Rameshwar P
An in vitro method to select malignant cells from surgical biopsies of breast cancer patients.
Biol Proced Online. 2005;78-16.
To date, breast cancer (BC) research is mainly studied with cell lines. These cells were passaged multiple times, acquiring phenotypes, additional mutations and epigenetic changes. These changes make the passaged cell lines different from the original malignancy. Thus cell lines, although useful as models could be improved with additional studies with primary BC. It is difficult to obtain malignant cells from breast tissues without contamination from surrounding healthy cells. Selection and expansion of malignant cells from surgical tissues have proved to be daunting tasks. This study describes a reliable and reproducible method for isolating and expanding malignant cells from surgical breast tissues. The method uses co-cultures with BM stroma to select for the cancer cells while the healthy cells undergo rapid cell death. Studies are described to show the cloning efficiencies and sensitivity of the method using surgical samples of varying sizes, different stages of BC, and samples from needle biopsies. [Abstract/Link to Full Text]

Smart N, Scambler PJ, Riley PR
A rapid and sensitive assay for quantification of siRNA efficiency and specificity.
Biol Proced Online. 2005;71-7.
RNA Interference has rapidly emerged as an efficient procedure for knocking down gene expression in model systems. However, cross-reactivity, whereby multiple genes may be simultaneously targeted by a single short interfering RNA (siRNA), can potentially jeopardize correct interpretation of gene function. As such, it is essential to test the specificity of a siRNA prior to a full phenotypic analysis. To this end, we have adapted a reporter-based assay harnessing the sensitivity of luciferase activity to provide a quantitative readout of relative RNAi efficacy and specificity. We have tested different siRNAs directed against Thymosin beta4 (Tbeta4); determined their effectiveness at silencing Tbeta4 and have both excluded off-target silencing of the Tbeta4 homologue Thymosin beta10 (Tbeta10) and demonstrated partial knockdown of Tbeta10 despite significant (12/23; 52%) sequence mismatch. This assay system is applicable to any RNAi study where there is a risk of targeting homologous genes and to the monitoring of off-target effects at the genome level following microarray expression profiling. [Abstract/Link to Full Text]

Shiner EK, Reddy S, Timmons C, Li G, Williams SC, Rumbaugh KP
Construction of a bacterial autoinducer detection system in mammalian cells.
Biol Proced Online. 2004;6268-276.
Quorum sensing (QS) is a cell density-dependent signaling system used by bacteria to coordinate gene expression within a population. QS systems in Gram negative bacteria consist of transcription factors of the LuxR family and their acyl homoserine lactone (AHL) ligands. We describe here a method for examining QS signaling systems in mammalian cells that uses engineered LuxR-type proteins from the opportunistic pathogen, Pseudomonas aeruginosa, which can function as AHL-dependent transcription factors. The engineered proteins respond to their cognate ligands and display sequence specific DNA binding properties. This system has several potential biotechnological and biological applications. It may be used to characterize any LuxR-type protein, screen animal and plant cell extracts or exudates for compounds that mimic or interfere with AHL signaling or to screen different cell types for AHL inactivating activities. [Abstract/Link to Full Text]

Garimella R, Sipe JB, Anderson HC
A simple and non-radioactive technique to study the effect of monophosphoesters on matrix vesicle-mediated calcification.
Biol Proced Online. 2004;6263-267.
A simple and non-radioactive technique based on O-cresolpthalein complexone assay was developed to study in vitro non-radioactive calcium ((40)Ca) deposition by isolated matrix vesicles. Using this technique, the effect of various phosphoester substrates including ATP, AMP and beta-GP on in vitro MV-calcification was studied. O-cresolpthalein complexone assay with non-radioactive calcium demonstrated that AMP or beta-GP were more effective in promoting calcium deposition by isolated MVs than ATP. The application of this non-radioactive technique, which is highly sensitive and simple, would offer a useful alternative approach to the routinely used radiometric biomineralization assay which employs radioactive (45)Ca. [Abstract/Link to Full Text]

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Recent Articles in BMC Developmental Biology

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Filippi A, Duerr K, Ryu S, Willaredt M, Holzschuh J, Driever W
Expression and function of nr4a2, lmx1b, and pitx3 in zebrafish dopaminergic and noradrenergic neuronal development.
BMC Dev Biol. 2007 Dec 5;7(1):135.
ABSTRACT: BACKGROUND: Dopaminergic neurons form in diverse areas of the vertebrate di- and mesencephalon to constitute several major neuromodulatory systems. While much is known about mammalian mesencephalic dopaminergic neuron development, little is known about the specification of the diencephalic dopaminergic groups. The transcription factors Pitx3 and Lmx1b play an important role in mammalian mesencephalic dopaminergic specification, and Nurr1/Nr4a2 has been shown to contribute to specification of the dopaminergic neurotransmitter phenotype. We use zebrafish to analyze potentially evolutionarily conserved roles of these transcription factors in a vertebrate brain that lacks a mesencephalic dopaminergic system, but has an ascending dopaminergic system in the ventral diencephalon. RESULTS: We use a combination of fluorescent in situ hybridization and immunohistochemistry to determine whether nr4a2, lmx1b, and pitx3 genes are expressed in mature dopaminergic neurons or in potential precursor populations. We identify a second nr4a2 paralogue, nr4a2a, and find it co-expressed with Tyrosine hydroxylase in preoptic, pretectal and retinal amacrine dopaminergic neurons, while nr4a2b is only expressed in preoptic and retinal dopaminergic neurons. Both zebrafish nr4a2 paralogues are not expressed in ventral diencephalic dopaminergic neurons with ascending projections. Combined morpholino antisense oligo mediated knock-down of both nr4a2a and nr4a2b transcripts reveals that all zebrafish dopaminergic neurons expressing nr4a2a depend on Nr4a2 activity for tyrosine hydroxylase and dopamine transporter expression. Zebrafish lmx1b.1 is expressed in noradrenergic neurons of the locus coeruleus and medulla oblongata, but knock-down reveals that it is specifically required for tyrosine hydroxylase expression only in the medulla oblongata area postrema noradrenergic neurons. Both lmx1b genes and pitx3 are not expressed in dopaminergic neurons, but in a diencephalic territory that might contain precursor cells for ventral diencephalic dopaminergic neurons. Upon morpholino knock-down of both lmx1b paralogues, the number of neurons in diencephalic dopaminergic clusters with ascending projections appears specifically reduced. Thus lmx1b paralogues may contribute to generation of diencephalic dopaminergic precursors. Conversely, knock-down of pitx3 does not specifically affect any diencephalic DA cluster. CONCLUSIONS: Our data indicate a conserved evolutionary role of Nr4a2 proteins in specification of the neurotransmitter phenotype, albeit it appears to be only one of several regulatory modules of dopaminergic differentiation, as most ventral diencephalic dopaminergic neurons do not express nr4a2 genes in zebrafish. For zebrafish lmx1b genes, which are not expressed in mature dopaminergic neurons, our data suggest a role in diencephalic precursor populations contributing to the ascending dopaminergic systems. A di-mesencephalic longitudinal domain of lmx1b expression may be the basis for the expansion and posterior shift of ventral di-/mesencephalic dopaminergic populations with ascending projections during evolution. [Abstract/Link to Full Text]

Amin S, Matalova E, Simpson C, Yoshida H, Tucker AS
Incudomalleal joint formation: the roles of apoptosis, migration and downregulation.
BMC Dev Biol. 2007 Dec 5;7(1):134.
ABSTRACT: BACKGROUND: The middle ear of mammals is composed of three endochondrial ossicles, the stapes, incus and malleus. Joints link the malleus to the incus and the incus to the stapes. In the mouse the first arch derived malleus and incus are formed from a single Sox9 and Type II collagen expressing condensation that later subdivides to give rise to two separate ossicles. In contrast the stapes forms from a separate condensation derived from the second branchial arch. Fusion of the malleus and incus is observed in a number of human syndromes and results in conductive hearing loss. Understanding how this joint forms during normal development is thus an important step in furthering our understanding of such defects. RESULTS: We show that the developing incudomalleal joint is characterised by a lack of proliferation and discrete areas of apoptosis. Apoptosis has been suggested to aid in the removal of pre-cartilaginous cells from the joint region, allowing for the physical separation of the cartilaginous elements, however, we show that joint initiation is unaffected by blocking apoptosis. There is also no evidence of cell migration out of the presumptive joint region, as observed by labelling of joint and ossicle cells in culture. Using Type II collagen lacZ reporter mice, however, it is evident that cells in the presumptive joint region remain in place and downregulate cartilage markers. CONCLUSION: The malleus and incus first appear as a single united condensation expressing early cartilage markers. The incudomalleal joint region forms by cells in the presumptive joint region switching off cartilage markers and turning on joint markers. Failure in this process may result in fusion of this joint, as observed in human syndromes such as Branchio-Oto-Renal Syndrome or Treacher Collins Syndrome. [Abstract/Link to Full Text]

Shapovalova Z, Tabunshchyk K, Greer PA
The Fer tyrosine kinase regulates an axon retraction response to Semaphorin 3A in dorsal root ganglion neurons.
BMC Dev Biol. 2007 Nov 30;7(1):133.
ABSTRACT: BACKGROUND: Fps/Fes and Fer are the only two members of a distinct subclass of cytoplasmic protein tyrosine kinases. Fps/Fes was previously implicated in Semaphorin 3A (Sema3A)-induced growth cone collapse signaling in neurons from the dorsal root ganglion (DRG) through interaction with and phosphorylation of the Sema3A receptor component PlexinA1, and members of the collapsin response mediator protein (CRMP) family of microtubule regulators. However, the potential role of the closely related Fer kinase has not been examined. RESULTS: Here we provide novel biochemical and genetic evidence that Fer plays a prominent role in microtubule regulation in DRG neurons in response to Sema3A. Although Fps/Fes and Fer were both expressed in neonatal brains and isolated DRGs, Fer was expressed at higher levels; and Fer, but not Fps/Fes kinase activity was detected in vivo. Fer also showed higher in vitro kinase activity toward tubulin, as an exogenous substrate; and this activity was higher when the kinases were isolated from perinatal relative to adult brain stages. CRMP2 was a substrate for both kinases in vitro, but both CRMP2 and PlexinA1 inhibited their autophosphorylation activities. Cultured mouse DRG neurons retracted their axons upon exposure to Sema3A, and this response was significantly diminished in Fer-deficient, but only slightly attenuated in Fps/Fes-deficient DRG neurons. CONCLUSIONS: Fps/Fes and Fer are both capable of phosphorylating tubulin and the microtubule regulator CRMP2 in vitro; and their in vitro kinase activities were both inhibited by CRMP2 or PlexinA1, suggesting a possible regulatory interaction. Furthermore, Fer plays a more prominent role than Fps/Fes in regulating the axon retraction response to Sema3A in DRG neurons. Therefore, Fps/Fes and Fer may play important roles in developmental or regenerative axon pathfinding through signalling from Sema3A to the microtubule cytoskeleton. [Abstract/Link to Full Text]

Favetta LA, Madan P, Mastromonaco GF, St John EJ, King WA, Betts DH
The oxidative stress adaptor p66shc is required for permanent embryo arrest in vitro.
BMC Dev Biol. 2007 Nov 29;7(1):132.
ABSTRACT: BACKGROUND: Excessive developmental failure occurs during the first week of in vitro embryo development due to elevated levels of cell death and arrest. We hypothesize that permanently arrested embryos enter a stress-induced "senescence-like" state that is dependent on the oxidative stress-adaptor and lifespan determinant protein p66Shc. The aim of this study was to selectively diminish p66Shc gene expression in bovine oocytes and embryos using post-transcriptional gene silencing by RNA-mediated interference to study the effects of p66Shc knockdown on in vitro fertilized bovine embryos. RESULTS: Approximately 12,000-24,000 short hairpin (sh)RNAi molecules specific for p66Shc were microinjected into bovine germinal vesicle stage oocytes or zygotes. Experiments were comprised of a control group undergoing IVF alone and two groups microinjected with and without p66Shc shRNAi molecules prior to IVF. The amount of p66Shc mRNA quantified by Real Time PCR was significantly (P<0.001) lowered upon p66Shc shRNAi microinjection. This reduction was selective for p66Shc mRNA, as both histone H2a and p53 mRNA levels were not altered. The relative signal strength of p66Shc immuno-fluorescence revealed a significant reduction in the number of pixels for p66Shc shRNAi microinjected groups compared to controls (P<0.05). A significant decrease (P<0.001) in the incidence of arrested embryos upon p66Shc shRNAi microinjection was detected compared to IVF and microinjected controls along with significant reductions (P<0.001) in both cleavage divisions and blastocyst development. No significant differences in p66Shc mRNA levels (P=0.314) were observed among the three groups at the blastocyst stage. CONCLUSIONS: These results show that p66Shc is involved in the regulation of embryo development specifically in mediating early cleavage arrest and facilitating development to the blastocyst stage for in vitro produced bovine embryos. [Abstract/Link to Full Text]

Mensah A, Mulligan C, Linehan J, Ruf S, O'Doherty A, Grygalewicz B, Shipley J, Groet J, Tybulewicz V, Fisher E, Brandner S, Nizetic D
An additional human chromosome 21 causes suppression of neural fate of pluripotent mouse embryonic stem cells in a teratoma model.
BMC Dev Biol. 2007 Nov 29;7(1):131.
ABSTRACT: BACKGROUND: Down syndrome (DS), caused by trisomy of human chromosome 21 (HSA21), is the most common genetic cause of mental retardation in humans. Among complex phenotypes, it displays a number of neural pathologies including smaller brain size, reduced numbers of neurons, reduced dendritic spine density and plasticity, and early Alzheimer-like neurodegeneration. Mouse models for DS show behavioural and cognitive defects, synaptic plasticity defects, and reduced hippocampal and cerebellar neuron numbers. Early postnatal development of both human and mouse-model DS shows the reduced capability of neuronal precursor cells to generate neurons. The exact molecular cause of this reduction, and the role played by increased dosage of individual HSA21 genes, remain unknown. RESULTS: We have subcutaneously injected mouse pluripotent ES cells containing a single freely segregating supernumerary human chromosome 21 (HSA21) into syngeneic mice, to generate transchromosomic teratomas. Transchromosomic cells and parental control cells were injected into opposite flanks of thirty mice in three independent experiments. Tumours were grown for 30 days, a time-span equivalent to combined intra-uterine, and early post-natal mouse development. When paired teratomas from the same animals were compared, transchromosomic tumours showed a three-fold lower percentage of neuroectodermal tissue, as well as significantly reduced mRNA levels for neuron specific (Tubb3) and glia specific (Gfap) genes, relative to euploid controls. Two thirds of transchromosomic tumours also showed a lack of PCR amplification with multiple primers specific for HSA21, which were present in the ES cells at the point of injection, thus restricting a commonly retained trisomy to less than a third of HSA21 genes. CONCLUSIONS: We demonstrate that a supernumerary chromosome 21 causes Inhibition of Neuroectodermal DIfferentiation (INDI) of pluripotent ES cells. The data suggest that trisomy of less than a third of HSA21 genes, in two chromosomal regions, might be sufficient to cause this effect. [Abstract/Link to Full Text]

Harrington MJ, Hong E, Fasanmi O, Brewster R
Cadherin-mediated adhesion regulates posterior body formation.
BMC Dev Biol. 2007 Nov 28;7(1):130.
ABSTRACT: BACKGROUND: The anterior-posterior axis of the vertebrate embryo undergoes a dramatic elongation during early development. Convergence and extension of the mesoderm, occurring during gastrulation, initiates the narrowing and lengthening of the embryo. However the lengthening of the axis continues during post-gastrula stages in the tailbud region, and is thought to involve convergent extension movements as well as other cell behaviors specific to posterior regions. RESULTS: We demonstrate here, using a semi-dominant N-cadherin allele, that members of the classical cadherin subfamily of cell-cell adhesion molecules are required for tailbud elongation in the zebrafish. In vivo imaging of cell behaviors suggests that the extension of posterior axial mesodermal cells is impaired in embryos that carry the semi-dominant N-cadherin allele. This defect most likely results from a general loss of cell-cell adhesion in the tailbud region. Consistent with these observations, N-cadherin is expressed throughout the tailbud during post-gastrulation stages. In addition, we show that N-cadherin interacts synergistically with Strabismus, a member of the non-canonical Wnt signaling/planar cell polarity pathway, to mediate tail morphogenesis. CONCLUSIONS: We provide the first evidence here that N-cadherin and other members of the classical cadherin subfamily function in parallel with the planar cell polarity pathway to shape the posterior axis during post-gastrulation stages. These findings further highlight the central role that adhesion molecules play in the cellular rearrangements that drive morphogenesis in vertebrates and identify classical cadherins as major contributors to tail development. [Abstract/Link to Full Text]

Svensson P, Williams C, Lundeberg J, Ryden P, Bergqvist I, Edlund H
Gene array identification of Ipf1/Pdx1 -/- regulated genes in pancreatic progenitor cells.
BMC Dev Biol. 2007 Nov 23;7(1):129.
ABSTRACT: BACKGROUND: The homeodomain transcription factor IPF1/PDX1 exerts a dual role in the pancreas; Ipf1/Pdx1 global null mutants fail to develop a pancreas whereas conditional inactivation of Ipf1/Pdx1 in beta-cells leads to impaired beta-cell function and diabetes. Although several putative target genes have been linked to the beta-cell function of Ipf1/Pdx1, relatively little is known with respect to genes regulated by IPF1/PDX1 in early pancreatic progenitor cells. RESULTS: Microarray analyses identified a total of 111 genes that were differentially expressed in e10.5 pancreatic buds of Ipf1/Pdx1- /- embryos. The expression of one these, Spondin 1, which encodes an extracellular matrix protein, has not previously been described in the pancreas. Quantitative real-time RT-PCR analyses and immunohistochemical analyses also revealed that the expression of FgfR2IIIb, that encodes the receptor for FGF10, was down-regulated in Ipf1/Pdx1-/- pancreatic progenitor cells. CONCLUSION: This microarray analysis has identified a number of candidate genes that are differentially expressed in Ipf1/Pdx1-/- pancreatic buds. Several of the differentially expressed genes were known to be important for pancreatic progenitor cell proliferation and differentiation whereas others have not previously been associated with pancreatic development. [Abstract/Link to Full Text]

Zhu H, Cabrera R, Wlodarczyk BJ, Bozinov D, Wang D, Schwartz RJ, Finnell RH
Differentially expressed genes in embryonic cardiac tissues of mice lacking Folr1 gene activity.
BMC Dev Biol. 2007 Nov 20;7(1):128.
ABSTRACT: BACKGROUND: Heart anomalies are the most frequently observed among all human congenital defects. As with the situation for neural tube defects (NTDs), it has been demonstrated that women who use multivitamins containing folic acid peri-conceptionally have a reduced risk for delivering offspring with conotruncal heart defects [1-3]. Cellular folate transport is mediated by a receptor or binding protein and by an anionic transporter protein system. Defective function of the Folr1 (also known as Folbp1; homologue of human FRalpha) gene in mice results in inadequate transport, accumulation, or metabolism of folate during cardiovascular morphogenesis. RESULTS: We have observed cardiovascular abnormalities including outflow tract and aortic arch arterial defects in genetically compromised Folr1 knockout mice. In order to investigate the molecular mechanisms underlying the failure to complete development of outflow tract and aortic arch arteries in the Folr1 knockout mouse model, we examined tissue-specific gene expression difference between Folr1 nullizygous embryos and morphologically normal heterozygous embryos during early cardiac development (14-somite stage), heart tube looping (28-somite stage), and outflow track septation (38-somite stage). Microarray analysis was performed as a primary screening, followed by investigation using quantitative real-time PCR assays. Gene ontology analysis highlighted the following ontology groups: cell migration, cell motility and localization of cells, structural constituent of cytoskeleton, cell-cell adhesion, oxidoreductase, protein folding and mRNA processing. This study provided preliminary data and suggested potential candidate genes for further description and investigation. CONCLUSIONS: The results suggested that Folr1 gene ablation and abnormal folate homeostasis altered gene expression in developing heart and conotruncal tissues. These changes affected normal cytoskeleton structures, cell migration and motility as well as cellular redox status, which may contribute to cardiovascular abnormalities in mouse embryos lacking Folr1 gene activity. [Abstract/Link to Full Text]

Candiani S, Pestarino M, Cattaneo E, Tartari M
Characterization, developmental expression and evolutionary features of the huntingtin gene in the amphioxus Branchiostoma floridae.
BMC Dev Biol. 2007 Nov 15;7(1):127.
ABSTRACT: BACKGROUND: Huntington's disease is an inherited neurodegenerative disorder that is caused by the expansion of an N terminal polyQ stretch in the huntingtin protein. In order to investigate the hypothesis that huntingtin was already involved in development of the nervous system in the last common ancestor of chordates, we isolated and characterised the huntingtin homologue from the amphioxus Branchiostoma floridae. In the present paper the amphioxus general term must be referred to Branchiostoma floridae. RESULTS: In this report, we show that the exon-intron organization of the amphioxus huntingtin gene is highly conserved with that of other vertebrates species. The AmphiHtt protein has two glutamine residues in the position of the typical vertebrate polyQ tract. Sequence conservation is greater along the entire length of the protein than in a previously identified Ciona huntingtin. The first three N terminal HEAT repeats are highly conserved in vertebrates and amphioxus, although exon rearrangement has occurred in this region. AmphiHtt expression is detectable by in situ hybridization starting from the early neurula stage, where it is found in cells of the neural plate. At later stages, it is retained in the neural compartment but also it appears in limited and well-defined groups of non-neural cells. At subsequent larval stages, AmphiHtt expression is detected in the neural tube, with the strongest signal being present in the most anterior part. CONCLUSIONS: The cloning of amphioxus huntingtin allows to infer that the polyQ in huntingtin was already present 540 million years ago and provides a further element for the study of huntingtin function and its evolution along the deuterostome branch. [Abstract/Link to Full Text]

Aquilina-Beck A, Ilagan K, Liu Q, Liang JO
Nodal signaling is required for closure of the anterior neural tube in zebrafish.
BMC Dev Biol. 2007 Nov 8;7(1):126.
ABSTRACT: BACKGROUND: Nodals are secreted signaling proteins with many roles in vertebrate development. Here, we identify a new role for Nodal signaling in regulating closure of the rostral neural tube of zebrafish. RESULTS: We find that the neural tube in the presumptive forebrain fails to close in zebrafish Nodal signaling mutants. For instance, the cells that will give rise to the pineal organ fail to move from the lateral edges of the neural plate to the midline of the diencephalon. The open neural tube in Nodal signaling mutants may be due in part to reduced function of N-cadherin, a cell adhesion molecule expressed in the neural tube and required for neural tube closure. N-cadherin expression and localization to the membrane are reduced in fish that lack Nodal signaling. Further, N-cadherin mutants and morphants have a pineal phenotype similar to that of mutants with deficiencies in the Nodal pathway. Overexpression of an activated form of the TGFbeta Type I receptor Taram-A (Taram-A*) cell autonomously rescues mesendoderm formation in fish with a severe decrease in Nodal signaling. We find that overexpression of Taram-A* also corrects their open neural tube defect. This suggests that, as in mammals, the mesoderm and endoderm have an important role in regulating closure of the anterior neural tube of zebrafish. CONCLUSIONS: This work helps establish a role for Nodal signals in neurulation, and suggests that defects in Nodal signaling could underlie human neural tube defects such as exencephaly, a fatal condition characterized by an open neural tube in the anterior brain. [Abstract/Link to Full Text]

Thelie A, Papillier P, Pennetier S, Perreau C, Traverso JM, Uzbekova S, Mermillod P, Joly C, Humblot P, Dalbies-Tran R
Differential regulation of abundance and deadenylation of maternal transcripts during bovine oocyte maturation in vitro and in vivo.
BMC Dev Biol. 2007 Nov 7;7(1):125.
ABSTRACT: BACKGROUND: In bovine maturing oocytes and cleavage stage embryos, gene expression is mostly controlled at the post-transcriptional level, through degradation and deadenylation/polyadenylation. We have investigated how post transcriptional control of maternal transcripts was affected during in vitro and in vivo maturation, as a model of differential developmental competence. RESULTS: Using real time PCR, we have analyzed variation of maternal transcripts, in terms of abundance and polyadenylation, during in vitro or in vivo oocyte maturation and in vitro embryo development. Four genes are characterized here for the first time in bovine: ring finger protein 18 (RNF18) and breast cancer anti-estrogen resistance 4 (BCAR4), whose oocyte preferential expression was not previously reported in any species, as well as Maternal embryonic leucine zipper kinase (MELK) and STELLA. We included three known oocyte marker genes (Maternal antigen that embryos require (MATER), Zygote arrest 1 (ZAR1), NACHT, leucine rich repeat and PYD containing 9 (NALP9)). In addition, we selected transcripts previously identified as differentially regulated during maturation, peroxiredoxin 1 and 2 (PRDX1, PRDX2), inhibitor of DNA binding 2 and 3 (ID2, ID3), cyclin B1 (CCNB1), cell division cycle 2 (CDC2), as well as Aurora A (AURKA). Most transcripts underwent a moderate degradation during maturation. But they displayed sharply contrasted deadenylation patterns that account for variations observed previously by DNA array and correlated with the presence of a putative cytoplasmic polyadenylation element in their 3' untranslated region. Similar variations in abundance and polyadenylation status were observed during in vitro maturation or in vivo maturation, except for PRDX1, that appears as a marker of in vivo maturation. Throughout in vitro development, oocyte restricted transcripts were progressively degraded until the morula stage, except for MELK ; and the corresponding genes remained silent after major embryonic genome activation. CONCLUSIONS: Altogether, our data emphasize the extent of post-transcriptional regulation during oocyte maturation. They do not evidence a general alteration of this phenomenon after in vitro maturation as compared to in vivo maturation, but indicate that some individual messenger RNA can be affected. [Abstract/Link to Full Text]

Rolfe KJ, Cambrey AD, Richardson J, Irvine LM, Grobbelaar AO, Linge C
Dermal fibroblasts derived from fetal and postnatal humans exhibit distinct responses to insulin like growth factors.
BMC Dev Biol. 2007 Nov 7;7(1):124.
ABSTRACT: BACKGROUND: It has been well established that human fetuses will heal cutaneous wounds with perfect regeneration. Insulin-like growth factors are pro-fibrotic fibroblast mitogens that have important roles in both adult wound healing and during development, although their relative contribution towards fetal wound healing is currently unknown. We have compared responses to IGF-I and -II in human dermal fibroblast strains derived from early gestational age fetal (<14 weeks) and developmentally mature postnatal skin to identify any differences that might relate to their respective wound healing responses of regeneration or fibrosis. RESULTS: We have established that the mitogenic response of fetal cells to both IGF-I and -II is much lower than that seen in postnatal dermal fibroblasts. Further, unlike postnatal cells, fetal cells fail to synthesise collagen in response to IGF-I, whereas they do increase synthesis in response to IGF-II. This apparent developmentally regulated difference in response to these related growth factors is also reflected in changes in the tyrosine phosphorylation pattern of a number of proteins. Postnatal cells exhibit a significant increase in phosphorylation of ERK 1 (p44) in response to IGF-I and conversely the p46 isoform of Shc on IGF-II stimulation. Fetal cells however only show a significant increase in an unidentified 100kDa tyrosine-phosphorylated protein on stimulation with IGF-II. CONCLUSION: Dermal fibroblasts exhibit different responses to the two forms of IGF depending on their developmental maturity. This may relate to the developmental transition in cutaneous wound healing from regeneration to fibrosis. [Abstract/Link to Full Text]

McGraw S, Morin G, Vigneault C, Leclerc P, Sirard MA
Investigation of MYST4 histone acetyltransferase and its involvement in mammalian gametogenesis.
BMC Dev Biol. 2007 Nov 2;7(1):123.
ABSTRACT: BACKGROUND: Various histone acetylases (HATs) play a critical role in the regulation of gene expression, but the precise functions of many of those HATs are still unknown. Here we provide evidence that MYST4, a known HAT, may be involved in early mammalian gametogenesis. RESULTS: Although MYST4 mRNA transcripts are ubiquitous, protein expression was restricted to select extracts (including ovary and testis). Immunohistochemistry experiments performed on ovary sections revealed that the MYST4 protein is confined to oocytes, granulosa and theca cells, as well as to cells composing the blood vessels. The transcripts for MYST4 and all-MYST4-isoforms were present in oocytes and in in vitro produced embryos. In oocytes and embryos the MYST4 protein was localized in both the cytoplasm and nucleus. Within testis sections, the MYST4 protein was specific to only one cell type, the elongating spermatids, where it was exclusively nuclear. CONCLUSIONS: We established that MYST4 is localized into specialized cells of the ovary and testis. Because the majority of these cells are involved in male and female gametogenesis, MYST4 may contribute to important and specific acetylation events occurring during gametes and embryo development. [Abstract/Link to Full Text]

Fossat N, Le Greneur C, Beby F, Vincent S, Godement P, Chatelain G, Lamonerie T
A new GFP-tagged line reveals unexpected Otx2 protein localization in retinal photoreceptors.
BMC Dev Biol. 2007 Nov 2;7(1):122.
ABSTRACT: BACKGROUND: Dynamic monitoring of protein expression and localization is fundamental to understand biological processes. The paired-class homeodomain-containing transcription factor Otx2 is essential for normal head and brain development in vertebrates. Recent conditional knockout studies have pointed to multiple roles of this protein during late development and post-natal life. Yet, these later expression and functions remain poorly characterized as specific reagents to detect the protein at any stage of development are still missing. RESULTS: We generated a new mouse line harbouring an insertion of the GFP gene within the Otx2 coding sequence to monitor the gene activity while preserving its function. Our results demonstrate that this line represents a convenient tool to capture the dynamics of Otx2 gene expression from early embryonic stages to adulthood. In addition, we could visualize the intracellular location of Otx2 protein. In the retina, we reinterpret the former view of protein distribution and show a further level of regulation of intranuclear protein localization, which depends on the cell type. CONCLUSIONS: The GFP-tagged Otx2 mouse line fully recapitulates previously known expression pattern and brings additional accuracy and easiness of detection of Otx2 gene activity. It opens up the way to live imaging of a highly dynamic actor of brain development and can be adapted to any mutant background to probe for genetic interaction between Otx2 and the mutated gene. [Abstract/Link to Full Text]

Lindner JR, Hillman PR, Barrett AL, Jackson MC, Perry TL, Park Y, Datta S
The Drosophila Perlecan gene trol regulates multiple signaling pathways in different developmental contexts.
BMC Dev Biol. 2007 Nov 2;7(1):121.
ABSTRACT: BACKGROUND: Heparan sulfate proteoglycans modulate signaling by a variety of growth factors. The mammalian proteoglycan Perlecan binds and regulates signaling by Sonic Hedgehog, Fibroblast Growth Factors (FGFs), Vascular Endothelial Growth Factor (VEGF) and Platelet Derived Growth Factor (PDGF), among others, in contexts ranging from angiogenesis and cardiovascular development to cancer progression. The Drosophila Perlecan homolog trol has been shown to regulate the activity of Hedgehog and Branchless (an FGF homolog) to control the onset of stem cell proliferation in the developing brain during first instar. Here we extend the analysis of trol mutant phenotypes to show that trol is required for a variety of developmental events and modulates signaling by multiple growth factors in different situations. RESULTS: Different mutations in trol allow developmental progression to varying extents, suggesting that trol is involved in multiple cell-fate and patterning decisions. Analysis of the initiation of neuroblast proliferation at second instar demonstrated that trol regulates this event by modulating signaling by Hedgehog and Branchless, as it does during first instar. Trol protein is distributed over the surface of the larval brain, near the regulated neuroblasts that reside on the cortical surface. Mutations in trol also decrease the number of circulating plasmatocytes. This is likely to be due to decreased expression of pointed, the response gene for VEGF/PDGF signaling that is required for plasmatocyte proliferation. Trol is found on plasmatocytes, where it could regulate VEGF/PDGF signaling. Finally, we show that in second instar brains but not third instar brain lobes and eye discs, mutations in trol affect signaling by Decapentaplegic (a Transforming Growth Factor family member), Wingless (a Wnt growth factor) and Hedgehog. CONCLUSION: These studies extend the known functions of the Drosophila Perlecan homolog trol in both developmental and signaling contexts. These studies also highlight the fact that Trol function is not dedicated to a single molecular mechanism, but is capable of regulating different growth factor pathways depending on the cell-type and event underway. [Abstract/Link to Full Text]

Londin ER, Mentzer L, Sirotkin HI
Churchill regulates cell movement and mesoderm specification by repressing Nodal signaling.
BMC Dev Biol. 2007 Nov 2;7(1):120.
ABSTRACT: BACKGROUND: Cell movements are essential to the determination of cell fates during development. The zinc-finger transcription factor, Churchill (ChCh) has been proposed to regulate cell fate by regulating cell movements during gastrulation in the chick. However, the mechanism of action of ChCh is not understood. RESULTS: We demonstrate that ChCh acts to repress the response to Nodal-related signals in zebrafish. When ChCh function is abrogated the expression of mesodermal markers is enhanced while ectodermal markers are expressed at decreased levels. In cell transplant assays, we observed that ChCh-deficient cells are more motile than wild-type cells. When placed in wild-type hosts, ChCh-deficient cells often leave the epiblast, migrate to the germ ring and are later found in mesodermal structures. We demonstrate that both movement of ChCh-compromised cells to the germ ring and acquisition of mesodermal character depend on the ability of the donor cells to respond to Nodal signals. Blocking Nodal signaling in the donor cells at the levels of Oep, Alk receptors or Fast1 inhibited migration to the germ ring and mesodermal fate change in the donor cells. We also detect additional unusual movements of transplanted ChCh-deficient cells which suggests that movement and acquisition of mesodermal character can be uncoupled. Finally, we demonstrate that ChCh is required to limit the transcriptional response to Nodal. CONCLUSIONS: These data establish a broad role for ChCh in regulating both cell movement and Nodal signaling during early zebrafish development. We show that chch is required to limit mesodermal gene expression, inhibit Nodal-dependant movement of presumptive ectodermal cells and repress the transcriptional response to Nodal signaling. These findings reveal a dynamic role for chch in regulating cell movement and fate during early development. [Abstract/Link to Full Text]

Wroble BN, Finkielstein CV, Sible JC
Wee1 kinase alters cyclin E/Cdk2 and promotes apoptosis during the early embryonic development of Xenopus laevis.
BMC Dev Biol. 2007 Oct 25;7(1):119.
ABSTRACT: BACKGROUND: The cell cycles of the Xenopus laevis embryo undergo extensive remodeling beginning at the midblastula transition (MBT) of early development. Cell divisions 2-12 consist of rapid cleavages without gap phases or cell cycle checkpoints. Some remodeling events depend upon a critical nucleo-cytoplasmic ratio, whereas others rely on a maternal timer controlled by cyclin E/Cdk2 activity. One key event that occurs at the MBT is the degradation of maternal Wee1, a negative regulator of cyclin-dependent kinase (Cdk) activity. RESULTS: In order to assess the effect of Wee1 on embryonic cell cycle remodeling, Wee1 mRNA was injected into one-cell stage embryos. Overexpression of Wee1 caused cell cycle delay and tyrosine phosphorylation of Cdks prior to the MBT. Furthermore, overexpression of Wee1 disrupted key developmental events that normally occur at the MBT such as the degradation of Cdc25A, cyclin E, and Wee1. Overexpression of Wee1 also resulted in post-MBT apoptosis, tyrosine phosphorylation of Cdks and persistence of cyclin E/Cdk2 activity. To determine whether Cdk2 was required specifically for the survival of the embryo, the cyclin E/Cdk2 inhibitor, delta34-Xic1, was injected in embryos and also shown to induce apoptosis. CONCLUSIONS: Taken together, these data suggest that Wee1 triggers apoptosis through the disruption of the cyclin E/Cdk2 timer. In contrast to Wee1 and delta34-Xic1, altering Cdks by expression of Chk1 and Chk2 kinases blocks rather than promotes apoptosis and causes premature degradation of Cdc25A. Collectively, these data implicate Cdc25A as a key player in the developmentally regulated program of apoptosis in X. laevis embryos. [Abstract/Link to Full Text]

Infante C, Manchado M, Asensio E, Canavate JP
Molecular characterization, gene expression and dependence on thyroid hormones of two type I keratin genes (sseKer1 and sseKer2) in the flatfish Senegalese sole (Solea senegalensis Kaup).
BMC Dev Biol. 2007 Oct 23;7(1):118.
ABSTRACT: BACKGROUND: Keratins make up the largest subgroup of intermediate filaments, and, in chordates, represent the most abundant proteins in epithelial cells. They have been associated with a wide range of functions in the cell, but little information is still available about their expression profile and regulation during flatfish metamorphosis. Senegalese sole (Solea senegalensis) is a commercially important flatfish in which no keratin gene has been described yet. RESULTS: The development of large-scale genomics on Senegalese sole has facilitated the identification of two different type I keratin genes referred to as sseKer1 and sseKer2. Main characteristics and sequence identities with other fish and mammal keratins are described. Phylogenetic analyses grouped sseKer1 and sseKer2 in a significant clade with other teleost epidermal type I keratins, and have allowed for the identification of sseKer2 as a novel keratin. The expression profile of both genes was studied during larval development and in tissues using a real-time approach. sseKer1 and sseKer2 mRNA levels were significantly higher in skin than in other tissues examined. During metamorphosis, sseKer1 transcripts increased significantly at first stages, and reduced thereafter. In contrast, sseKer2 mRNA levels did not change during early metamorphosis although a significant drop at metamorphosis climax and late metamorphosis was also detected. To study the possible regulation of sseKer gene expressions by thyroid hormones (THs), larvae were exposed to the goitrogen thiourea (TU). TU-treated larvae exhibited higher sseKer1 and sseKer2 mRNA levels than untreated control at both 11 and 15 days after treatment. Moreover, addition of exogenous T4 hormone to TU-treated larvae restored or even reduced the steady-state levels with respect to the untreated control, demonstrating that expression of both genes is negatively regulated by THs. CONCLUSION: We have identified two keratin genes, referred to as sseKer1 and sseKer2, in Senegalese sole. Phylogenetic analyses revealed sseKer2 as a novel keratin. Although they exhibit different expression patterns during larval development, both of them are negatively regulated by THs. The co-regulation by THs could explain the reduction of both keratin transcripts after the metamorphosis climax, suggesting their role in the tissue remodelling processes that occur during metamorphosis. [Abstract/Link to Full Text]

Fauque P, Jouannet P, Lesaffre C, Ripoche MA, Dandolo L, Vaiman D, Jammes H
Assisted Reproductive Technology affects developmental kinetics, H19 Imprinting Control Region methylation and H19 gene expression in individual mouse embryos.
BMC Dev Biol. 2007 Oct 18;7(1):116.
ABSTRACT: BACKGROUND: In the last few years, an increase in imprinting anomalies has been reported in children born from Assisted Reproductive Technology (ART). Various clinical and experimental studies also suggest alterations of embryo development after ART. Therefore, there is a need for studying early epigenetic anomalies which could result from ART manipulations, especially on single embryos. In this study, we evaluated the impact of superovulation, in vitro fertilization (IVF) and embryo culture conditions on proper genomic imprinting and blastocyst development in single mouse embryos. In this study, different experimental groups were established to obtain embryos from superovulated and non-superovulated females, either from in vivo or in vitro fertilized oocytes, themselves grown in vitro or not. The embryos were cultured either in M16 medium or in G1.2/G2.2 sequential medium. The methylation status of H19 Imprinting Control Region (ICR) and H19 proximal promoter (PP) was assessed, as well as the gene expression level of H19, in individual blastocysts. In parallel, we have evaluated embryo cleavage kinetics and recorded morphological data. RESULTS: We show that: 1. the culture medium influences early embryo development with faster cleavage kinetics for culture in G1.2/G2.2 medium compared to M16 medium. 2. Epigenetic alterations of the H19 ICR and H19 PP are influenced by the fertilization method since methylation anomalies were observed only in the in vitro fertilized subgroup, however to different degrees according to the culture medium. 3. Superovulation clearly disrupted H19 gene expression in individual blastocysts. Moreover, when embryos were cultured in vitro after either in vivo or in vitro fertilization, the percentage of blastocysts which expressed H19 was higher in G1.2/G2.2 medium compared to M16. CONCLUSIONS: Compared to previous reports utilizing pools of embryos, our study enables to emphasize a high individual variability of blastocysts in the H19 ICR and H19 proximal promoter methylation and H19 gene expression, with a striking effect of each manipulation associated to ART practices. Our results suggest that H19 could be used as a sensor of the invasiveness of the utilized techniques. [Abstract/Link to Full Text]

Li Z, Korzh V, Gong Z
Localized rbp4 expression in the yolk syncytial layer plays a role in yolk cell extension and early liver development.
BMC Dev Biol. 2007 Oct 19;7(1):117.
ABSTRACT: BACKGROUND: The number of genes characterized in liver development is steadily increasing, but the origin of liver precursor cells and the molecular control of liver formation remain poorly understood. Existing theories about formation of zebrafish visceral organs emphasize either their budding from the endodermal rod or formation of independent anlage followed by their later fusion, but none of these is completely satisfactory in explaining liver organogenesis in zebrafish. RESULTS: Expression of a gene encoding the retinol binding protein 4 (Rbp4) was analyzed in zebrafish. rbp4, which is expressed mainly in the liver in adults, was shown to be expressed in the yolk syncytial layer (YSL) during early embryogenesis. At 12-16 hpf rbp4 expression was restricted to the ventro-lateral YSL and later expanded to cover the posterior YSL. We demonstrated that rbp4 expression was negatively regulated by Nodal and Hedgehog (Hh) signalling and positively controlled by retinoic acid (RA). Knockdown of Rbp4 in the YSL resulted in shortened yolk extension as well as the formation of two liver buds, which could be due to impaired migration of liver progenitor cells. rbp4 appears also to regulate the extracellular matrix protein Fibronectin1 (Fn1) specifically in the ventro-lateral yolk, indicating a role of Fn1 in liver progenitor migration. Since exocrine pancreas, endocrine pancreas, intestine and heart developed normally in Rbp4 morphants, we suggest that rbp4 expression in the YSL is required only for liver development. CONCLUSION: The characteristic expression pattern of rbp4 suggests that the YSL is patterned despite its syncytial nature. YSL-expressed Rbp4 plays a role in formation of both yolk extension and liver bud, the latter may also require migration of liver progenitor cells. [Abstract/Link to Full Text]

San Miguel-Ruiz JE, Garcia-Arraras JE
Common cellular events occur during wound healing and organ regeneration in the sea cucumber Holothuria glaberrima.
BMC Dev Biol. 2007 Oct 18;7(1):115.
ABSTRACT: BACKGROUND: All animals possess some type of tissue repair mechanism. In some species, the capacity to repair tissues is limited to the healing of wounds. Other species, such as echinoderms, posses a striking repair capability that can include the replacement of entire organs. It has been reported that some mechanisms, namely extracellular matrix remodeling, appear to occur in most repair processes. However, it remains unclear to what extent the process of organ regeneration, particularly in animals where loss and regeneration of complex structures is a programmed natural event, is similar to wound healing. We have now used the sea cucumber Holothuria glaberrima to address this question. RESULTS: Animals were lesioned by making a 3-5 mm transverse incision between one of the longitudinal muscle pairs along the bodywall. Lesioned tissues included muscle, nerve, water canal and dermis. Animals were allowed to heal for up to four weeks (2, 6, 12, 20, and 28 days post-injury) before sacrificed. Tissues were sectioned in a cryostat and changes in cellular and tissue elements during repair were evaluated using classical dyes, immmuohistochemistry and phalloidin labeling. In addition, the temporal and spatial distribution of cell proliferation in the animals was assayed using BrdU incorporation. We found that cellular events associated with wound healing in H. glaberrima correspond to those previously shown to occur during intestinal regeneration. These include: (1) an increase in the number of spherule-containing cells, (2) remodeling of the extracellular matrix, (3) formation of spindle-like structures that signal dedifferentiation of muscle cells in the area flanking the lesion site and (4) intense cellular division occurring mainly in the coelomic epithelium after the first week of regeneration. CONCLUSIONS: Our data indicate that H. glaberrima employs analogous cellular mechanisms during wound healing and organ regeneration. Thus, it is possible that regenerative limitations in some organisms are due either to the absence of particular mechanisms associated with repair or the inability of activating the repair process in some tissues or stages. [Abstract/Link to Full Text]

Alvarez Y, Cederlund ML, Cottell DC, Bill BR, Ekker SC, Torres-Vazquez J, Weinstein BM, Hyde DR, Vihtelic TS, Kennedy BN
Genetic determinants of hyaloid and retinal vasculature in zebrafish.
BMC Dev Biol. 2007 Oct 15;7(1):114.
ABSTRACT: BACKGROUND: The retinal vasculature is a capillary network of blood vessels that nourishes the inner retina of most mammals. Developmental abnormalities or microvascular complications in the retinal vasculature result in severe human eye diseases that lead to blindness. To exploit the advantages of zebrafish for genetic, developmental and pharmacological studies of retinal vasculature, we characterised the intraocular vasculature in zebrafish. RESULTS: We show a detailed morphological and developmental analysis of the retinal blood supply in zebrafish. Similar to the transient hyaloid vasculature in mammalian embryos, vessels are first found attached to the zebrafish lens at 2.5 days post fertilisation. These vessels progressively lose contact with the lens and by 30 days post fertilisation adhere to the inner limiting membrane of the juvenile retina. Ultrastructure analysis shows these vessels to exhibit distinctive hallmarks of mammalian retinal vasculature. For example, smooth muscle actin-expressing pericytes are ensheathed by the basal lamina of the blood vessel, and vesicle vacuolar organelles (VVO), subcellular mediators of vessel-retinal nourishment, are present. Finally, we identify 9 genes with cell membrane, extracellular matrix and unknown identity that are necessary for zebrafish hyaloid and retinal vasculature development. CONCLUSIONS: Zebrafish have a retinal blood supply with a characteristic developmental and adult morphology. Abnormalities of these intraocular vessels are easily observed, enabling application of genetic and chemical approaches in zebrafish to identify molecular regulators of hyaloid and retinal vasculature in development and disease. [Abstract/Link to Full Text]

Hempel LU, Oliver B
Sex-specific Doublesex[M] expression in subsets of Drosophila somatic gonad cells.
BMC Dev Biol. 2007 Oct 12;7(1):113.
ABSTRACT: BACKGROUND: In Drosophila melanogaster, a pre-mRNA splicing hierarchy controls sexual identity and ultimately leads to sex-specific Doublesex (DSX) transcription factor isoforms. The male-specific DSXM represses genes involved in female development and activates genes involved in male development. Spatial and temporal control of dsx during embryogenesis is not well documented. RESULTS: Here we show that DSXM is specifically expressed in subsets of male somatic gonad cells during embryogenesis. Following testis formation, germ cells remain in contact with DSXM-expressing cells, including hub cells and premeiotic somatic cyst cells that surround germ cells during spermatogenesis in larval and adult testes. CONCLUSIONS: We show that dsx is transcriptionally regulated in addition to being regulated at the pre-mRNA splicing level by the sex determination hierarchy. The dsx locus is spatially controlled by somatic gonad identity. The continuous expression of DSXM in cells contacting the germline suggests an ongoing short-range influence of the somatic sex determination pathway on germ cell development. [Abstract/Link to Full Text]

Oshima K, Teo DT, Senn P, Starlinger V, Heller S
LIF promotes neurogenesis and maintains neural precursors in cell populations derived from spiral ganglion stem cells.
BMC Dev Biol. 2007;7112.
BACKGROUND: Stem cells with the ability to form clonal floating colonies (spheres) were recently isolated from the neonatal murine spiral ganglion. To further examine the features of inner ear-derived neural stem cells and their derivatives, we investigated the effects of leukemia inhibitory factor (LIF), a neurokine that has been shown to promote self-renewal of other neural stem cells and to affect neural and glial cell differentiation. RESULTS: LIF-treatment led to a dose-dependent increase of the number of neurons and glial cells in cultures of sphere-derived cells. Based on the detection of developmental and progenitor cell markers that are maintained in LIF-treated cultures and the increase of cycling nestin-positive progenitors, we propose that LIF maintains a pool of neural progenitor cells. We further provide evidence that LIF increases the number of nestin-positive progenitor cells directly in a cell cycle-independent fashion, which we interpret as an acceleration of neurogenesis in sphere-derived progenitors. This effect is further enhanced by an anti-apoptotic action of LIF. Finally, LIF and the neurotrophins BDNF and NT3 additively promote survival of stem cell-derived neurons. CONCLUSION: Our results implicate LIF as a powerful tool to control neural differentiation and maintenance of stem cell-derived murine spiral ganglion neuron precursors. This finding could be relevant in cell replacement studies with animal models featuring spiral ganglion neuron degeneration. The additive effect of the combination of LIF and BDNF/NT3 on stem cell-derived neuronal survival is similar to their effect on primary spiral ganglion neurons, which puts forward spiral ganglion-derived neurospheres as an in vitro model system to study aspects of auditory neuron development. [Abstract/Link to Full Text]

Glassmann A, Molly S, Surchev L, Nazwar TA, Holst M, Hartmann W, Baader SL, Oberdick J, Pietsch T, Schilling K
Developmental expression and differentiation-related neuron-specific splicing of metastasis suppressor 1 (Mtss1) in normal and transformed cerebellar cells.
BMC Dev Biol. 2007 Oct 9;7(1):111.
ABSTRACT: BACKGROUND: Mtss1 encodes an actin-binding protein, dysregulated in a variety of tumors, that interacts with sonic hedgehog/Gli signaling in epidermal cells. Given the prime importance of this pathway for cerebellar development and tumorigenesis, we assessed expression of Mtss1 in the developing murine cerebellum and human medulloblastoma specimens. RESULTS: During development, Mtss1 is transiently expressed in granule cells, from the time point they cease to proliferate to their synaptic integration. It is also expressed by granule cell precursor-derived medulloblastomas. In the adult CNS, Mtss1 is found exclusively in cerebellar Purkinje cells. Neuronal differentiation is accompanied by a switch in Mtss1 splicing. Whereas immature granule cells express a Mtss1 variant observed also in peripheral tissues and comprising exon 12, this exon is replaced by a CNS-specific exon, 12a, in more mature granule cells and in adult Purkinje cells. Bioinformatic analysis of Mtss1 suggests that differential exon usage may affect interaction with Fyn and Src, two tyrosine kinases previously recognized as critical for cerebellar cell migration and histogenesis. Further, this approach led to the identification of two evolutionary conserved nuclear localization sequences. These overlap with the actin filament binding site of Mtss1, and one also harbors a potential PKA and PKC phosphorylation site. CONCLUSIONS: Both the pattern of expression and splicing of Mtss1 is developmentally regulated in the murine cerebellum. These findings are discussed with a view on the potential role of Mtss1 for cytoskeletal dynamics in developing and mature cerebellar neurons. [Abstract/Link to Full Text]

Dullin JP, Locker M, Robach M, Henningfeld KA, Parain K, Afelik S, Pieler T, Perron M
Ptf1a triggers GABAergic neuronal cell fates in the retina.
BMC Dev Biol. 2007 Oct 2;7(1):110.
ABSTRACT: BACKGROUND: In recent years, considerable knowledge has been gained on the molecular mechanisms underlying retinal cell fate specification. However, hitherto studies focused primarily on the six major retinal cell classes (five types of neurons of one type of glial cell), and paid little attention to the specification of different neuronal subtypes within the same cell class. In particular, the molecular machinery governing the specification of the two most abundant neurotransmitter phenotypes in the retina, GABAergic and glutamatergic, is largely unknown. In the spinal cord and cerebellum, the transcription factor Ptf1a is essential for GABAergic neuron production. In the mouse retina, Ptf1a has been shown to be involved in horizontal and most amacrine neurons differentiation. RESULTS: In this study, we examined the distribution of neurotransmitter subtypes following Ptf1a gain and loss of function in the Xenopus retina. We found cell-autonomous dramatic switches between GABAergic and glutamatergic neuron production, concomitant with profound defects in the genesis of amacrine and horizontal cells, which are mainly GABAergic. Therefore, we investigated whether Ptf1a promotes the fate of these two cell types or acts directly as a GABAergic subtype determination factor. In ectodermal explant assays, Ptf1a was found to be a potent inducer of the GABAergic subtype. Moreover, clonal analysis in the retina revealed that Ptf1a overexpression leads to an increased ratio of GABAergic subtypes among the whole amacrine and horizontal cell population, highlighting its instructive capacity to promote this specific subtype of inhibitory neurons. Finally, we also found that within bipolar cells, which are typically glutamatergic interneurons, Ptf1a is able to trigger a GABAergic fate. CONCLUSIONS: Altogether, our results reveal for the first time in the retina a major player in the GABAergic versus glutamatergic cell specification genetic pathway. [Abstract/Link to Full Text]

Murani E, Muraniova M, Ponsuksili S, Schellander K, Wimmers K
Identification of genes differentially expressed during prenatal development of skeletal muscle in two pig breeds differing in muscularity.
BMC Dev Biol. 2007 Oct 1;7(1):109.
ABSTRACT: BACKGROUND: Postnatal muscle growth is largely depending on the number and size of muscle fibers. The number of myofibers and to a large extent their metabolic and contractile properties, which also influence their size, are determined prenatally during the process of myogenesis. Hence identification of genes and their networks governing prenatal development of skeletal muscles will provide insight into the control of muscle growth and facilitate finding the source of its variation. So far most of the genes involved in myogenesis were identified by in vitro studies using gene targeting and transgenesis. Profiling of transcriptome changes during the myogenesis in vivo promises to obtain a more complete picture. In order to address this, we performed transcriptome profiling of prenatal skeletal muscle using differential display RT-PCR as on open system with the potential to detect novel transcripts. Seven key stages of myogenesis (days 14, 21, 35, 49, 63, 77 and 91 post conception) were studied in two breeds, Pietrain and Duroc, differing markedly in muscularity and muscle structure. RESULTS: Eighty prominent cDNA fragments were sequenced, 43 showing stage-associated and 37 showing breed-associated differences in the expression, respectively. Out of the resulting 85 unique expressed sequence tags, EST, 52 could be assigned to known genes. The most frequent functional categories represented genes encoding myofibrillar proteins (8), genes involved in cell adhesion, cell-cell signaling and extracellular matrix synthesis/remodeling (8), genes regulating gene expression (8), and metabolism genes (8). Some of the EST that showed no identity to any known transcripts in the databases are located in introns of known genes and most likely represent novel exons (e.g. HMGA2). Expression of thirteen transcripts along with five reference genes was further analyzed by means of real-time quantitative PCR. Nine of the target transcripts showed higher than twofold differences in the expression between the two breeds (GATA3, HMGA2, NRAP, SMC6L1, SPP1, RAB6IP2, TJP1 and two EST). CONCLUSION: The present study revealed several genes and novel transcripts not previously associated with myogenesis and expands our knowledge of genetic factors operating during myogenesis. Genes that exhibited differences between the divergent breeds represent candidate genes for muscle growth and structure. [Abstract/Link to Full Text]

Katsantoni EZ, Anghelescu NE, Rottier R, Moerland M, Antoniou M, de Crom R, Grosveld F, Strouboulis J
Ubiquitous expression of the rtTA2S-M2 inducible system in transgenic mice driven by the human hnRNPA2B1/CBX3 CpG island.
BMC Dev Biol. 2007;7108.
BACKGROUND: A sensitive, ubiquitously expressed tetracycline inducible system would be a valuable tool in mouse transgenesis. However, this has been difficult to obtain due to position effects observed at different chromosomal sites of transgene integration, which negatively affect expression in many tissues. The aim of this study was to test the utility of a mammalian methylation-free CpG island to drive ubiquitous expression of the sensitive doxycycline (Dox) inducible rtTA2S-M2 Tet-transactivator in transgenic mice. RESULTS: An 8 kb genomic fragment from the methylation-free CpG island of the human hnRNPA2B1-CBX3 housekeeping gene locus was tested. In a number of transgenic mouse lines obtained, rtTA2S-M2 expression was detected in many tissues examined. Characterisation of the highest expressing rtTA2S-M2 transgenic mouse line demonstrated Dox-inducible GFP transgene expression in many tissues. Using this line we also show highly sensitive quantitative induction with low doses of Dox of an assayable plasma protein transgene under the control of a Tet Responsive Element (TRE). The utility of this rtTA2S-M2 line for inducible expression in mouse embryos was also demonstrated using a GATA-6 Tet-inducible transgene to show specific phenotypes in the embryonic lung, as well as broader effects resulting from the inducible widespread overexpression of the transgene. CONCLUSION: The ubiquitously expressing rtTA2S-M2 transgenic mouse line described here provides a very useful tool for studying the effects of the widespread, inducible overexpression of genes during embryonic development and in adult mice. [Abstract/Link to Full Text]

Falk J, Drinjakovic J, Leung KM, Dwivedy A, Regan AG, Piper M, Holt CE
Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in Xenopus.
BMC Dev Biol. 2007 Sep 27;7(1):107.
ABSTRACT: BACKGROUND: Blastomere injection of mRNA or antisense oligonucleotides has proven effective in analyzing early gene function in Xenopus. However, functional analysis of genes involved in neuronal differentiation and axon pathfinding by this method is often hampered by earlier function of these genes during development. Therefore, fine spatio-temporal control of over-expression or knock-down approaches is required to specifically address the role of a given gene in these processes. RESULTS: We describe here an electroporation procedure that can be used with high efficiency and low toxicity for targeting DNA and antisense morpholino oligonucleotides (MOs) into spatially restricted regions of the Xenopus CNS at a critical time-window of development (22-50 hour post-fertilization) when axonal tracts are first forming. The approach relies on the design of "electroporation chambers" that enable reproducible positioning of fixed-spaced electrodes coupled with accurate DNA/MO injection. Simple adjustments can be made to the electroporation chamber to suit the shape of different aged embryos and to alter the size and location of the targeted region. This procedure can be used to electroporate distinct regions of the CNS in the same embryo allowing separate manipulation of growing axons and their intermediate and final targets in the brain. CONCLUSION: Our study demonstrates that electroporation can be used as a versatile tool to investigate molecular pathways involved in axon extension during Xenopus embryogenesis. Electroporation enables gain or loss of function studies to be performed with easy monitoring of electroporated cells. Double-targeted transfection provides a unique opportunity to monitor axon-target interaction in vivo. Finally, electroporated embryos represent a valuable source of MO-loaded or DNA transfected cells for in vitro analysis. The technique has broad applications as it can be tailored easily to other developing organ systems and to other organisms by making simple adjustments to the electroporation chamber. [Abstract/Link to Full Text]

Villa-Cuesta E, González-Pérez E, Modolell J
Apposition of iroquois expressing and non-expressing cells leads to cell sorting and fold formation in the Drosophila imaginal wing disc.
BMC Dev Biol. 2007;7106.
BACKGROUND: The organization of the different tissues of an animal requires mechanisms that regulate cell-cell adhesion to promote and maintain the physical separation of adjacent cell populations. In the Drosophila imaginal wing disc the iroquois homeobox genes are expressed in the notum anlage and contribute to the specification of notum identity. These genes are not expressed in the adjacent wing hinge territory. These territories are separated by an approximately straight boundary that in the mature disc is associated with an epithelial fold. The mechanism by which these two cell populations are kept separate is unclear. RESULTS: Here we show that the Iro-C genes participate in keeping the notum and wing cell populations separate. Indeed, within the notum anlage, cells not expressing Iro-C tend to join together and sort out from their Iro-C expressing neighbours. We also show that apposition of Iro-C expressing and non-expressing cells induces invagination and apico-basal shortening of the Iro-C- cells. This effect probably underlies formation of the fold that separates the notum and wing hinge territories. In addition, cells overexpressing a member of the Iro-C contact one another and become organized in a network of thin strings that surrounds and isolates large groups of non-overexpressing cells. The strings appear to exert a pulling force along their longitudinal axis. CONCLUSION: Apposition of cells expressing and non-expressing the Iro-C, as it occurs in the notum-wing hinge border of the Drosophila wing disc, influences cell behaviour. It leads to cell sorting, and cellular invagination and apical-basal shortening. These effects probably account for keeping the prospective notum and wing hinge cell populations separate and underlie epithelial fold formation. Cells that overexpress a member of the Iro-C and that confront non-expressing cells establish contacts between themselves and become organized in a network of thin strings. This is a complex and unique phenotype that might be important for the generation of a straight notum-wing hinge border. [Abstract/Link to Full Text]

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Recent Articles in BMC Evolutionary Biology

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Patil PB, Bogdanove AJ, Sonti RV
The role of horizontal transfer in the evolution of a highly variable lipopolysaccharide biosynthesis locus in xanthomonads that infect rice, citrus and crucifers.
BMC Evol Biol. 2007 Dec 6;7(1):243.
ABSTRACT: BACKGROUND: Lipopolysaccharide (LPS) is a pathogen associated molecular pattern (PAMP) of animal and plant pathogenic bacteria. Variation at the interstrain level is common in LPS biosynthetic gene clusters of animal pathogenic bacteria. This variation has been proposed to play a role in evading the host immune system. Even though LPS is a modulator of plant defense responses, reports of interstrain variation in LPS gene clusters of plant pathogenic bacteria are rare. RESULTS: In this study we report the complete sequence of a variant 19.9 kb LPS locus present in the BXO8 strain of Xanthomonas oryzae pv. oryzae (Xoo), the bacterial blight pathogen of rice. This region is completely different in size, number and organization of genes from the canonical LPS locus present in most other strains of Xoo from India and Asia. Surprisingly, except for one ORF, all the other ORFs at the BXO8 LPS locus are orthologous to the genes present at this locus in a sequenced strain of X. axonopodis pv. citri (Xac; a pathogen of citrus plants). One end of the BXO8 LPS gene cluster, comprised of ten genes, is also present in the related rice pathogen, X. oryzae pv. oryzicola (Xoc). In Xoc, the remainder of the LPS gene cluster, consisting of seven genes, is novel and unrelated to LPS gene clusters of any of the sequenced xanthomonads. We also report substantial interstrain variation suggestive of very recent horizontal gene transfer (HGT) at the LPS biosynthetic locus of Xanthomonas campestris pv. campestris (Xcc), the black rot pathogen of crucifers. CONCLUSION: Our analyses indicate that HGT has altered the LPS locus during the evolution of Xanthomonas oryzae pathovars and suggest that the ancestor of all Xanthomonas oryzae pathovars had an Xac type of LPS gene cluster. Our finding of interstrain variation in two major xanthomonad pathogens infecting different hosts suggests that the LPS locus in plant pathogenic bacteria, as in animal pathogens, is under intense diversifying selection. [Abstract/Link to Full Text]

Dutheil J, Galtier N
Detecting groups of co-evolving positions in a molecule: a clustering approach.
BMC Evol Biol. 2007 Nov 30;7(1):242.
ABSTRACT: BACKGROUND: Although the patterns of co-substitutions in RNA is now well characterized, detection of co-evolving positions in proteins remains a difficult task. It has been recognized that the signal is typically weak, due to the fact that (i) amino-acid are characterized by various biochemical properties, so that distinct amino-acids changes are not functionally equivalent, and (ii) a given mutation can be compensated by more than one mutation, at more than one position. RESULTS: We present a new method based on phylogenetic substitution mapping. The two above-mentioned problems are addressed by (i) the introduction of a weighted mapping, which accounts for the biochemical effects (volume, polarity, charge) of amino-acid changes, (ii) the use of a clustering approach to detect groups of co-evolving sites of virtually any size, and (iii) the distinction between biochemical compensation and other coevolutionary mechanisms. We apply this methodology to a previously studied data set of bacterial ribosomal RNA, and to three protein data sets (myoglobin of vertebrates, S-locus Receptor Kinase and Methionine Amino-Peptidase). CONCLUSIONS: We succeed in detecting groups of sites which significantly depart the null hypothesis of independence. Group sizes range from pairs to groups of size ~10, depending on the substitution weights used. The structural and functional relevance of these groups of sites are assessed, and the various evolutionary processes potentially generating correlated substitution patterns are discussed. [Abstract/Link to Full Text]

Ranwez V, Delsuc F, Ranwez S, Belkir K, Tilak MK, Douzery EJ
OrthoMaM: A database of orthologous genomic markers for placental mammal phylogenetics.
BMC Evol Biol. 2007 Nov 30;7(1):241.
ABSTRACT: BACKGROUND: Molecular sequence data have become the standard in modern day phylogenetics. In particular, several long-standing questions of mammalian evolutionary history have been recently resolved thanks to the use of molecular characters. Yet, most studies have focused on only a handful of standard markers. The availability of an ever increasing number of whole genome sequences is a golden mine for modern systematics. Genomic data now provides the opportunity to select new markers that are potentially relevant for further resolving branches of the mammalian phylogenetic tree at various taxonomic levels. Description: The EnsEMBL database was used to determine a set of orthologous genes from 12 available complete mammalian genomes. As targets for possible amplification and sequencing in additional taxa, more than 3,000 exons of length > 400 bp have been selected, among which 118, 368, 608, and 674 are respectively retrieved for 12, 11, 10, and 9 species. A bioinformatic pipeline has been developed to provide evolutionary descriptors for these candidate markers in order to assess their potential phylogenetic utility. The resulting OrthoMaM (Orthologous Mammalian Markers) database can be queried and alignments can be downloaded through a dedicated web interface (http://kimura.univ-montp2.fr/orthomam). CONCLUSION: The importance of marker choice in phylogenetic studies has long been stressed. Our database centered on complete genome information now makes possible to select promising markers to a given phylogenetic question or a systematic framework by querying a number of evolutionary descriptors. The usefulness of the database is illustrated with two biological examples. First, two potentially useful markers were identified for rodent systematics based on relevant evolutionary parameters and sequenced in additional species. Second, a complete, gapless 94 kb supermatrix of 118 orthologous exons was assembled for 12 mammals. Phylogenetic analyses using probabilistic methods unambiguously supported the new placental phylogeny by retrieving the monophyly of Glires, Euarchontoglires, Laurasiatheria, and Boreoeutheria. Muroid rodents thus do not represent a basal placental lineage as it was mistakenly reasserted in some recent phylogenomic analyses based on fewer taxa. We expect the OrthoMaM database to be useful for further resolving the phylogenetic tree of placental mammals and for better understanding the evolutionary dynamics of their genomes, i.e., the forces that shaped coding sequences in terms of selective constraints. [Abstract/Link to Full Text]

Andras P, Lazarus J, Roberts G
Environmental adversity and uncertainty favour cooperation.
BMC Evol Biol. 2007 Nov 30;7(1):240.
ABSTRACT: BACKGROUND: A major cornerstone of evolutionary biology theory is the explanation of the emergence of cooperation in communities of selfish individuals. There is an unexplained tendency in the plant and animal world - with examples from alpine plants, worms, fish, mole-rats, monkeys and humans - for cooperation to flourish where the environment is more adverse (harsher) or more unpredictable. RESULTS: Using mathematical arguments and computer simulations we show that in more adverse environments individuals perceive their resources to be more unpredictable, and that this unpredictability favours cooperation. First we show analytically that in a more adverse environment the individual experiences greater perceived uncertainty. Second we show through a simulation study that more perceived uncertainty produces a higher level of cooperation in communities of selfish individuals. CONCLUSIONS: This study captures the essential features of the natural examples: the positive impact of resource adversity or uncertainty on cooperation. These newly discovered connections between environmental adversity, uncertainty and cooperation help to explain the emergence and evolution of cooperation in animal and human societies. [Abstract/Link to Full Text]

Abbasi AA, Grzeschik KH
An insight into the phylogenetic history of HOX linked gene families in vertebrates.
BMC Evol Biol. 2007 Nov 30;7(1):239.
ABSTRACT: BACKGROUND: The human chromosomes 2q, 7, 12q and 17q show extensive intra-genomic homology, containing duplicate, triplicate and quadruplicate paralogous regions centered on the HOX gene clusters. The fact that two or more representatives of different gene families are linked with HOX clusters is taken as evidence that these paralogous gene sets might have arisen from a single chromosomal segment through block or whole chromosome duplication events. This would imply that the constituent genes including the HOX clusters reflect the architecture of a single ancestral block (before vertebrate origin) where all of these genes were linked in a single copy. RESULTS: In the present study we have employed the currently available set of protein data for a wide variety of vertebrate and invertebrate genomes to analyze the phylogenetic history of 11 multigene families with three or more of their representatives linked to human HOX clusters. A topology comparison approach revealed four discrete co-duplicated groups: group 1 involves the genes from GLI, HH, INHB, IGFBP (cluster-1), and SLC4A families; group 2 involves ERBB, ZNFN1A, and IGFBP (cluster-2) gene families; group 3 involves the HOX clusters and the SP gene family; group 4 involves the integrin beta chain and myosine light chain families. The distinct genes within each co-duplicated group share the same evolutionary history and are duplicated in concert with each other, while the constituent genes of two different co-duplicated groups may not share their evolutionary history and may not have duplicated simultaneously. CONCLUSIONS: We conclude that co-duplicated groups may themselves be remnants of ancient small-scale duplications (involving chromosomal segments or gene-clusters) which occurred at different time points during chordate evolution. Whereas the recent combination of genes from distinct co-duplicated groups on different chromosomal regions (human chromosomes 2q, 7, 12q, and 17q) is probably the outcome of subsequent rearrangement of genomic segments, including syntenic groups of genes. [Abstract/Link to Full Text]

Tinsley MC, Majerus ME
Small steps or giant leaps for male-killers? Phylogenetic constraints to male-killer host shifts.
BMC Evol Biol. 2007 Nov 29;7(1):238.
ABSTRACT: BACKGROUND: Arthropods are infected by a wide diversity of maternally transmitted microbes. Some of these manipulate host reproduction to facilitate population invasion and persistence. Such parasites transmit vertically on an ecological timescale, but rare horizontal transmission events have permitted colonisation of new species. Here we report the first systematic investigation into the influence of the phylogenetic distance between arthropod species on the potential for reproductive parasite interspecific transfer. RESULTS: We employed a well characterised reproductive parasite, a coccinellid beetle male-killer, and artificially injected the bacterium into a series of novel species. Genetic distances between native and novel hosts were ascertained by sequencing sections of the 16S and 12S mitochondrial rDNA genes. The bacterium colonised host tissues and transmitted vertically in all cases tested. However, whilst transmission efficiency was perfect within the native genus, this was reduced following some transfers of greater phylogenetic distance. The bacterium's ability to distort offspring sex ratios in novel hosts was negatively correlated with the genetic distance of transfers. Male-killing occurred with full penetrance following within-genus transfers; but whilst sex ratio distortion generally occurred, it was incomplete in more distantly related species. CONCLUSIONS: This study indicates that the natural interspecific transmission of reproductive parasites might be constrained by their ability to tolerate the physiology or genetics of novel hosts. Our data suggest that horizontal transfers are more likely between closely related species. Successful bacterial transfer across large phylogenetic distances may require rapid adaptive evolution in the new species. This finding has applied relevance regarding selection of suitable bacteria to manipulate insect pest and vector populations by symbiont gene-drive systems. [Abstract/Link to Full Text]

Lemoine F, Lespinet O, Labedan B
Assessing the evolutionary rate of positional orthologous genes in prokaryotes using synteny data.
BMC Evol Biol. 2007 Nov 29;7(1):237.
ABSTRACT: BACKGROUND: Comparison of completely sequenced microbial genomes has revealed how fluid these genomes are. Detecting synteny blocks requires reliable methods to determining the orthologs among the whole set of homologs detected by exhaustive comparisons between each pair of completely sequenced genomes. This is a complex and difficult problem in the field of comparative genomics but will help to better understand the way prokaryotic genomes are evolving. RESULTS: We have developed a suite of programs that automate three essential steps to study conservation of gene order, and validated them with a set of 107 bacteria and archaea that cover the majority of the prokaryotic taxonomic space. We identified the whole set of shared homologs between two or more species and computed the evolutionary distance separating each pair of homologs. We applied two strategies to extract from the set of homologs a collection of valid orthologs shared by at least two genomes. The first computes the Reciprocal Smallest Distance (RSD) using the PAM distances separating pairs of homologs. The second method groups homologs in families and reconstructs each family's evolutionary tree, distinguishing bona fide orthologs as well as paralogs created after the last speciation event. Although the phylogenetic tree method often succeeds where RSD fails, the reverse could occasionally be true. Accordingly, we used the data obtained with either methods or their intersection to number the orthologs that are adjacent in for each pair of genomes, the Positional Orthologous Genes (POGs), and to further study their properties. Once all these synteny blocks have been detected, we showed that POGs are subject to more evolutionary constraints than orthologs outside synteny groups, whichever the taxonomic distance separating the compared organisms. CONCLUSIONS: The suite of programs described in this paper allows a reliable detection of orthologs and is useful for evaluating gene order conservation in prokaryotes whichever their taxonomic distance. Thus, our approach will make easy the rapid identification of POGS in the next few years as we are expecting to be inundated with thousands of completely sequenced microbial genomes. [Abstract/Link to Full Text]

Wunder T, Martin R, Loffelhardt W, Schleiff E, Steiner JM
The invariant phenylalanine of precursor proteins discloses the importance of Omp85 for protein translocation into cyanelles.
BMC Evol Biol. 2007 Nov 28;7(1):236.
ABSTRACT: BACKGROUND: Today it is widely accepted that plastids are of cyanobacterial origin. During their evolutionary integration into the metabolic and regulatory networks of the host cell the engulfed cyanobacteria lost their independency. This process was paralleled by a massive gene transfer from symbiont to the host nucleus challenging the development of a retrograde protein translocation system to ensure plastid functionality. Such a system includes specific targeting signals of the proteins needed for the function of the plastid and membrane-bound machineries performing the transfer of these proteins across the envelope membranes. At present, most informations on protein translocation are obtained by the analysis of land plants. However, the analysis of protein import into the primitive plastids of glaucocystophyte algae, revealed distinct features placing this system as a tool to understand the evolutionary development of translocation systems. Here, bacterial outer membrane proteins of the Omp85 family have recently been discussed as evolutionary seeds for the development of translocation systems. RESULTS: To further explore the initial mode of protein translocation, the observed phenylalanine dependence for protein translocation into glaucophyte plastids was pursued in detail. We document that indeed the phenylalanine has an impact on both, lipid binding and binding to proteoliposomes hosting an Omp85 homologue. Comparison to established import experiments, however, unveiled a major importance of the phenylalanine for recognition by Omp85. This finding is placed into the context of the evolutionary development of the plastid translocon. CONCLUSION: The phenylalanine in the N-terminal domain signs as a prerequisite for protein translocation across the outer membrane assisted by a primitive translocon. This amino acid appears to be optimized for specifically targeting the Omp85 protein without enforcing aggregation on the membrane surface. The phenylalanine has subsequently been lost in the transit sequence, but can be found at the C-terminal position of the translocating pore. Thereby, the current hypothesis of Omp85 being the prokaryotic contribution to the ancestral Toc translocon can be supported. [Abstract/Link to Full Text]

Connallon T, Knowles LL
Recombination rate and protein evolution in yeast.
BMC Evol Biol. 2007 Nov 27;7(1):235.
ABSTRACT: BACKGROUND: Theory and artificial selection experiments show that recombination can promote adaptation by enhancing the efficacy of natural selection, but the extent to which recombination affects levels of adaptation across the genome is still an open question. Because patterns of molecular evolution reflect long-term processes of mutation and selection in nature, interactions between recombination rate and genetic differentiation between species can be used to test the benefits of recombination. However, this approach faces a major difficulty: different evolutionary processes (i.e. negative versus positive selection) produce opposing relationships between recombination rate and genetic divergence, and obscure patterns predicted by individual benefits of recombination. RESULTS: We use a combination of polymorphism and genomic data from the yeast Saccharomyces cerevisiae to infer the relative importance of nearly-neutral (i.e. slightly deleterious) evolution in different gene categories. For genes with high opportunities for slightly deleterious substitution, recombination substantially reduces the rate of molecular evolution, whereas divergence in genes with little opportunity for slightly deleterious substitution is not strongly affected by recombination. CONCLUSION: These patterns indicate that adaptation throughout the genome can be strongly influenced by each gene's recombinational environment, and suggest substantial long-term fitness benefits of enhanced purifying selection associated with sexual recombination. [Abstract/Link to Full Text]

Deng X, Havukkala I, Deng X
Large-scale genomic 2D visualization reveals extensive CG-AT skew correlation in bird genomes.
BMC Evol Biol. 2007 Nov 23;7(1):234.
ABSTRACT: BACKGROUND: The bird genome has very different compositional structure compared with other warm-blood animals. The variation in the base skew rules in the vertebrate genomes remains puzzling, but it must be somehow related to large-scale genome evolution. Current research inclines to relate base skew to mutations and their fixation. Here we wish to explore base skew correlations in bird genomes, develop methods to display and quantify such correlations at different scales, and to discuss possible explanations to the peculiarities of the bird genomes in skew correlation. RESULTS: We have developed a method (called Base Skew Double Triangle, BSDT) for exhibiting the genome-scale change of AT/CG skew as a two-dimensional square picture, showing base skews at many scales simultaneously in a single image. By this method we found that most chicken chromosomes have high AT/CG skew correlation (symmetry in 2D picture), except for some microchromosomes. No other organisms studied (19 species) reach the high skew correlation level of chicken. This visualized high correlation was validated by three kinds of overlapping and nonoverlapping quantitative calculations, all indicating that chicken and birds in general have a special genome structure, presumably evolved after birds separated from other vertebrate lineages. When we eliminated the repeat sequences from the genomes, the AT and CG skews correlation increased for some mammal genomes, but were still clearly lower than in chicken. CONCLUSIONS: Our results suggest that BSDT is an expressive visualization method for AT and CG skew and enabled discovery of the very high skew correlation in bird genomes; this peculiarity is worth further study. Computational analysis indicated that this correlation might be a compositional characteristic, not only in chicken, but also in some mammals during evolution. Special aspects of bird metabolism related to e.g. flight may be the reason why birds evolved or retained the skew correlation. Analysis also indicated that repetitive DNA sequence elements need to be taken into account in the evolution of the correlation between AT and CG skews. [Abstract/Link to Full Text]

Naydenov K, Senneville S, Beaulieu J, Tremblay F, Bousquet J
Glacial vicariance in Eurasia: mitochondrial DNA evidence from Scots pine for a complex heritage involving genetically distinct refugia at mid-northern latitudes and in Asia Minor.
BMC Evol Biol. 2007 Nov 22;7(1):233.
ABSTRACT: BACKGROUND: At the last glacial maximum, Fennoscandia was covered by an ice sheet while the tundra occupied most of the rest of northern Eurasia. More or less disjunct refugial populations of plants were dispersed in southern Europe, often trapped between mountain ranges and seas. Genetic and paleobotanical evidences indicate that these populations have contributed much to Holocene recolonization of more northern latitudes. Less supportive evidence has been found for the existence of glacial populations located closer to the ice margin. Scots pine (Pinus sylvestris L.) is a nordic conifer with a wide natural range covering much of Eurasia. Fractures in its extant genetic structure might be indicative of glacial vicariance and how different refugia contributed to the current distribution at the continental level. The population structure of Scots pine was investigated on much of its Eurasian natural range using maternally inherited mitochondrial DNA polymorphisms. RESULTS: A novel polymorphic region of the Scots pine mitochondrial genome has been identified, the intron 1 of nad7, with three variants caused by insertions-deletions. From 986 trees distributed among 54 populations, four distinct multi-locus mitochondrial haplotypes (mitotypes) were detected based on the three nad7 intron 1 haplotypes and two previously reported size variants for nad1 intron B/C. Population differentiation was high (GST = 0.657) and the distribution of the mitotypes was geographically highly structured, suggesting at least four genetically distinct ancestral lineages. A cosmopolitan lineage was widely distributed in much of Europe throughout eastern Asia. A previously reported lineage limited to the Iberian Peninsula was confirmed. A new geographically restricted lineage was found confined to Asia Minor. A new lineage was restricted to more northern latitudes in northeastern Europe and the Baltic region. CONCLUSION: The contribution of the various ancestral lineages to the current distribution of Scots pine was asymmetric and extant endemism reflected the presence of large geographic barriers to migration. The results suggest a complex biogeographical history with glacial refugia shared with temperate plant species in southern European Peninsulas and Asia Minor, and a genetically distinct glacial population located more North. These results confirm recent observations for cold tolerant species about the possible existence of refugial populations at mid-northern latitudes contributing significantly to the recolonization of northern Europe. Thus, Eurasian populations of nordic plant species might not be as genetically homogenous as assumed by simply considering them as offsets of glacial populations located in southern peninsulas. As such, they might have evolved distinctive genetic adaptations during glacial vicariance, worth evaluating and considering for conservation. [Abstract/Link to Full Text]

Sharma S, Rai E, Bhat AK, Bhanwer AS, Bamezai RN
A novel subgroup Q5 of human Y-chromosomal haplogroup Q in India.
BMC Evol Biol. 2007 Nov 19;7(1):232.
ABSTRACT: BACKGROUND: Y-chromosomal haplogroup (Y-HG) Q is suggested to originate in Asia and represent recent founder paternal Native American radiation into the Americas. This group is delineated into Q1, Q2 and Q3 subgroups defined by biallelic markers M120, M25/M143 and M3, respectively. Recently, a novel subgroup Q4 has been identified which is defined by bi-allelic marker M346, representing HG Q (0.41%, 3/728) in Indian population. With scanty details of HG Q in Asia, especially India, it was pertinent to explore the status of the Y-HG Q in Indian population to gather an insight to determine the extent of diversity within this region. RESULTS: We observed 15/630 (2.38%) Y-HG Q individuals in India with an ancestral state at M120, M25, M3 and M346 markers, indicating an absence of already known Q1, Q2, Q3 and Q4 sub-haplogroups. Interestingly, we further observed a novel 4bp deletion / insertion polymorphism (ss4bp, rs41352448) at 72,314 position of human arylsulfatase D pseudogene, defining a novel sub-lineage Q5 (in 5/15 individuals, i.e., 33.3 % of the observed Y-HG Q) with distributions independent of the social, cultural, linguistic and geographical affiliations in India. CONCLUSIONS: The study adds another sublineage Q5 in the already existing arrangement of Y-HG Q in literature. It was quite interesting to observe an ancestral state Q* and a novel sub-branch Q5, not reported elsewhere, in Indian subcontinent, though in low frequency. A novel subgroup Q4 was identified recently which is also restricted to Indian subcontinent. The most plausible explanation for these observations could be an ancestral migration of individuals bearing ancestral lineage Q* to Indian subcontinent followed by an autochthonous differentiation to Q4 and Q5 sublineages later on. However, other explanations of, either the presence of both the sub haplogroups (Q4 and Q5) in ancestral migrants or recent migrations from central Asia, cannot be ruled out till the distribution and diversity of these subgroups is explored extensively in Central Asia and other regions. [Abstract/Link to Full Text]

Eugster M, Roten CA, Greub G
Analyses of six homologous proteins of Protochlamydia amoebophila UWE25 encoded by large GC-rich genes (lgr): a model of evolution and concatenation of leucine-rich repeats.
BMC Evol Biol. 2007 Nov 16;7(1):231.
ABSTRACT: BACKGROUND: Along the chromosome of the obligate intracellular bacteria Protochlamydia amoebophila UWE25, we recently described a genomic island Pam100G. It contains a tra unit likely involved in conjugative DNA transfer and lgrE, a 5.6-kb gene similar to five others of P. amoebophila: lgrA to lgrD, lgrF. We describe here the structure, regulation and evolution of these proteins termed LGRs since encoded by "Large G+C-Rich" genes. RESULTS: No homologs to the whole protein sequence of LGRs were found in other organisms. Phylogenetic analyses suggest that serial duplications producing the six LGRs occurred relatively recently and nucleotide usage analyses show that lgrB, lgrE and lgrF were relocated on the chromosome. The C-terminal part of LGRs is homologous to Leucine-Rich Repeats domains (LRRs). Defined by a cumulative alignment score, the 5 to 18 concatenated octacosapeptidic (28-meric) LRRs of LGRs present all a predicted alpha-helix conformation. Their closest homologs are the 28-residue RI-like LRRs of mammalian NODs and the 24-meres of some Ralstonia and Legionella proteins. Interestingly, LgrE, which is present on Pam100G like the tra operon, exhibits Pfam domains related to DNA metabolism. CONCLUSIONS: Comparison of the LRRs, enable us to propose a parsimonious evolutionary scenario of these domains driven by adjacent concatenations of LRRs. Our model established on bacterial LRRs can be challenged in eucaryotic proteins carrying less conserved LRRs, such as NOD proteins and Toll-like receptors. [Abstract/Link to Full Text]

Boxma B, Ricard G, van Hoek AH, Severing E, Moon-Vander Staay SY, van der Staay GW, van Alen TA, de Graaf RM, Cremers G, Kwantes M, McEwan NR, Newbold CJ, Jouany JP, Michalowski T, Pristas P, Huynen MA, Hackstein JH
The [FeFe] hydrogenase of Nyctotherus ovalis has a chimeric origin.
BMC Evol Biol. 2007 Nov 16;7(1):230.
ABSTRACT: BACKGROUND: The hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis show how mitochondria can evolve into hydrogenosomes because they possess a mitochondrial genome and parts of an electron-transport chain on the one hand, and a hydrogenase on the other hand. The hydrogenase permits direct reoxidation of NADH because it consists of a [FeFe] hydrogenase module that is fused to two modules, which are functionally homologous to the 24 kDa and the 51kDa subunits of a mitochondrial complex I RESULTS: The [FeFe] hydrogenase belongs to a cluster of hydrogenases that are different from well-known eukaryotic hydrogenases. The 24 kDa and the 51 kDa modules are most closely related to homologous modules that function in bacterial [NiFe] hydrogenases. Paralogous, mitochondrial 24 kDa and 51 kDa modules function in the mitochondrial complex I in N. ovalis. The different hydrogenase modules have been fused to form a polyprotein that is targeted into the hydrogenosome. CONCLUSION: The hydrogenase and their associated modules have most likely been acquired by independent lateral gene transfer from different sources. This scenario for a concerted lateral gene transfer is in agreement with the evolution of the hydrogenosome from a genuine ciliate mitochondrion by evolutionary tinkering. [Abstract/Link to Full Text]

Frank SA, Bush RM
Barriers to antigenic escape by pathogens: trade-off between reproductive rate and antigenic mutability.
BMC Evol Biol. 2007 Nov 15;7(1):229.
ABSTRACT: BACKGROUND: A single measles vaccination provides lifelong protection. No antigenic variants that escape immunity have been observed. By contrast, influenza continually evolves new antigenic variants, and the vaccine has to be updated frequently with new strains. Both measles and influenza are RNA viruses with high mutation rates, so the mutation rate alone cannot explain the differences in antigenic variability. RESULTS: We develop a new hypothesis to explain antigenic stasis versus change. We first note that the antigenically static viruses tend to have high reproductive rates and to concentrate infection in children, whereas antigenically variable viruses such as influenza tend to spread more widely across age classes. We argue that, for pathogens in a naive host population that spread more rapidly in younger individuals than in older individuals, natural selection weights more heavily a rise in reproductive rate. By contrast, pathogens that spread more readily among older individuals gain more by antigenic escape, so natural selection weights more heavily antigenic mutability. CONCLUSIONS: These divergent selective pressures on reproductive rate and antigenic mutability may explain some of the observed differences between pathogens in age-class bias, reproductive rate, and antigenic variation. [Abstract/Link to Full Text]

Catanzaro D, Pesenti R, Milinkovitch MC
An ant colony optimization algorithm for phylogenetic estimation under the minimum evolution principle.
BMC Evol Biol. 2007 Nov 15;7(1):228.
ABSTRACT: BACKGROUND: Distance matrix methods constitute a major family of phylogenetic estimation methods, and the minimum evolution (ME) principle (aiming at recovering the phylogeny with shortest length) is one of the most commonly used optimality criteria for estimating phylogenetic trees. The major difficulty for its application is that the number of possible phylogenies grows exponentially with the number of taxa analyzed and the minimum evolution principle is known to belong to the NP-hard class of problems. RESULTS: In this paper, we introduce an Ant Colony Optimization (ACO) algorithm to estimate phylogenies under the minimum evolution principle. ACO is an optimization technique inspired from the foraging behavior of real ant colonies. This behavior is exploited in artificial ant colonies for the search of approximate solutions to discrete optimization problems. CONCLUSION: We show that the ACO algorithm is potentially competitive in comparison with state-of-the-art algorithms for the minimum evolution principle. This is the first application of an ACO algorithm to the phylogenetic estimation problem. [Abstract/Link to Full Text]

Hughes J, Page RD
Comparative tests of ectoparasite species richness in seabirds.
BMC Evol Biol. 2007 Nov 15;7(1):227.
ABSTRACT: BACKGROUND: The diversity of parasites attacking a host varies substantially among different host species. Understanding the factors that explain these patterns of parasite diversity is critical to identifying the ecological principles underlying biodiversity. Seabirds (Charadriiformes, Pelecaniformes and Procellariiformes) and their ectoparasitic lice (Insecta: Phthiraptera) are ideal model groups in which to study correlates of parasite species richness. We evaluated the relative importance of morphological (body size, body weight, wingspan, bill length), life-history (longevity, clutch size ), ecological (population size, geographical range) and behavioural (diving versus non-diving) variables as predictors of louse diversity on 413 seabird hosts species. Diversity was measured at the level of louse suborder, genus, and species, and uneven sampling of hosts was controlled for using literature citations as a proxy for sampling effort. RESULTS: The only variable consistently correlated with louse diversity was host population size and to a lesser extent geographic range. Other variables such as clutch size, longevity, morphological and behavioural variables including body mass showed inconsistent patterns dependent on the method of analysis. CONCLUSIONS: The comparative analysis presented herein is (to our knowledge) the first to test correlates of parasite species richness in seabirds. We believe that the comparative data and phylogeny provide a valuable framework for testing future evolutionary hypotheses relating to the diversity and distribution of parasites on seabirds. [Abstract/Link to Full Text]

Vicario S, Moriyama EN, Powell JR
Codon usage in twelve species of Drosophila.
BMC Evol Biol. 2007 Nov 15;7(1):226.
ABSTRACT: BACKGROUND: Codon usage bias (CUB), the uneven use of synonymous codons, is a ubiquitous observation in virtually all organisms examined. The pattern of codon usage is generally similar among closely related species, but differs significantly among distantly related organisms, e.g., bacteria, yeast, and Drosophila. Several explanations for CUB have been offered and some have been supported by observations and experiments, although a thorough understanding of the evolutionary forces (random drift, mutation bias, and selection) and their relative importance remains to be determined. The recently available complete genome DNA sequences of twelve phylogenetically defined species of Drosophila offer a hitherto unprecedented opportunity to examine these problems. We report here the patterns of codon usage in the twelve species and offer insights on possible evolutionary forces involved. RESULTS: (1) Codon usage is quite stable across 11/12 of the species: G- and especially C-ending codons are used most frequently, thus defining the preferred codons. (2) The only amino acid that changes in preferred codon is Serine with six species of the melanogaster group favoring TCC while the other species, particularly subgenus Drosophila species, favor AGC. (3) D. willistoni is an exception to these generalizations in having a shifted codon usage for seven amino acids toward A/T in the wobble position. (4) Amino acids differ in their contribution to overall CUB, Leu having the greatest and Asp the least. (5) Among two-fold degenerate amino acids, A/G ending amino acids have more selection on codon usage than T/C ending amino acids. (6) Among the different chromosome arms or elements, genes on the non-recombining element F (dot chromosome) have the least CUB, while genes on the element A (X chromosome) have the most. (7) Introns indicate that mutation bias in all species is approximately 2:1, AT:GC, the opposite of codon usage bias. (8) There is also evidence for some overall regional bias in base composition that may influence codon usage. CONCLUSIONS: Overall, these results suggest that natural selection has acted on codon usage in the genus Drosophila, at least often enough to leave a footprint of selection in modern genomes. However, there is evidence in the data that random forces (drift and mutation) have also left patterns in the data, especially in genes under weak selection for codon usage for example genes in regions of low recombination. The documentation of codon usage patterns in each of these twelve genomes also aids in ongoing annotation efforts. [Abstract/Link to Full Text]

Mills S, Lunt DH, Gomez A
Global isolation by distance despite strong regional phylogeography in a small metazoan.
BMC Evol Biol. 2007 Nov 14;7(1):225.
ABSTRACT: BACKGROUND: Small vagile eukaryotic organisms, which comprise a large proportion of the Earth's biodiversity, have traditionally been thought to lack the extent of population structuring and geographic speciation observed in larger taxa. Here we investigate the patterns of genetic diversity, amongst populations of the salt lake microscopic metazoan Brachionus plicatilis s. s. (sensu stricto) (Rotifera: Monogononta) on a global scale. We examine the phylogenetic relationships of geographic isolates from four continents using a 603 bp fragment of the mitochondrial COI gene to investigate patterns of phylogeographic subdivision in this species. In addition we investigate the relationship between genetic and geographic distances on a global scale to try and reconcile the paradox between the high vagility of this species and the previously reported patterns of restricted gene flow, even over local spatial scales. RESULTS: Analysis of global sequence diversity of B. plicatilis s. s. reveals the presence of four allopatric genetic lineages: North American-Far East Asian, Western Mediterranean, Australian, and an Eastern Mediterranean lineage represented by a single isolate. Geographically orientated substructure is also apparent within the three best sampled lineages. Surprisingly, given this strong phylogeographic structure, B. plicatilis s. s. shows a significant correlation between geographic and genetic distance on a global scale ('isolation by distance' - IBD). CONCLUSIONS: Despite its cosmopolitan distribution and potential for high gene flow, B. plicatilis s. s. is strongly structured at a global scale. IBD patterns have traditionally been interpreted to indicate migration-drift equilibrium, although in this system equilibrium conditions are incompatible with the observed genetic structure. Instead, we suggest the pattern may have arisen through persistent founder effects, acting in a similar fashion to geographic barriers for larger organisms. Our data indicates that geographic speciation, contrary to historical views, is likely to be very important in microorganisms. By presenting compelling evidence for geographic speciation in a small eukaryote we add to the growing body of evidence that is forcing us to rethink our views of global biodiversity. [Abstract/Link to Full Text]

Seiffert ER
A new estimate of afrotherian phylogeny based on simultaneous analysis of genomic, morphological, and fossil evidence.
BMC Evol Biol. 2007 Nov 13;7(1):224.
ABSTRACT: BACKGROUND: The placental mammalian clade Afrotheria is now supported by diverse forms of genomic data, but interordinal relationships within, and morphological support for, the group remains elusive. As a means for addressing these outstanding problems, competing hypotheses of afrotherian interordinal relationships were tested through simultaneous parsimony analysis of a large data set (>4,590 parsimony informative characters) containing genomic data (>17kb of nucleotide data, chromosomal associations, and retroposons) and 400 morphological characters scored across 16 extant and 35 extinct afrotherians. RESULTS: Parsimony analysis of extant taxa alone recovered the interordinal topology (Afrosoricida, ((Macroscelidea, Tubulidentata), (Hyracoidea, (Proboscidea, Sirenia)))). Analysis following addition of extinct taxa instead supported Afroinsectivora (Afrosoricida + Macroscelidea) and Pseudoungulata (Tubulidentata + Paenungulata), as well as Tethytheria (Proboscidea + Sirenia). This latter topology is, however, sensitive to taxon deletion and different placements of the placental root, and numerous alternative interordinal arrangements within Afrotheria could not be statistically rejected. Relationships among extinct stem members of each afrotherian clade were more stable, but one alleged stem macroscelidean (Herodotius) never grouped with that clade and instead consistently joined pseudoungulates or paenungulates. When character transformations were optimized onto a less resolved afrotherian tree that reflects uncertainty about the group's interordinal phylogeny, a total of 21 morphological features were identified as possible synapomorphies of crown Afrotheria, 9 of which optimized unambiguously across all character treatments and optimization methods. CONCLUSIONS: Instability in afrotherian interordinal phylogeny presumably reflects rapid divergences during two pulses of cladogenesis - the first in the Late Cretaceous, at and just after the origin of crown Afrotheria, and the second in the early Cenozoic, with the origin of crown Paenungulata. Morphological evidence for divergences during these two pulses either never existed or has largely been "erased" by subsequent evolution along long ordinal branches. There may, nevertheless, be more morphological character support for crown Afrotheria than is currently assumed; the features identified here as possible afrotherian synapomorphies can be further scrutinized through future phylogenetic analyses with broader taxon sampling, as well as recovery of primitive fossil afrotherians from the Afro-Arabian landmass, where the group is likely to have first diversified. [Abstract/Link to Full Text]

Shu W, Bo X, Ni M, Zheng Z, Wang S
In silico genetic robustness analysis of microRNA secondary structures: potential evidence of congruent evolution in microRNA.
BMC Evol Biol. 2007 Nov 13;7(1):223.
ABSTRACT: BACKGROUND: Robustness is a fundamental property of biological systems and is defined as the ability to maintain stable functioning in the face of various perturbations. Understanding how robustness has evolved has become one of the most attractive areas of research for evolutionary biologists, as it is still unclear whether genetic robustness evolved as a direct consequence of natural selection, as an intrinsic property of adaptations, or as congruent correlate of environment robustness. Recent studies have demonstrated that the stem-loop structures of microRNA (miRNA) are tolerant to some structural changes and show thermodynamic stability. We therefore hypothesize that genetic robustness may evolve as a correlated side effect of the evolution for environmental robustness. RESULTS: We examine the robustness of 1,082 miRNA genes covering six species. Our data suggest the stem-loop structures of miRNA precursors exhibit a significantly higher level of genetic robustness, which goes beyond the intrinsic robustness of the stem-loop structure and is not a byproduct of the base composition bias. Furthermore, we demonstrate that the phenotype of miRNA buffers against genetic perturbations, and at the same time is also insensitive to environmental perturbations. CONCLUSION: The results suggest that the increased robustness of miRNA stem-loops may result from congruent evolution for environment robustness. Potential applications of our findings are also discussed. [Abstract/Link to Full Text]

Reschly EJ, Bainy AC, Mattos JJ, Hagey LR, Bahary N, Mada SR, Ou J, Venkataramanan R, Krasowski MD
Functional evolution of the vitamin D and pregnane X receptors.
BMC Evol Biol. 2007 Nov 12;7(1):222.
ABSTRACT: BACKGROUND: The vitamin D receptor (VDR) and pregnane X receptor (PXR) are nuclear hormone receptors of the NR1I subfamily that show contrasting patterns of cross-species variation. VDR and PXR are thought to have arisen from duplication of an ancestral gene, evident now as a single gene in the genome of the chordate invertebrate Ciona intestinalis (sea squirt). VDR genes have been detected in a wide range of vertebrates including jawless fish. To date, PXR genes have not been found in cartilaginous fish. In this study, the ligand selectivities of VDRs were compared in detail across a range of vertebrate species and compared with those of the Ciona VDR/PXR. In addition, several assays were used to search for evidence of PXR-mediated hepatic effects in three model non-mammalian species: sea lamprey (Petromyzon marinus), zebrafish (Danio rerio), and African clawed frog (Xenopus laevis). RESULTS: Human, mouse, frog, zebrafish, and lamprey VDRs were found to have similar ligand selectivities for vitamin D derivatives. In contrast, using cultured primary hepatocytes, only zebrafish showed evidence of PXR-mediated induction of enzyme expression, with increases in testosterone 6beta-hydroxylation activity (a measure of cytochrome P450 3A activity in other species) and flurbiprofen 4-hydroxylation activity (measure of cytochrome P450 2C activity) following exposure to known PXR activators. A separate assay in vivo using zebrafish demonstrated increased hepatic transcription of another PXR target, multidrug resistance gene (ABCB5), following injection of the major zebrafish bile salt, 5alpha-cyprinol 27-sulfate. The PXR target function, testosterone hydroxylation, was detected in frog and sea lamprey primary hepatocytes, but was not inducible in these two species by a wide range of PXR activators in other animals. Analysis of the sea lamprey draft genome also did not show evidence of a PXR gene. CONCLUSIONS: Our results show tight conservation of ligand selectivity of VDRs across vertebrate species from Agnatha to mammals. Using a functional approach, we demonstrate classic PXR-mediated effects in zebrafish, but not in sea lamprey or African clawed frog liver cells. Using a genomic approach, we failed to find evidence of a PXR gene in lamprey, suggesting that VDR may be the original NR1I gene. [Abstract/Link to Full Text]

Cardoso JC, de Vet EC, Louro B, Elgar G, Clark MS, Power DM
Persistence of duplicated PAC1 receptors in the teleost, Sparus auratus.
BMC Evol Biol. 2007 Nov 12;7(1):221.
ABSTRACT: BACKGROUND: Duplicated genes are common in vertebrate genomes. Their persistence is assumed to be either a consequence of gain of novel function (neofunctionalisation) or partitioning of the function of the ancestral molecule (sub-functionalisation). Surprisingly few studies have evaluated the extent of such modifications despite the numerous duplicated receptor and ligand genes identified in vertebrate genomes to date. In order to study the importance of function in the maintenance of duplicated genes, sea bream (Sparus auratus) PAC1 receptors, sequence homologues of the mammalian receptor specific for PACAP (Pituitary Adenylate Cyclase-Activating Polypeptide), were studied. These receptors belong to family 2 GPCRs and most of their members are duplicated in teleosts although the reason why both persist in the genome is unknown. RESULTS: Duplicate sea bream PACAP receptor genes (sbPAC1A and sbPAC1B), members of family 2 GPCRs, were isolated and share 77% amino acid sequence identity. RT-PCR with specific primers for each gene revealed that they have a differential tissue distribution which overlaps with the distribution of the single mammalian receptor. Furthermore, in common with mammals, the teleost genes undergo alternative splicing and a PAC1Ahop1 isoform has been characterised. Duplicated orthologous receptors have also been identified in other teleost genomes and their distribution profile suggests that function may be species specific. Functional analysis of the paralogue sbPAC1s in Cos7 cells revealed that they are strongly stimulated in the presence of mammalian PACAP27 and PACAP38 and far less with VIP (Vasoactive Intestinal Peptide). The sbPAC1 receptors are equally stimulated (LOGEC50 values for maximal cAMP production) in the presence of PACAP27 (-8.74+/-0.29M and -9.15+/-0.21M, respectively for sbPAC1A and sbPAC1B, P>0.05) and PACAP38 (-8.54+/-0.18M and -8.92+/-0.24M, respectively for sbPAC1A and sbPAC1B, P>0.05). Human VIP was found to stimulate sbPAC1A (-7.23+/-0.20M) more strongly than sbPAC1B (-6.57+/-0.14M, P<0.05) and human secretin (SCT), which has not so far been identified in fish genomes, caused negligible stimulation of both receptors. CONCLUSIONS: The existence of functionally divergent duplicate sbPAC1 receptors is in line with previously proposed theories about the origin and maintenance of duplicated genes. Sea bream PAC1 duplicate receptors resemble the typical mammalian PAC1, and PACAP peptides were found to be more effective than VIP in stimulating cAMP production, although sbPAC1A was more responsive for VIP than sbPAC1B. These results together with the highly divergent pattern of tissue distribution suggest that a process involving neofunctionalisation occurred after receptor duplication within the fish lineage and probably accounts for their persistence in the genome. The characterisation of further duplicated receptors and their ligands should provide insights into the evolution and function of novel protein-protein interactions associated with the vertebrate radiation. [Abstract/Link to Full Text]

Janko K, Lecointre G, Devries A, Couloux A, Cruaud C, Marshall C
Did glacial advances during the Pleistocene influence differently the demographic histories of benthic and pelagic Antarctic shelf fishes? - Inferences from intraspecific mitochondrial and nuclear DNA sequence diversity.
BMC Evol Biol. 2007 Nov 12;7(1):220.
ABSTRACT: BACKGROUND: Circum-antarctic waters harbour a rare example of a marine species flock - the Notothenioid fishes, most of which are restricted to the continental shelf. It remains an open question how they survived Pleistocene climatic fluctuations characterised by repeated advances of continental glaciers as far as the shelf break resulting in a probable loss of habitat for benthic organisms. Pelagic ecosystems, on the other hand, might have flourished during glacial maxima due to the northward expansion of Antarctic polar waters. In order to better understand the role of ecological traits in Quaternary climatic fluctuations, we performed demographic analyses of populations of four fish species from the tribe Trematominae, including fully benthic species as well as pelagic, using the mitochondrial cytochrome b gene and an intron from the nuclear S7 gene. RESULTS: We observed different results from nuclear and cytoplasmic markers with respect to the rate and time of population expansions as well as population structure. According to neutrality tests, we suggest that such discordance comes from different coalescence dynamics of both markers rather than from selective pressure. Demographic analyses based on intraspecific DNA diversity suggested a recent population expansion in both benthic species dated by the cyt b locus to the last glacial cycle, whereas the population structure of pelagic feeders either did not deviate from constant-size model or suggested that the onset of major population expansion by far predated those of the benthic species. Similar pattern was apparent even when comparing previously published data on other Southern Ocean organisms, but we observed considerable heterogeneities within both groups as to the onset of major demographic events and their rates. CONCLUSIONS: Our data suggest different reactions of benthic and pelagic species to Pleistocene ice-sheet expansions, which probably significantly reduced the suitable habitat for benthic species. However, the asynchronicity of major demographic events observed in different species, suggest that more species should be analysed in order to more precisely assess the role of life history in the response of organisms to climatic changes. [Abstract/Link to Full Text]

Swennen D, Beckerich JM
Yarrowia lipolytica vesicle-mediated protein transport pathways.
BMC Evol Biol. 2007 Nov 12;7(1):219.
ABSTRACT: BACKGROUND: Protein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Genolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y.lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y.lipolytica and is largely co-translational as in the mammalian protein secretion pathway. RESULTS: We identified S.cerevisiae proteins involved in vesicular secretion and these protein sequences were used for the BLAST searches against Genolevures protein database (Y.lipolytica, C.glabrata, K.lactis and D.hansenii). These proteins are well conserved between these yeasts and Saccharomyces cerevisiae. We note several specificities of Y.lipolytica which may be related to its good protein secretion capacities and to its dimorphic aspect. An expansion of the Y.lipolytica Rab protein family was observed with autoBLAST and the Rab2- and Rab4-related members were identified with BLAST against NCBI protein database. An expansion of this family is also found in filamentous fungi and may reflect the greater complexity of the Y.lipolytica secretion pathway. The Rab4p-related protein may play a role in membrane recycling as rab4 deleted strain shows a modification of colony morphology, dimorphic transition and permeability. Similarly, we find three copies of the gene (SSO) encoding the plasma membrane SNARE protein. Quantification of the percentages of proteins with the greatest homology between S.cerevisiae, Y.lipolytica and animal homologues involved in vesicular transport shows that 40% of Y.lipolytica proteins are closer to animal ones, whereas they are only 13% in the case of S.cerevisiae. CONCLUSION: These results provide further support for the idea, previously noted about the endoplasmic reticulum translocation pathway, that Y.lipolytica is more representative of vesicular secretion of animals and other fungi than is S.cerevisiae. [Abstract/Link to Full Text]

Curtu AL, Gailing O, Finkeldey R
Evidence for hybridization and introgression within a species-rich oak (Quercus spp.) community.
BMC Evol Biol. 2007 Nov 10;7(1):218.
ABSTRACT: BACKGROUND: Analysis of interspecific gene flow is crucial for the understanding of speciation processes and maintenance of species integrity. Oaks (genus Quercus, Fagaceae) are among the model species for the study of hybridization. Natural co-occurrence of four closely related oak species is a very rare case in the temperate forests of Europe. We used both morphological characters and genetic markers to characterize hybridization in a natural community situated in west-central Romania and which consists of Quercus robur, Q. petraea, Q. pubescens, and Q. frainetto, respectively. RESULTS: On the basis of pubescence and leaf morphological characters ~94% of the sampled individuals were assigned to pure species. Only 16 (~6%) individual trees exhibited intermediate morphologies or a combination of characters of different species. Four chloroplast DNA haplotypes were identified in the study area. The distribution of haplotypes within the white oak complex showed substantial differences among species. However, the most common haplotypes were present in all four species. Furthermore, based on a set of 7 isozyme and 6 microsatellite markers and using a Bayesian admixture analysis without any a priori information on morphology we found that four genetic clusters best fit the data. There was a very good correspondence of each species with one of the inferred genetic clusters. The estimated introgression level varied markedly between pairs of species ranging from 1.7% between Q. robur and Q. frainetto to 16.2% between Q. pubescens and Q. frainetto. Only nine individuals (3.4%) appeared to be first-generation hybrids. CONCLUSIONS: Our data indicate that natural hybridization has occurred at relatively low rates. The different levels of gene flow among species might be explained by differences in flowering time and spatial position within the stand. In addition, a partial congruence between phenotypically and genetically intermediate individuals was found, suggesting that intermediate appearance does not necessarily mean hybridization. However, it appears that natural hybridization did not seriously affect the species identity in this area of sympatry. [Abstract/Link to Full Text]

Zhu XY, Chase MW, Qiu YL, Kong HZ, Dilcher DL, Li JH, Chen ZD
Mitochondrial matR sequences help to resolve deep phylogenetic relationships in rosids.
BMC Evol Biol. 2007 Nov 10;7(1):217.
ABSTRACT: BACKGROUND: Rosids are a major clade in the angiosperms containing 13 orders and about one-third of angiosperm species. Recent molecular analyses recognized two major groups (i.e., fabids with seven orders and malvids with three orders). However, phylogenetic relationships within the two groups and among fabids, malvids, and potentially basal rosids including Geraniales, Myrtales, and Crossosomatales remain to be resolved with more data and a broader taxon sampling. In this study, we obtained DNA sequences of the mitochondrial matR gene from 174 species representing 72 families of putative rosids and examined phylogenetic relationships and phylogenetic utility of matR in rosids. We also inferred phylogenetic relationships within the "rosid clade" based on a combined data set of 91 taxa and four genes including matR, two plastid genes (rbcL, atpB), and one nuclear gene (18S rDNA). RESULTS: Comparison of mitochondrial matR and two plastid genes (rbcL and atpB) showed that the synonymous substitution rate in matR was approximately four times slower than those of rbcL and atpB; however, the nonsynonymous substitution rate in matR was relatively high, close to its synonymous substitution rate, indicating that the matR has experienced a relaxed evolutionary history. Analyses of our matR sequences supported the monophyly of malvids and most orders of the rosids. However, fabids did not form a clade; instead, the COM clade of fabids (Celastrales, Oxalidales, Malpighiales, and Huaceae) was sister to malvids. Analyses of the four-gene data set suggested that Geraniales and Myrtales were successively sister to other rosids, and that Crossosomatales were sister to malvids. CONCLUSIONS: Compared to plastid genes such as rbcL and atpB, slowly evolving matR produced less homoplasious but not less informative substitutions. Thus, matR appears useful in higher-level angiosperm phylogenetics. Analysis of matR alone identified a novel deep relationship within rosids, the grouping of the COM clade of fabids and malvids, which was not resolved by any previous molecular analyses but recently suggested by floral structural features. Our four-gene analysis supported the placements of Geraniales, Myrtales at basal nodes of the rosid clade and placed Crossosomatales as sister to malvids. We also suggest that the core part of rosids should include fabids, malvids and Crossosomatales. [Abstract/Link to Full Text]

Higdon JW, Bininda-Emonds OR, Beck RM, Ferguson SH
Phylogeny and divergence of the pinnipeds (Carnivora: Mammalia) assessed using a multigene dataset.
BMC Evol Biol. 2007 Nov 9;7(1):216.
ABSTRACT: Background Phylogenetic comparative methods are often improved by complete phylogenies with meaningful branch lengths (e.g., divergence dates). This study presents a dated molecular supertree for all 34 world pinniped species derived from a weighted matrix representation with parsimony (MRP) supertree analysis of 50 gene trees, each determined under a maximum likelihood (ML) framework. Divergence times were determined by mapping the same sequence data (plus two additional genes) on to the supertree topology and calibrating the ML branch lengths against a range of fossil calibrations. We assessed the sensitivity of our supertree topology in two ways: 1) a second supertree with all mtDNA genes combined into a single source tree, and 2) likelihood-based supermatrix analyses. Divergence dates were also calculated using a Bayesian relaxed molecular clock with rate autocorrelation to test the sensitivity of our supertree results further. Results The resulting phylogenies all agreed broadly with recent molecular studies, in particular supporting the monophyly of Phocidae, Otariidae, and the two phocid subfamilies, as well as an Odobenidae + Otariidae sister relationship; areas of disagreement were limited to four more poorly supported regions. Neither the supertree nor supermatrix analyses supported the monophyly of the two traditional otariid subfamilies, supporting suggestions for the need for taxonomic revision in this group. Phocid relationships were similar to other recent studies and deeper branches were generally well-resolved. Halichoerus grypus was nested within a paraphyletic Pusa, although relationships within Phocina tend to be poorly supported. Divergence date estimates for the supertree were in good agreement with other studies and the available fossil record; however, the Bayesian relaxed molecular clock divergence date estimates were significantly older. Conclusions Our results join other recent studies and highlight the need for a re-evaluation of pinniped taxonomy, especially as regards the subfamilial classification of otariids and the generic nomenclature of Phocina. Even with the recent publication of new sequence data, the available genetic sequence information for several species, particularly those in Arctocephalus, remains very limited, especially for nuclear markers. However, resolution of parts of the tree will probably remain difficult, even with additional data, due to apparent rapid radiations. Our study addresses the lack of a recent pinniped phylogeny that includes all species and robust divergence dates for all nodes, and will therefore prove indispensable to comparative and macroevolutionary studies of this group of carnivores. [Abstract/Link to Full Text]

Drummond AJ, Rambaut A
BEAST: Bayesian evolutionary analysis by sampling trees.
BMC Evol Biol. 2007 Nov 8;7(1):214.
ABSTRACT: BACKGROUND: The evolutionary analysis of molecular sequence variation is a statistical enterprise. This is reflected in the increased use of probabilistic models for phylogenetic inference, multiple sequence alignment, and molecular population genetics. Here we present BEAST: a fast, flexible software architecture for Bayesian analysis of molecular sequences related by an evolutionary tree. A large number of popular stochastic models of sequence evolution are provided and tree-based models suitable for both within- and between-species sequence data are implemented. RESULTS: BEAST version 1.4.6 consists of 81000 lines of Java source code, 779 classes and 81 packages. It provides models for DNA and protein sequence evolution, highly parametric coalescent analysis, relaxed clock phylogenetics, non-contemporaneous sequence data, statistical alignment and a wide range of options for prior distributions. BEAST source code is object-oriented, modular in design and freely available at http://beast-mcmc.googlecode.com/ under the GNU LGPL license. CONCLUSIONS: BEAST is a powerful and flexible evolutionary analysis package for molecular sequence variation. It also provides a resource for the further development of new models and statistical methods of evolutionary analysis. [Abstract/Link to Full Text]

Maccarthy T, Bergman A
The limits of subfunctionalization.
BMC Evol Biol. 2007 Nov 7;7(1):213.
ABSTRACT: BACKGROUND: The duplication-degeneration-complementation (DDC) model has been proposed as an explanation for the unexpectedly high retention of duplicate genes. The hypothesis proposes that, following gene duplication, the two gene copies degenerate to perform complementary functions that jointly match that of the single ancestral gene, a process also known as subfunctionalization. We distinguish between subfunctionalization at the regulatory level and at the product level (e.g within temporal or spatial expression domains). RESULTS: In contrast to what is expected under the DDC model, we use in silico modeling to show that regulatory subfunctionalization is expected to peak and then decrease significantly. At the same time, neofunctionalization (recruitment of novel interactions) increases monotonically, eventually affecting the regulatory elements of the majority of genes. Furthermore, since this process occurs under conditions of stabilizing selection, there is no need to invoke positive selection. At the product level, the frequency of subfunctionalization is no higher than would be expected by chance, a finding that was corroborated using yeast microarray time-course data. We also find that product subfunctionalization is not necessarily caused by regulatory subfunctionalization. CONCLUSIONS: Our results suggest a more complex picture of post-duplication evolution in which subfunctionalization plays only a partial role in conjunction with redundancy and neofunctionalization. We argue that this behavior is a consequence of the high evolutionary plasticity in gene networks. [Abstract/Link to Full Text]

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Recent Articles in BMC Structural Biology

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Faure G, Gowda VT, Maroun RC
Characterization of human coagulation factor Xa-binding site on Viperidae snake venom phospholipases A2 by affinity binding studies and molecular bioinformatics.
BMC Struct Biol. 2007 Dec 6;7(1):82.
ABSTRACT: BACKGROUND: The snake venom group IIA secreted phospholipases A2 (SVPLA2), present in the Viperidae snake family exhibit a wide range of toxic and pharmacological effects. They exert their different functions by catalyzing the hydrolysis of phospholipids (PL) at the membrane/water interface and by highly specific direct binding to: (i) presynaptic membrane-bound or intracellular receptors; (ii) natural PLA2-inhibitors from snake serum; and (iii) coagulation factors present in human blood. RESULTS: Using surface plasmon resonance (SPR) protein-protein interaction measurements and an in vitro biological test of inhibition of prothrombinase activity, we identify a number of Viperidae venom SVPLA2s that inhibit blood coagulation through direct binding to human blood coagulation factor Xa (FXa) via a non-catalytic, PL-independent mechanism. We classify the SVPLA2s in four groups, depending on the strength of their binding. Molecular electrostatic potentials calculated at the surface of 3D homology-modeling models show a correlation with inhibition of prothrombinase activiy. In addition, molecular docking simulations between SVPLA2 and FXa guided by the experimental data identify the potential FXa binding site on the SVPLA2s. This site is composed of the following regions: helices A and B, the Ca2+ loop, the helix C-beta wing loop, and the C-terminal fragment. Some of the SVPLA2 binding site residues belong also to the interfacial binding site (IBS). The interface in FXa involves both, the light and heavy chains. CONCLUSION: We have experimentally identified several strong FXa-binding SVPLA2s that disrupt the function of the coagulation cascade by interacting with FXa by the non-catalytic PL-independent mechanism. By theoretical methods we mapped the interaction sites on both, the SVPLA2s and FXa. Our findings may lead to the design of novel, non-competitive FXa inhibitors. [Abstract/Link to Full Text]

Lemaster DM, Anderson JS, Wang L, Guo Y, Li H, Hernandez G
NMR and X-ray analysis of structural additivity in metal binding site-swapped hybrids of rubredoxin.
BMC Struct Biol. 2007 Dec 5;7(1):81.
ABSTRACT: BACKGROUND: Chimeric hybrids derived from the rubredoxins of Pyrococcus furiosus (Pf) and Clostridium pasteurianum (Cp) provide a robust system for the characterization of protein conformational stability and dynamics in a differential mode. Interchange of the seven nonconserved residues of the metal binding site between the Pf and Cp rubredoxins yields a complementary pair of hybrids, for which the sum of the thermodynamic stabilities is equal to the sum for the parental proteins. Furthermore, the increase in amide hydrogen exchange rates for the hyperthermophile-derived metal binding site hybrid is faithfully mirrored by a corresponding decrease for the complementary hybrid that is derived from the less thermostable rubredoxin, indicating a degree of additivity in the conformational fluctuations that underlie these exchange reactions. RESULTS: Initial NMR studies indicated that the structures of the two complementary hybrids closely resemble "cut-and-paste" models derived from the parental Pf and Cp rubredoxins. This protein system offers a robust opportunity to characterize differences in solution structure, permitting the quantitative NMR chemical shift and NOE peak intensity data to be analyzed without recourse to the conventional conversion of experimental NOE peak intensities into distance restraints. The intensities for 1573 of the 1652 well-resolved NOE crosspeaks from the hybrid rubredoxins were statistically indistinguishable from the intensities of the corresponding parental crosspeaks, to within the baseplane noise level of these high sensitivity data sets. The difference in intensity for the remaining 79 NOE crosspeaks were directly ascribable to localized dynamical processes. Subsequent X-ray analysis of the metal binding site-swapped hybrids, to resolution limits of 0.79 A and 1.04 A, demonstrated that the backbone and sidechain heavy atoms in the NMR-derived structures lie within the range of structural variability exhibited among the individual molecules in the crystallographic asymmetric unit (~0.3 A), indicating consistency with the "cut-and-paste" structuring of the hybrid rubredoxins in both crystal and solution. CONCLUSION: Each of the significant energetic interactions in the metal binding site-swapped hybrids appears to exhibit a 1-to-1 correspondence with the interactions present in the corresponding parental rubredoxin structure, thus providing a structural basis for the observed additivity in conformational stability and dynamics. The congruence of these X-ray and NMR experimental data offers additional support for the interpretation that the conventional treatment of NOE distance restraints contributes substantially to the systematic differences that are commonly reported between NMR- and X-ray-derived protein structures. [Abstract/Link to Full Text]

Jackson SG, Zhang Y, Haslam RJ, Junop MS
Structural analysis of the carboxy terminal PH domain of pleckstrin bound to D-myo-inositol 1,2,3,5,6-pentakisphosphate.
BMC Struct Biol. 2007 Nov 22;7(1):80.
ABSTRACT: BACKGROUND: Pleckstrin homology (PH) domains are one of the most prevalent domains in the human proteome and represent the major phosphoinositide-binding module. These domains are often found in signaling proteins and function predominately by targeting their host proteins to the cell membrane. Inositol phosphates, which are structurally similar to phosphoinositides, are not only known to play a role as signaling molecules but are also capable of being bound by PH domains. RESULTS: In the work presented here it is shown that the addition of commercial myo-inositol hexakisphosphate (IP6) inhibited the binding of the carboxy terminal PH domain of pleckstrin (C-PH) to phosphatidylinositol 3,4-bisphosphate with an IC50 of 7.5 micromolar. In an attempt to characterize this binding structurally, C-PH was crystallized in the presence of IP6 and the structure was determined to 1.35 angstrom. Examination of the resulting electron density unexpectedly revealed the bound ligand to be D-myo-inositol 1,2,3,5,6-pentakisphosphate. CONCLUSIONS: The discovery of D-myo-inositol 1,2,3,5,6-pentakisphosphate in the crystal structure suggests that the inhibitory effects observed in the binding studies may be due to this ligand rather than IP6. Analysis of the protein-ligand interaction demonstrated that this myo-inositol pentakisphosphate isomer interacts specifically with protein residues known to be involved in phosphoinositide binding. In addition to this, a structural alignment of other PH domains bound to inositol phosphates containing either four or five phosphate groups revealed that the majority of phosphate groups occupy conserved locations in the binding pockets of PH domains. These findings, taken together with other recently reported studies suggest that myo-inositol pentakisphosphates could act to regulate PH domain-phosphoinositide interactions by directly competing for binding, thus playing an important role as signaling molecules. [Abstract/Link to Full Text]

Noirel J, Simonson T
Neutral evolution of protein-protein interactions: a computational study using simple models.
BMC Struct Biol. 2007 Nov 19;7(1):79.
ABSTRACT: BACKGROUND: Protein-protein interactions are central to cellular organization, and must have appeared at an early stage of evolution. To understand better their role, we consider a simple model of protein evolution and determine the effect of an explicit selection for protein-protein interactions. RESULTS: In the model, viable sequences all have the same fitness, following the neutral evolution theory. A very simple, two-dimensional lattice representation of the protein structures is used, and the model only considers two kinds of amino acids: hydrophobic and polar. With these approximations, exact calculations are performed. The results do not depend too strongly on these assumptions, since a model using a 3D, off-lattice representation of the proteins gives results in qualitative agreement with the 2D one. With both models, the evolutionary dynamics lead to a steady state population that is enriched in sequences that dimerize with a high affinity, well beyond the minimal level needed to survive. Correspondingly, sequences close to the viability threshold are less abundant in the steady state, being subject to a larger proportion of lethal mutations. The set of viable sequences has a "funnel" shape, consistent with earlier studies: sequences that are highly populated in the steady state are "close" to each other (with proximity being measured by the number of amino acids that differ). CONCLUSIONS: This bias in the the steady state sequences should lead to an increased resistance of the population to environmental change and an increased ability to evolve. [Abstract/Link to Full Text]

Abyzov A, Ilyin VA
A comprehensive analysis of non-sequential alignments between all protein structures.
BMC Struct Biol. 2007 Nov 16;7(1):78.
ABSTRACT: BACKGROUND: The majority of relations between proteins can be represented as a conventional sequential alignment. Nevertheless, unusual non-sequential alignments with different connectivity of the aligned fragments in compared proteins have been reported by many researchers. It is interesting to understand those non-sequential alignments; are they unique, sporadic cases or they occur frequently; do they belong to a few specific folds or spread among many different folds, as a common feature of protein structure. We present here a comprehensive large-scale study of non-sequential alignments between available protein structures in Protein Data Bank. RESULTS: The study has been conducted on a non-redundant set of 8,865 protein structures aligned with the aid of the TOPOFIT method. It has been estimated that between 17.4% and 35.2% of all alignments are non-sequential depending on variations in the parameters. Analysis of the data revealed that non-sequential relations between proteins do occur systematically and in large quantities. Various sizes and numbers of non-sequential fragments have been observed with all possible complexities of fragment rearrangements found for alignments consisting of up to 12 fragments. It has been found that non-sequential alignments are not limited to proteins of any particular fold and are present in more than two hundred of them. Moreover, many of them are found between proteins with different fold assignments. It has been shown that protein structure symmetry does not explain non-sequential alignments. Therefore, compelling evidences have been provided that non-sequential alignments between proteins are systematic and widespread across the protein universe. CONCLUSIONS: The phenomenon of the widespread occurrence of non-sequential alignments between proteins might represent a missing rule of protein structure organization. More detailed study of this phenomenon will enhance our understanding of protein stability, folding, and evolution. [Abstract/Link to Full Text]

Silverman BD
Using molecular principal axes for structural comparison: determining the tertiary changes of a FAB antibody domain induced by antigenic binding.
BMC Struct Biol. 2007 Nov 9;7(1):77.
ABSTRACT: BACKGROUND: Comparison of different protein x-ray structures has previously been made in a number of different ways; for example, by visual examination, by differences in the locations of secondary structures, by explicit superposition of structural elements, e.g. alpha-carbon atom locations, or by procedures that utilize a common symmetry element or geometrical feature of the structures to be compared. RESULTS: A new approach is applied to determine the structural changes that an antibody protein domain experiences upon its interaction with an antigenic target. These changes are determined with the use of two different, however comparable, sets of principal axes that are obtained by diagonalizing the second-order tensors that yield the moments-of-geometry as well as an ellipsoidal characterization of domain shape, prior to and after interaction. Determination of these sets of axes for structural comparison requires no internal symmetry features of the domains, depending solely upon their representation in three-dimensional space. This representation may involve atomic, Carbon-alpha , or residue centroid coordinates. The present analysis utilizes residue centroids. When the structural changes are minimal, the principal axes of the domains, prior to and after interaction, are essentially comparable and consequently may be used for structural comparison. When the differences of the axes cannot be neglected, but are nevertheless slight, a smaller relatively invariant substructure of the domains may be utilized for comparison. The procedure yields two distance metrics for structural comparison. First, the displacements of the residue centroids due to antigenic binding, referenced to the ellipsoidal principal axes, are noted. Second, changes in the ellipsoidal distances with respect to the non-interacting structure provide a direct measure of the spatial displacements of the residue centroids, towards either the interior or exterior of the domain. CONCLUSIONS: With use of x-ray data from the protein data bank (PDB), these two metrics are shown to highlight, in a manner different from before, the structural changes that are induced in the overall domains as well as in the H3 loops of the complementarity-determining regions (CDR) upon FAB antibody binding to a truncated and to a synthetic hemagglutinin viral antigenic target. [Abstract/Link to Full Text]

Ramaswamy A, Ioshikhes I
Global dynamics of newly constructed oligonucleosomes of conventional and variant H2A.Z histone.
BMC Struct Biol. 2007 Nov 8;7(1):76.
ABSTRACT: BACKGROUND: Complexes of nucleosomes, which often occur in the gene promoter areas, are one of the fundamental levels of chromatin organization and thus are important for transcription regulation. Investigating the dynamic structure of a single nucleosome as well as nucleosome complexes is important for understanding transcription within chromatin. In a previous work, we highlighted the influence of histone variants on the functional dynamics of a single nucleosome using normal mode analysis developed by Bahar et al. The present work further analyzes the dynamics of nucleosome complexes (nucleosome oligomers or oligonucleosomes) such as dimer, trimer and tetramer (beads on a string model) with conventional core histones as well as with the H2A.Z histone variant using normal mode analysis. RESULTS: The global dynamics of oligonucleosomes reveal larger amplitude of motion within the nucleosomes that contain the H2A.Z variant with in-planar and out-of-planar fluctuations as the common mode of relaxation. The docking region of H2A.Z and the L1:L1 interactions between H2A.Z monomers of nucleosome (that are responsible for the highly stable nucleosome containing variant H2A.Z-histone) are highly dynamic throughout the first two dynamic modes. CONCLUSIONS: Dissection of the dynamics of oligonucleosomes discloses in-plane as well as out-of-plane fluctuations as the common mode of relaxation throughout the global motions. The dynamics of individual nucleosomes and the combination of the relaxation mechanisms expressed by the individual nucleosome are quite interesting and highly dependent on the number of nucleosome fragments present in the complexes. Distortions generated by the non-planar dynamics influence the DNA conformation, and hence the histone-DNA interactions significantly alter the dynamics of the DNA. The variant H2A.Z histone is a major source of weaker intra- and inter-molecular correlations resulting in more disordered motions. [Abstract/Link to Full Text]

Pallares I, Berenguer C, Aviles FX, Vendrell J, Ventura S
Self-assembly of human latexin into amyloid-like oligomers.
BMC Struct Biol. 2007 Nov 8;7(1):75.
ABSTRACT: BACKGROUND: In conformational disorders, it is not evident which amyloid aggregates affect specific molecular mechanisms or cellular pathways, which cause disease because of their quantity and mechanical features and which states in aggregate formation are pathogenic. Due to the increasing consensus that prefibrillar oligomers play a major role in conformational diseases, there is a growing interest in understanding the characteristics of metastable polypeptide associations. RESULTS: Here, we show that human latexin, a protein that shares the same fold with cystatin C, assembles into stable spherical amyloid-like oligomers that bind thioflavin-T and congo red similarly to common amyloid structures but do not evolve into fibrils. Latexin self-assembly correlates with the formation of a mostly denaturated state rather than with the population of partially structured intermediates during the unfolding process. The results suggest that unfolding of alpha-helix 3 might be involved in the transition of latexin toward amyloidotic species, supporting the notion of the protective role of the native protein structure against polymerization. CONCLUSIONS: Overall the data herein indicate that latexin could be a good model for the study of the structural and sequential determinants of oligomeric assemblies in protein aggregation processes. [Abstract/Link to Full Text]

Maslennikov I, Kefala G, Johnson C, Riek R, Choe S, Kwiatkowski W
NMR spectroscopic and analytical ultracentrifuge analysis of membrane protein detergent complexes.
BMC Struct Biol. 2007 Nov 8;7(1):74.
ABSTRACT: BACKGROUND: Structural studies of integral membrane proteins (IMPs) are hampered by inherent difficulties in their heterologous expression and in the purification of solubilized protein-detergent complexes (PDCs). The choice and concentrations of detergents used in an IMP preparation play a critical role in protein homogeneity and are thus important for successful crystallization. RESULTS: Seeking an effective and standardized means applicable to genomic approaches for the characterization of PDCs, we chose 1D-NMR spectroscopic analysis to monitor the detergent content throughout their purification: protein extraction, detergent exchange, and sample concentration. We demonstrate that a single NMR measurement combined with a SDS-PAGE of a detergent extracted sample provides a useful gauge of the detergent's extraction potential for a given protein. Furthermore, careful monitoring of the detergent content during the process of IMP production allows for a high level of reproducibility. We also show that in many cases a simple sedimentation velocity measurement provides sufficient data to estimate both the oligomeric state and the detergent-to-protein ratio in PDCs, as well as to evaluate the homogeneity of the samples prior to crystallization screening. CONCLUSIONS: The techniques presented here facilitate the screening and selection of the extraction detergent, as well as help to maintain reproducibility in the detergent exchange and PDC concentration procedures. Such reproducibility is particularly important for the optimization of initial crystallization conditions, for which multiple purifications are routinely required. [Abstract/Link to Full Text]

Falconi M, Biocca S, Novelli G, Desideri A
Molecular dynamics simulation of human LOX-1 provides an explanation for the lack of OxLDL binding to the Trp150Ala mutant.
BMC Struct Biol. 2007 Nov 7;7(1):73.
ABSTRACT: BACKGROUND: Dimeric lectin-like oxidized low-density lipoprotein receptor-1 LOX-1 is the target receptor for oxidized low density lipoprotein in endothelial cells. In vivo assays revealed that in LOX-1 the basic spine arginine residues are important for binding, which is lost upon mutation of Trp150 with alanine. Molecular dynamics simulations of the wild-type LOX-1 and of the Trp150Ala mutant C-type lectin-like domains, have been carried out to gain insight into the severe inactivating effect. RESULTS: The mutation does not alter the dimer stability, but a different dynamical behaviour differentiates the two proteins. As described by the residues fluctuation, the dynamic cross correlation map and the principal component analysis in the wild-type the two monomers display a symmetrical motion that is not observed in the mutant. CONCLUSIONS: The symmetrical motion of monomers is completely damped by the structural rearrangement caused by the Trp150Ala mutation. An improper dynamical coupling of the monomers and different fluctuations of the basic spine residues are observed, with a consequent altered binding affinity. [Abstract/Link to Full Text]

Carra JH, McHugh CA, Mulligan S, Machiesky LM, Soares AS, Millard CB
Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site.
BMC Struct Biol. 2007 Nov 6;7(1):72.
ABSTRACT: BACKGROUND: Ricin is a potent toxin and known bioterrorism threat with no available antidote. The ricin A-chain (RTA) acts enzymatically to cleave a specific adenine base from ribosomal RNA, thereby blocking translation. To understand better the relationship between ligand binding and RTA active site conformational change, we used a fragment-based approach to find a minimal set of bonding interactions able to induce rearrangements in critical side-chain positions. RESULTS: We found that the smallest ligand stabilizing an open conformer of the RTA active site pocket was an amide group, bound weakly by only a few hydrogen bonds to the protein. Complexes with small amide-containing molecules also revealed a switch in geometry from a parallel towards a splayed arrangement of an arginine-tryptophan cation-pi interaction that was associated with an increase and red-shift in tryptophan fluorescence upon ligand binding. Using the observed fluorescence signal, we determined the thermodynamic changes of adenine binding to the RTA active site, as well as the site-specific binding of urea. Urea binding had a favorable enthalpy change and unfavorable entropy change, with a deltaH of -13 +/-2 kJ/mol and a deltaS of -0.04 +/-0.01 kJ/K*mol. The side-chain position of residue Tyr80 in a complex with adenine was reexamined and found not to involve as large an overlap of rings with the purine as previously considered, suggesting a smaller role for aromatic stacking at the RTA active site. CONCLUSIONS: We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function. Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding to a protein, results that are relevant to understanding the physical mechanisms of protein denaturation. [Abstract/Link to Full Text]

Schonitzer V, Weiss IM
The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z.
BMC Struct Biol. 2007 Nov 6;7(1):71.
ABSTRACT: BACKGROUND: Chitin self-assembly provides a dynamic extracellular biomineralization interface. The insoluble matrix of larval shells of the marine bivalve mollusc Mytilus galloprovincialis consists of chitinous material that is distributed and structured in relation to characteristic shell features. Mollusc shell chitin is synthesized via a complex transmembrane chitin synthase with an intracellular myosin motor domain. RESULTS: Enzymatic mollusc chitin synthesis was investigated in vivo by using the small-molecule drug NikkomycinZ, a structural analogue to the sugar donor substrate UDP-N-acetyl-D-glucosamine (UDP-GlcNAc). The impact on mollusc shell formation was analyzed by binocular microscopy, polarized light video microscopy in vivo, and scanning electron microscopy data obtained from shell material formed in the presence of NikkomycinZ. The partial inhibition of chitin synthesis in vivo during larval development by NikkomycinZ (5 micro-molar to 10 micro-molar) dramatically alters the structure and thus the functionality of the larval shell at various growth fronts, such as the bivalve hinge and the shell's edges. CONCLUSIONS: Provided that NikkomycinZ mainly affects chitin synthesis in molluscs, the presented data suggest that the mollusc chitin synthase fulfils an important enzymatic role in the coordinated formation of larval bivalve shells. It can be speculated that chitin synthesis bears the potential to contribute via signal transduction pathways to the implementation of hierarchical patterns into chitin mineral-composites such as prismatic, nacre, and crossed-lamellar shell types. [Abstract/Link to Full Text]

Ku SY, Cornell KA, Howell PL
Structure of A. thaliana 5-methylthioribose kinase in complex with ADP and MTR reveals a more occluded active site than its bacterial homolog.
BMC Struct Biol. 2007 Oct 25;7(1):70.
ABSTRACT: BACKGROUND: Metabolic variations exist between the methionine salvage pathway of humans and a number of plants and microbial pathogens. 5-Methylthioribose (MTR) kinase is a key enzyme required for methionine salvage in plants and many bacteria. The absence of a mammalian homolog suggests that MTR kinase is a good target for the design of specific herbicides or antibiotics. RESULTS: The structure of Arabidopsis thaliana MTR kinase co-crystallized with ATPgammaS and MTR has been determined at 1.9 A resolution. The structure is similar to B. subtilis MTR kinase and has the same protein kinase fold observed in other evolutionarily related protein kinase-like phosphotransferases. The active site is comparable between the two enzymes with the DXE-motif coordinating the nucleotide-Mg, the D238 of the HGD catalytic loop polarizing the MTR O1 oxygen, and the RR-motif interacting with the substrate MTR. Unlike its bacterial homolog, however, the Gly-rich loop (G-loop) of A. thaliana MTR kinase has an extended conformation, which shields most of the active site from solvent, a feature that resembles eukaryotic protein kinases more than the bacterial enzyme. The G- and W-loops of A. thaliana and B. subtilis MTR kinase adopt different conformations despite high sequence similarity. The ATPgammaS analog was hydrolyzed during the co-crystallization procedure, resulting in ADP in the active site. This suggests that the A. thaliana enzyme, like its bacterial homolog, may have significant ATPase activity in the absence of MTR. CONCLUSIONS: The structure of A. thaliana MTR kinase provides a template for structure-based design of agrochemicals, particularly herbicides whose effectiveness could be regulated by nutrient levels. Features of the MTR binding site offer an opportunity for a simple organic salt of an MTR analog to specifically inhibit MTR kinase. [Abstract/Link to Full Text]

Nascimento AS, Catalano-Dupuy DL, Bernardes A, de Oliveira Neto M, Santos MA, Ceccarelli EA, Polikarpov I
Crystal structures of Leptospira interrogans FAD-containing ferredoxin-NADP+ reductase and its complex with NADP+
BMC Struct Biol. 2007 Oct 24;7(1):69.
ABSTRACT: BACKGROUND: Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron transfer between NADP(H) and the proteins ferredoxin or flavodoxin. A number of structural features distinguish plant and bacterial FNRs, one of which is the mode of the cofactor FAD binding. Leptospira interrogans is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. Leptospira interrogans FNR (LepFNR) displays low sequence identity with plant (34% with Zea mays) and bacterial (31% with Escherichia coli) FNRs. However, LepFNR contains all consensus sequences that define the plastidic class FNRs. RESULTS: The crystal structures of the FAD-containing LepFNR and the complex of the enzyme with NADP+, were solved and compared to known FNRs. The comparison reveals significant structural similarities of the enzyme with the plastidic type FNRs and differences with the bacterial enzymes. Our small angle X-ray scattering experiments show that LepFNR is a monomeric enzyme. Moreover, our biochemical data demonstrate that the LepFNR has an enzymatic activity similar to those reported for the plastidic enzymes and that is significantly different from bacterial flavoenzymes, which display lower turnover rates. CONCLUSION: LepFNR is the first plastidic type FNR found in bacteria and, despite of its low sequence similarity with plastidic FNRs still displays high catalytic turnover rates. The typical structural and biochemical characteristics of plant FNRs unveiled for LepFNR support a notion of a putative lateral gene transfer which presumably offers Leptospira interrogans evolutionary advantages. The wealth of structural information about LepFNR provides a molecular basis for advanced drugs developments against leptospirosis. [Abstract/Link to Full Text]

Buetow L, Brown AC, Parish T, Hunter WN
The structure of Mycobacteria 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, an essential enzyme, provides a platform for drug discovery.
BMC Struct Biol. 2007 Oct 23;7(1):68.
ABSTRACT: BACKGROUND: The prevalence of tuberculosis, the prolonged and expensive treatment that this disease requires and an increase in drug resistance indicate an urgent need for new treatments. The 1-deoxy-D-xylulose 5-phosphate pathway of isoprenoid precursor biosynthesis is an attractive chemotherapeutic target because it occurs in many pathogens, including Mycobacterium tuberculosis, and is absent from humans. To underpin future drug development it is important to assess which enzymes in this biosynthetic pathway are essential in the actual pathogens and to characterize them. RESULTS: The fifth enzyme of this pathway, encoded by ispF, is 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF). A two-step recombination strategy was used to construct ispF deletion mutants in M. tuberculosis but only wild-type double crossover strains were isolated. The chromosomal copy could be deleted when a second functional copy was provided on an integrating plasmid, demonstrating that ispF is an essential gene under the conditions tested thereby confirming its potential as a drug target. We attempted structure determination of the M. tuberculosis enzyme (MtIspF), but failed to obtain crystals. We instead analyzed the orthologue M. smegmatis IspF (MsIspF), sharing 73% amino acid sequence identity, at 2.2 A resolution. The high level of sequence conservation is particularly pronounced in and around the active site. MsIspF is a trimer with a hydrophobic cavity at its center that contains density consistent with diphosphate-containing isoprenoids. The active site, created by two subunits, comprises a rigid CDP-Zn2+ binding pocket with a flexible loop to position the 2C-methyl-D-erythritol moiety of substrate. Sequence-structure comparisons indicate that the active site and interactions with ligands are highly conserved. CONCLUSIONS: Our study genetically validates MtIspF as a therapeutic target and provides a model system for structure-based ligand design. [Abstract/Link to Full Text]

Tripathi T, Rahlfs S, Becker K, Bhakuni V
Glutathione mediated regulation of oligomeric structure and functional activity of Plasmodium falciparum glutathione S-transferase.
BMC Struct Biol. 2007;767.
BACKGROUND: In contrast to many other organisms, the malarial parasite Plasmodium falciparum possesses only one typical glutathione S-transferase. This enzyme, PfGST, cannot be assigned to any of the known GST classes and represents a most interesting target for antimalarial drug development. The PfGST under native conditions forms non-covalently linked higher aggregates with major population (approximately 98%) being tetramer. However, in the presence of 2 mM GSH, a dimer of PfGST is observed. Recently reported study on binding and catalytic properties of PfGST indicated a GSH dependent low-high affinity transition with simultaneous binding of two GSH molecules to PfGST dimer suggesting that GSH binds to low affinity inactive enzyme dimer converting it to high affinity functionally active dimer. In order to understand the role of GSH in tetramer-dimer transition of PfGST as well as in modulation of functional activity of the enzyme, detailed structural, functional and stability studies on recombinant PfGST in the presence and absence of GSH were carried out. RESULTS: Our data indicate that the dimer - and not the tetramer - is the active form of PfGST, and that substrate saturation is directly paralleled by dissociation of the tetramer. Furthermore, this dissociation is a reversible process indicating that the tetramer-dimer equilibrium of PfGST is defined by the surrounding GSH concentration. Equilibrium denaturation studies show that the PfGST tetramer has significantly higher stability compared to the dimer. The enhanced stability of the tetramer is likely to be due to stronger ionic interactions existing in it. CONCLUSION: This is the first report for any GST where an alteration in oligomeric structure and not just small conformational change is observed upon GSH binding to the enzyme. Furthermore we also demonstrate a reversible mechanism of regulation of functional activity of Plasmodium falciparum glutathione S-transferase via GSH induced dissociation of functionally inactive tetramer into active dimers. [Abstract/Link to Full Text]

Winnig M, Bufe B, Kratochwil NA, Slack JP, Meyerhof W
The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor.
BMC Struct Biol. 2007;766.
BACKGROUND: Differences in sweet taste perception among species depend on structural variations of the sweet taste receptor. The commercially used isovanillyl sweetener neohesperidin dihydrochalcone activates the human but not the rat sweet receptor TAS1R2+TAS1R3. Analysis of interspecies combinations and chimeras of rat and human TAS1R2+TAS1R3 suggested that the heptahelical domain of human TAS1R3 is crucial for the activation of the sweet receptor by neohesperidin dihydrochalcone. RESULTS: By mutational analysis combined with functional studies and molecular modeling we identified a set of different amino acid residues within the heptahelical domain of human TAS1R3 that forms the neohesperidin dihydrochalcone binding pocket. Sixteen amino acid residues in the transmembrane domains 2 to 7 and one in the extracellular loop 2 of hTAS1R3 influenced the receptor's response to neohesperidin dihydrochalcone. Some of these seventeen residues are also part of the binding sites for the sweetener cyclamate or the sweet taste inhibitor lactisole. In line with this observation, lactisole inhibited activation of the sweet receptor by neohesperidin dihydrochalcone and cyclamate competitively, whereas receptor activation by aspartame, a sweetener known to bind to the N-terminal domain of TAS1R2, was allosterically inhibited. Seven of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are thought to play a role in the binding of allosteric modulators of other class C GPCRs, further supporting our model of the neohesperidin dihydrochalcone pharmacophore. CONCLUSION: From our data we conclude that we identified the neohesperidin dihydrochalcone binding site at the human sweet taste receptor, which overlaps with those for the sweetener cyclamate and the sweet taste inhibitor lactisole. This readily delivers a molecular explanation of our finding that lactisole is a competitive inhibitor of the receptor activation by neohesperidin dihydrochalcone and cyclamate. Some of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are involved in the binding of allosteric modulators in other class C GPCRs, suggesting a general role of these amino acid positions in allosterism and pointing to a common architecture of the heptahelical domains of class C GPCRs. [Abstract/Link to Full Text]

Hegyi H, Schad E, Tompa P
Structural disorder promotes assembly of protein complexes.
BMC Struct Biol. 2007 Oct 8;7(1):65.
ABSTRACT: The idea that the assembly of protein complexes is linked with protein disorder has been inferred from only a few large complexes, such as the viral capsid or bacterial flagellar system. The relationship, which suggests that larger complexes have more disorder, has never been systematically tested. The recent high-throughput analyses of protein-protein interactions and protein complexes in the cell generated data that enable to address this issue by bioinformatic means. RESULTS: In this work we predicted structural disorder in proteins involved in complexes, as reported in high-throughput TAP-tag/MS analyses for both E. coli and S. cerevisiae, and correlated it with the size of complexes. Using IUPred to predict the disorder for each complex, we found a statistically significant correlation between disorder and the number of proteins assembled into complexes. The distribution of disorder has a median value of 10% in yeast for complexes of 2-4 components (6% in E. coli), but 18% for complexes in the size range of 11-100 proteins (12% in E. coli). The level of disorder as assessed for regions longer than 30 consecutive disordered residues shows an even stronger division between small and large complexes (median values about 4% for complexes of 2-4 components, but 12% for complexes of 11-100 components in yeast). The predicted correlation is also supported by experimental evidence, by observing the structural disorder in protein components of complexes that can be found in the Protein Data Bank (median values 1. 5% for complexes of 2 to 4 components, and 9.6% for complexes of 11 to 100 components in yeast). Further analysis shows that this correlation is not directly linked with the increased disorder in hub proteins, but reflects a genuine systemic property of the proteins that make up the complexes. CONCLUSIONS: Overall, it is suggested and discussed that the assembly of protein-protein complexes is enabled and probably promoted by protein disorder. [Abstract/Link to Full Text]

Ponomarenko JV, Bourne PE
Antibody-protein interactions: benchmark datasets and prediction tools evaluation.
BMC Struct Biol. 2007 Oct 2;7(1):64.
ABSTRACT: BACKGROUND: The ability to predict antibody binding sites (aka antigenic determinants or B-cell epitopes) for a given protein is a precursor to new vaccine design and diagnostics. Among the various methods of B-cell epitope identification X-ray crystallography is one of the most reliable methods. Using these experimental data computational methods exist for B-cell epitope prediction. As the number of structures of antibody-protein complexes grows, further interest in prediction methods using 3D structure is anticipated. This work aims to establish a benchmark for 3D structure-based epitope prediction methods. RESULTS: Two B-cell epitope benchmark datasets inferred from the 3D structures of antibody-protein complexes were defined. The first is a dataset of 62 representative 3D structures of protein antigens with inferred structural epitopes. The second is a dataset of 82 structures of antibody-protein complexes containing different structural epitopes. Using these datasets, eight web-servers developed for antibody and protein binding sites prediction have been evaluated. In no method did performance exceed a 40% precision and 46% recall. The values of the area under the receiver operating characteristic curve for the evaluated methods were about 0.6 for ConSurf, DiscoTope, and PPI-PRED methods and above 0.65 but not exceeding 0.70 for protein-protein docking methods when the best of the top ten models for the bound docking were considered; the remaining methods performed close to random. The benchmark datasets are included as a supplement to this paper. CONCLUSIONS: It may be possible to improve epitope prediction methods through training on datasets which include only immune epitopes and through utilizing more features characterizing epitopes, for example, the evolutionary conservation score. Notwithstanding, overall poor performance may reflect the generality of antigenicity and hence the inability to decipher B-cell epitopes as an intrinsic feature of the protein. It is an open question as to whether ultimately discriminatory features can be found. [Abstract/Link to Full Text]

Scotter AJ, Guo M, Tomczak MM, Daley ME, Campbell RL, Oko RJ, Bateman DA, Chakrabartty A, Sykes BD, Davies PL
Metal ion-dependent, reversible, protein filament formation by a designed beta-roll polypeptide.
BMC Struct Biol. 2007 Oct 1;7(1):63.
ABSTRACT: BACKGROUND: Here, we used a right-handed, calcium-dependent beta-roll fold found in secreted proteases and repeat-in-toxin proteins as a template for the design of minimal, soluble, monomeric, beta-roll polypeptides. Two peptides were made that contained two and four metal-binding sites, respectively, and exploited stacked tryptophan pairs to stabilise the fold and report on the conformational state of the polypeptide. RESULTS: Initial analysis of the two beta-roll peptides in the presence of calcium suggested the polypeptides were disordered. The addition of lanthanum to these peptides caused aggregation. Upon further study by light scattering and electron microscopy, the aggregates were identified as ordered protein filaments that required lanthanum to polymerize. These filaments could be disassembled by the addition of a chelating agent. A simple head-to-tail model is proposed for filament formation that explains their metal ion-dependency. The model is supported by the capping of a beta-roll polypeptide with biotin, which disrupts filament formation and provides the ability to control the average length of the filaments. CONCLUSIONS: Metal ion-dependent, reversible protein filament formation is demonstrated for two designed beta-roll polypeptides. The polypeptides form filaments that are 3 nm in diameter and several hundred nm in length. They are not amyloid-like in nature as demonstrated by their behaviour in the presence of Congo red and thioflavin T. A capping strategy allows for the control of filament length and for potential applications including the decoration of a protein filament with various functional moieties. [Abstract/Link to Full Text]

Tyagi R, Burley SK, Swaminathan S
X-ray structures of two proteins belonging to Pfam DUF178 revealed unexpected structural similarity to the DUF191 Pfam family.
BMC Struct Biol. 2007 Oct 1;7(1):62.
ABSTRACT: BACKGROUND: Pfam is a comprehensive collection of protein domains and families, with a range of well-established information including genome annotation. Pfam has two large series of functionally uncharacterized families, known as Domains of Unknown Function (DUFs) and Uncharacterized Protein Families (UPFs). RESULTS: Crystal structures of two proteins from Deinococcus radiodurans and Streptomyces coelicolor belonging to Pfam protein family DUF178 (ID: PF02621) have been determined using Selenium-Single-wavelength Anomalous Dispersion (Se-SAD). Based on the structure, we have identified the putative function for this family of protein. CONCLUSION: Unexpectedly, we found that DUF178 Pfam is remarkably similar to Pfam family DUF191 suggesting that the sequence-based classification alone may not be sufficient to classify proteins into Pfam families. [Abstract/Link to Full Text]

Moroni E, Caselle M, Fogolari F
Identification of DNA-binding protein target sequences by physical effective energy functions, free energy analysis of lambda repressor-DNA complexes.
BMC Struct Biol. 2007 Sep 27;7(1):61.
ABSTRACT: BACKGROUND: Specific binding of proteins to DNA is one of the most common ways in which gene expression is controlled. Although general rules for the DNA-protein recognition can be derived, the ambiguous and complex nature of this mechanism precludes a simple recognition code, therefore the prediction of DNA target sequences is not straightforward. DNA-protein interactions can be studied using computational methods which can complement the current experimental methods and offer some advantages. In the present work we use physical effective potentials to evaluate the DNA-protein binding affinities for the lambda repressor-DNA complex for which structural and thermodynamic experimental data are available. RESULTS: The binding free energy of two molecules can be expressed as the sum of an intermolecular energy (evaluated using a molecular mechanics forcefield), a solvation free energy term and an entropic term. Different solvation models are used including distance dependent dielectric constants, solvent accessible surface tension models and the Generalized Born model. The effect of conformational sampling by Molecular Dynamics simulations on the computed binding energy is assessed. The free energy of binding for non-specific complexes, estimated using the best energetic model, agrees with earlier theoretical suggestions. Moreover, as a results of these analyses, we propose a protocol for the prediction of DNA-binding target sequences. The possibility of searching regulatory elements within the bacteriophage-lambda genome using this protocol is explored. Our analysis shows good prediction capabilities, even in the absence of any thermodynamic data and information on the naturally recognized sequence. CONCLUSION: This study supports the conclusion that physics-based methods can offer a completely complementary methodology to sequence-based methods for the identification of DNA-binding protein target sequences. [Abstract/Link to Full Text]

Ferraroni M, Myasoedova NM, Schmatchenko V, Leontievsky AA, Golovleva LA, Scozzafava A, Briganti F
Crystal structure of a blue laccase from Lentinus tigrinus: evidences for intermediates in the molecular oxygen reductive splitting by multicopper oxidases.
BMC Struct Biol. 2007;760.
BACKGROUND: Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in pathogenesis, immunogenesis and morphogenesis of organisms and in the metabolic turnover of complex organic substances. They catalyze the coupling between the four one-electron oxidations of a broad range of substrates with the four-electron reduction of dioxygen to water. These catalytic processes are made possible by the contemporaneous presence of at least four copper ion sites, classified according to their spectroscopic properties: one type 1 (T1) site where the electrons from the reducing substrates are accepted, one type 2 (T2), and a coupled binuclear type 3 pair (T3) which are assembled in a T2/T3 trinuclear cluster where the electrons are transferred to perform the O2 reduction to H2O. RESULTS: The structure of a laccase from the white-rot fungus Lentinus (Panus) tigrinus, a glycoenzyme involved in lignin biodegradation, was solved at 1.5 A. It reveals a asymmetric unit containing two laccase molecules (A and B). The progressive reduction of the copper ions centers obtained by the long-term exposure of the crystals to the high-intensity X-ray synchrotron beam radiation under aerobic conditions and high pH allowed us to detect two sequential intermediates in the molecular oxygen reduction pathway: the "peroxide" and the "native" intermediates, previously hypothesized through spectroscopic, kinetic and molecular mechanics studies. Specifically the electron-density maps revealed the presence of an end-on bridging, micro-eta 1:eta 1 peroxide ion between the two T3 coppers in molecule B, result of a two-electrons reduction, whereas in molecule A an oxo ion bridging the three coppers of the T2/T3 cluster (micro3-oxo bridge) together with an hydroxide ion externally bridging the two T3 copper ions, products of the four-electrons reduction of molecular oxygen, were best modelled. CONCLUSION: This is the first structure of a multicopper oxidase which allowed the detection of two intermediates in the molecular oxygen reduction and splitting. The observed features allow to positively substantiate an accurate mechanism of dioxygen reduction catalyzed by multicopper oxidases providing general insights into the reductive cleavage of the O-O bonds, a leading problem in many areas of biology. [Abstract/Link to Full Text]

Monzani PS, Trapani S, Thiemann OH, Oliva G
Crystal structure of Leishmania tarentolae hypoxanthine-guanine phosphoribosyltransferase.
BMC Struct Biol. 2007 Sep 25;7(1):59.
ABSTRACT: BACKGROUND: Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) is a central enzyme of purine recycling pathway. Parasitic protozoa of the order Kinetoplastida are unable to synthesize purines de novo and use the salvage pathway for the synthesis of purine bases making this an attractive target for antiparasitic drug design. RESULTS: The glycosomal HGPRT from Leishmania tarentolae in a catalytically active form purified and co-crystallized with guanosine monophosphate (GMP) in the active site. The HGPRT dimer structure has been solved by molecular replacement and refined against data extending to 2.1 A resolution. The structure reveals the contacts of the active site residues with GMP. CONCLUSION: The comparative analysis between Leishmania and human HGPRT active sites revealed subtle differences in the ligand position and its interaction with the active site residues which could be responsible for the differential reactivity of both enzymes to allopurinol. The Leishmania HGPRT structure solved and analyzed may contribute to further investigations to fully understand this important enzyme family in protozoa parasites. [Abstract/Link to Full Text]

Porter CJ, Matthews JM, Mackay JP, Pursglove SE, Schmidberger JW, Leedman PJ, Pero SC, Krag DN, Wilce MC, Wilce JA
Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation.
BMC Struct Biol. 2007;758.
BACKGROUND: Human growth factor receptor bound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines. RESULTS: As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 A resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the muM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of Kd = approximately 35.7 microM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding. CONCLUSION: Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion. [Abstract/Link to Full Text]

Huang H, Zhang J, Shen W, Wang X, Wu J, Wu J, Shi Y
Solution structure of the second bromodomain of Brd2 and its specific interaction with acetylated histone tails.
BMC Struct Biol. 2007;757.
BACKGROUND: Brd2 is a transcriptional regulator and belongs to BET family, a less characterized novel class of bromodomain-containing proteins. Brd2 contains two tandem bromodomains (BD1 and BD2, 46% sequence identity) in the N-terminus and a conserved motif named ET (extra C-terminal) domain at the C-terminus that is also present in some other bromodomain proteins. The two bromodomains have been shown to bind the acetylated histone H4 and to be responsible for mitotic retention on chromosomes, which is probably a distinctive feature of BET family proteins. Although the crystal structure of Brd2 BD1 is reported, no structure features have been characterized for Brd2 BD2 and its interaction with acetylated histones. RESULTS: Here we report the solution structure of human Brd2 BD2 determined by NMR. Although the overall fold resembles the bromodomains from other proteins, significant differences can be found in loop regions, especially in the ZA loop in which a two amino acids insertion is involved in an uncommon pi-helix, termed piD. The helix piD forms a portion of the acetyl-lysine binding site, which could be a structural characteristic of Brd2 BD2 and other BET bromodomains. Unlike Brd2 BD1, BD2 is monomeric in solution. With NMR perturbation studies, we have mapped the H4-AcK12 peptide binding interface on Brd2 BD2 and shown that the binding was with low affinity (2.9 mM) and in fast exchange. Using NMR and mutational analysis, we identified several residues important for the Brd2 BD2-H4-AcK12 peptide interaction and probed the potential mechanism for the specific recognition of acetylated histone codes by Brd2 BD2. CONCLUSION: Brd2 BD2 is monomeric in solution and dynamically interacts with H4-AcK12. The additional secondary elements in the long ZA loop may be a common characteristic of BET bromodomains. Surrounding the ligand-binding cavity, five aspartate residues form a negatively charged collar that serves as a secondary binding site for H4-AcK12. We suggest that Brd2 BD1 and BD2 may possess distinctive roles and cooperate to regulate Brd2 functions. The structure basis of Brd2 BD2 will help to further characterize the functions of Brd2 and its BET members. [Abstract/Link to Full Text]

Khan S, Vihinen M
Spectrum of disease-causing mutations in protein secondary structures.
BMC Struct Biol. 2007;756.
BACKGROUND: Most genetic disorders are linked to missense mutations as even minor changes in the size or properties of an amino acid can alter or prevent the function of the protein. Further, the effect of a mutation is also dependent on the sequence and structure context of the alteration. RESULTS: We investigated the spectrum of disease-causing missense mutations in secondary structure elements in proteins with numerous known mutations and for which an experimentally defined three-dimensional structure is available. We obtained a comprehensive map of the differences in mutation frequencies, location and contact energies, and the changes in residue volume and charge - both in the mutated (original) amino acids and in the mutant amino acids in the different secondary structure types. We collected information for 44 different proteins involved in a large number of diseases. The studied proteins contained a total of 2413 mutations of which 1935 (80%) appeared in secondary structures. Differences in mutation patterns between secondary structures and whole proteins were generally not statistically significant whereas within the secondary structural elements numerous highly significant features were observed. CONCLUSION: Numerous trends in mutated and mutant amino acids are apparent. Among the original residues, arginine clearly has the highest relative mutability. The overall relative mutability among mutant residues is highest for cysteine and tryptophan. The mutability values are higher for mutated residues than for mutant residues. Arginine and glycine are among the most mutated residues in all secondary structures whereas the other amino acids have large variations in mutability between structure types. Statistical analysis was used to reveal trends in different secondary structural elements, residue types as well as for the charge and volume changes. [Abstract/Link to Full Text]

Brown AK, Meng G, Ghadbane H, Scott DJ, Dover LG, Nigou J, Besra GS, Fütterer K
Dimerization of inositol monophosphatase Mycobacterium tuberculosis SuhB is not constitutive, but induced by binding of the activator Mg2+.
BMC Struct Biol. 2007;755.
BACKGROUND: The cell wall of Mycobacterium tuberculosis contains a wide range of phosphatidyl inositol-based glycolipids that play critical structural roles and, in part, govern pathogen-host interactions. Synthesis of phosphatidyl inositol is dependent on free myo-inositol, generated through dephosphorylation of myo-inositol-1-phosphate by inositol monophosphatase (IMPase). Human IMPase, the putative target of lithium therapy, has been studied extensively, but the function of four IMPase-like genes in M. tuberculosis is unclear. RESULTS: We determined the crystal structure, to 2.6 A resolution, of the IMPase M. tuberculosis SuhB in the apo form, and analysed self-assembly by analytical ultracentrifugation. Contrary to the paradigm of constitutive dimerization of IMPases, SuhB is predominantly monomeric in the absence of the physiological activator Mg2+, in spite of a conserved fold and apparent dimerization in the crystal. However, Mg2+ concentrations that result in enzymatic activation of SuhB decisively promote dimerization, with the inhibitor Li+ amplifying the effect of Mg2+, but failing to induce dimerization on its own. CONCLUSION: The correlation of Mg2+-driven enzymatic activity with dimerization suggests that catalytic activity is linked to the dimer form. Current models of lithium inhibition of IMPases posit that Li+ competes for one of three catalytic Mg2+ sites in the active site, stabilized by a mobile loop at the dimer interface. Our data suggest that Mg2+/Li+-induced ordering of this loop may promote dimerization by expanding the dimer interface of SuhB. The dynamic nature of the monomer-dimer equilibrium may also explain the extended concentration range over which Mg2+ maintains SuhB activity. [Abstract/Link to Full Text]

Parthiban V, Gromiha MM, Abhinandan M, Schomburg D
Computational modeling of protein mutant stability: analysis and optimization of statistical potentials and structural features reveal insights into prediction model development.
BMC Struct Biol. 2007;754.
BACKGROUND: Understanding and predicting protein stability upon point mutations has wide-spread importance in molecular biology. Several prediction models have been developed in the past with various algorithms. Statistical potentials are one of the widely used algorithms for the prediction of changes in stability upon point mutations. Although the methods provide flexibility and the capability to develop an accurate and reliable prediction model, it can be achieved only by the right selection of the structural factors and optimization of their parameters for the statistical potentials. In this work, we have selected five atom classification systems and compared their efficiency for the development of amino acid atom potentials. Additionally, torsion angle potentials have been optimized to include the orientation of amino acids in such a way that altered backbone conformation in different secondary structural regions can be included for the prediction model. This study also elaborates the importance of classifying the mutations according to their solvent accessibility and secondary structure specificity. The prediction efficiency has been calculated individually for the mutations in different secondary structural regions and compared. RESULTS: Results show that, in addition to using an advanced atom description, stepwise regression and selection of atoms are necessary to avoid the redundancy in atom distribution and improve the reliability of the prediction model validation. Comparing to other atom classification models, Melo-Feytmans model shows better prediction efficiency by giving a high correlation of 0.85 between experimental and theoretical Delta Delta G with 84.06% of the mutations correctly predicted out of 1538 mutations. The theoretical Delta Delta G values for the mutations in partially buried beta-strands generated by the structural training dataset from PISCES gave a correlation of 0.84 without performing the Gaussian apodization of the torsion angle distribution. After the Gaussian apodization, the correlation increased to 0.92 and prediction accuracy increased from 80% to 88.89% respectively. CONCLUSION: These findings were useful for the optimization of the Melo-Feytmans atom classification system and implementing them to develop the statistical potentials. It was also significant that the prediction efficiency of mutations in the partially buried beta-strands improves with the help of Gaussian apodization of the torsion angle distribution. All these comparisons and optimization techniques demonstrate their advantages as well as the restrictions for the development of the prediction model. These findings will be quite helpful not only for the protein stability prediction, but also for various structure solutions in future. [Abstract/Link to Full Text]

Zotenko E, Dogan RI, Wilbur WJ, O'Leary DP, Przytycka TM
Structural footprinting in protein structure comparison: the impact of structural fragments.
BMC Struct Biol. 2007;753.
BACKGROUND: One approach for speeding-up protein structure comparison is the projection approach, where a protein structure is mapped to a high-dimensional vector and structural similarity is approximated by distance between the corresponding vectors. Structural footprinting methods are projection methods that employ the same general technique to produce the mapping: first select a representative set of structural fragments as models and then map a protein structure to a vector in which each dimension corresponds to a particular model and "counts" the number of times the model appears in the structure. The main difference between any two structural footprinting methods is in the set of models they use; in fact a large number of methods can be generated by varying the type of structural fragments used and the amount of detail in their representation. How do these choices affect the ability of the method to detect various types of structural similarity? RESULTS: To answer this question we benchmarked three structural footprinting methods that vary significantly in their selection of models against the CATH database. In the first set of experiments we compared the methods' ability to detect structural similarity characteristic of evolutionarily related structures, i.e., structures within the same CATH superfamily. In the second set of experiments we tested the methods' agreement with the boundaries imposed by classification groups at the Class, Architecture, and Fold levels of the CATH hierarchy. CONCLUSION: In both experiments we found that the method which uses secondary structure information has the best performance on average, but no one method performs consistently the best across all groups at a given classification level. We also found that combining the methods' outputs significantly improves the performance. Moreover, our new techniques to measure and visualize the methods' agreement with the CATH hierarchy, including the threshholded affinity graph, are useful beyond this work. In particular, they can be used to expose a similar composition of different classification groups in terms of structural fragments used by the method and thus provide an alternative demonstration of the continuous nature of the protein structure universe. [Abstract/Link to Full Text]