Morphology and escape performance of tiger salamander larvae (Ambystoma tigrinum mavortium)

The ability of an individual to escape predators is an important component of fitness. Several adaptive explanations of body shape variation in amphibians hypothesize relationships between swimming performance and morphology, but these ideas have rarely been tested. Here we investigate bivariate and multivariate relationships between natural variation in morphology and performance. We used high-speed video to examine fast-starts associated with escape responses in small tiger salamander larvae (Ambystoma tigrinum). Our results indicate that performance is influenced by interactions among aspects of morphology, physiology, and behavior. Relationships between morphometric variables and velocity could be detected with multivariate, but not bivariate statistical analyses. In particular, relationships between morphology and velocity depend on tail beat frequency (potentially a measure of effort or vigor). Relationships between morphology and acceleration were detected with bivariate analyses, but multivariate analysis suggests that acceleration performance, too, depends on interactions between morphology and tail beat frequency. We found a positive relationship between tail area and propulsive performance, which supports adaptive interpretations of variation in larval tail shape within and between amphibian species.     

Identification of the cyclamate interaction site within the transmembrane domain of the human sweet taste receptor subunit T1R3

The artificial sweetener cyclamate tastes sweet to humans, but not to mice. When expressed in vitro, the human sweet receptor (a heterodimer of two taste receptor subunits: hT1R2 + hT1R3) responds to cyclamate, but the mouse receptor (mT1R2 + mT1R3) does not. Using mixed-species pairings of human and mouse sweet receptor subunits, we determined that responsiveness to cyclamate requires the human form of T1R3. Using chimeras, we determined that it is the transmembrane domain of hT1R3 that is required for the sweet receptor to respond to cyclamate. Using directed mutagenesis, we identified several amino acid residues within the transmembrane domain of T1R3 that determine differential responsiveness to cyclamate of the human versus mouse sweet receptors. Alanine-scanning mutagenesis of residues predicted to line a transmembrane domain binding pocket in hT1R3 identified six residues specifically involved in responsiveness to cyclamate. Using molecular modeling, we docked cyclamate within the transmembrane domain of T1R3. Our model predicts substantial overlap in the hT1R3 binding pockets for the agonist cyclamate and the inverse agonist lactisole. The transmembrane domain of T1R3 is likely to play a critical role in the interconversion of the sweet receptor from the ground state to the active state.     

Metazoan Scc4 homologs link sister chromatid cohesion to cell and axon migration guidance

Saccharomyces cerevisiae Scc2 binds Scc4 to form an essential complex that loads cohesin onto chromosomes. The prevalence of Scc2 orthologs in eukaryotes emphasizes a conserved role in regulating sister chromatid cohesion, but homologs of Scc4 have not hitherto been identified outside certain fungi. Some metazoan orthologs of Scc2 were initially identified as developmental gene regulators, such as Drosophila Nipped-B, a regulator of cut and Ultrabithorax, and delangin, a protein mutant in Cornelia de Lange syndrome. We show that delangin and Nipped-B bind previously unstudied human and fly orthologs of Caenorhabditis elegans MAU-2, a non-axis-specific guidance factor for migrating cells and axons. PSI-BLAST shows that Scc4 is evolutionarily related to metazoan MAU-2 sequences, with the greatest homology evident in a short N-terminal domain, and protein–protein interaction studies map the site of interaction between delangin and human MAU-2 to the N-terminal regions of both proteins. Short interfering RNA knockdown of human MAU-2 in HeLa cells resulted in precocious sister chromatid separation and in impaired loading of cohesin onto chromatin, indicating that it is functionally related to Scc4, and RNAi analyses show that MAU-2 regulates chromosome segregation in C. elegans embryos. Using antisense morpholino oligonucleotides to knock down Xenopus tropicalis delangin or MAU-2 in early embryos produced similar patterns of retarded growth and developmental defects. Our data show that sister chromatid cohesion in metazoans involves the formation of a complex similar to the Scc2-Scc4 interaction in the budding yeast. The very high degree of sequence conservation between Scc4 homologs in complex metazoans is consistent with increased selection pressure to conserve additional essential functions, such as regulation of cell and axon migration during development.     

The structure of the Lingo-1 ectodomain, a module implicated in central nervous system repair inhibition

Nogo receptor (NgR)-mediated control of axon growth relies on the central nervous system-specific type I transmembrane protein Lingo-1. Interactions between Lingo-1 and NgR, along with a complementary co-receptor, result in neurite and axonal collapse. In addition, the inhibitory role of Lingo-1 is particularly important in regulation of oligodendrocyte differentiation and myelination, suggesting that pharmacological modulation of Lingo-1 function could be a novel approach for nerve repair and remyelination therapies. Here we report on the crystal structure of the ligand-binding ectodomain of human Lingo-1 and show it has a bimodular, kinked structure composed of leucine-rich repeat (LRR) and immunoglobulin (Ig)-like modules. The structure, together with biophysical analysis of its solution properties, reveals that in the crystals and in solution Lingo-1 persistently associates with itself to form a stable tetramer and that it is its LRR-Ig-composite fold that drives such assembly. Specifically, in the crystal structure protomers of Lingo-1 associate in a ring-shaped tetramer, with each LRR domain filling an open cleft in an adjacent protomer. The tetramer buries a large surface area (9,200 A2) and may serve as an efficient scaffold to simultaneously bind and assemble the NgR complex components during activation on a membrane. Potential functional binding sites that can be identified on the ectodomain surface, including the site of self-recognition, suggest a model for protein assembly on the membrane.     

Serum sphinganine and the sphinganine to sphingosine ratio as a biomarker of dietary fumonisins during chronic exposure in ducks

Sphinganine concentration (Sa) and sphinganine to sphingosine ratio (Sa/So) are sensitive biomarkers of fumonisin B1 (FB1) exposure in animals and have been proposed to reveal FB1 exposure in humans. They correlate with liver and kidney toxicity and often precede signs of toxicity. However, the use of Sa and Sa/So is confusing during chronic exposure. Indeed, some authors report altered sphingolipids metabolism, whereas others fail to demonstrate significant effect. The aim of this study was to investigate the kinetics of Sa and Sa/So in the serum of ducks over a 77-day exposure to 0, 2, 8, 32 and 128 mg FB1/kg feeds. Serum biochemistry was also investigated to reveal hepatotoxicity. The results obtained indicate that the kinetics of sphingolipids and serum biochemistry are closely linked with the duration of the exposure. After a strong and rapid increase Sa and Sa/So decrease then stabilize. The lowest investigated dose able to determine a detectable effect is 2 mg/kg feeds, the Sa/So ratio being the most sensitive biomarker of FB1 exposure.     

Effects of fumonisins on liver and kidney sphinganine and the sphinganine to sphingosine ratio during chronic exposure in ducks

Sa and the Sa/So ratio are very sensitive biomarkers of exposure to fumonisins in several species. We previously demonstrated that increases in Sa and in the Sa/So ratio in serum were less pronounced when ducks ingested fumonisins for more than 7 weeks than when animals were exposed for only 1-2 weeks [S.T. Tran, D. Tardieu, A. Auvergne, J.D. Bailly, R. Babilé, S. Durand, G. Benard, P. Guerre, Serum sphinganine and the sphinganine to sphingosine ratio as biomarker of dietary fumonisins during chronic exposure in ducks, Chem. Biol. Interact., in press]. The aim of this study was to investigate the kinetics of Sa and of the Sa/So in both liver and kidney of ducks that have been previously tested for Sa and the Sa/So ratio in serum. Analysis were performed on treatment days 0, 7, 14, 28 and 77 in five groups of ducks fed fumonisins obtained from an extract of Fusarium verticillioides culture material by daily gavage to obtain an exposure equal to 0, 2, 8, 32 and 128 mg FB1/kg feed. Sa and the Sa/So ratio in tissues were then correlated with Sa and the Sa/So ratio previously obtained in serum. The amounts on sphinganine 1-phosphate (Sa1P) and sphingosine1-phosphate (So1P) in the liver were also investigated. On day 7 of treatment, 2mg/kg FB1 in the feed were sufficient to increase Sa and the Sa/So ratio in liver (by 165 and 148%, respectively) and kidney (by 193 and 104%, respectively). At a rate of 128 mg/kg FB1 in the feed, a very high increase in Sa concentration was observed in both liver and kidney without mortality and/or signs of necrosis (respective increase of 2034 and 3768%). Although the precise mechanism of the resistance of ducks to fumonisin-induced hepatotoxicity is still uncertain, it might be linked to the rate at which the sphingoid bases sphinganine and sphingosine are converted to their 1-phosphate or other metabolite and eliminated from target tissues.     

Metabolic consequences of NDUFS4 gene deletion in immortalized mouse embryonic fibroblasts

Human mitochondrial complex I (CI) deficiency is associated with progressive neurological disorders. To better understand the CI pathomechanism, we here studied how deletion of the CI gene NDUFS4 affects cell metabolism. To this end we compared immortalized mouse embryonic fibroblasts (MEFs) derived from wildtype (wt) and whole-body NDUFS4 knockout (KO) mice. Mitochondria from KO cells lacked the NDUFS4 protein and mitoplasts displayed virtually no CI activity, moderately reduced CII, CIII and CIV activities and normal citrate synthase and CV (F(o)F(1)-ATPase) activity. Native electrophoresis of KO cell mitochondrial fractions revealed two distinct CI subcomplexes of ~830kDa (enzymatically inactive) and ~200kDa (active). The level of fully-assembled CII-CV was not affected by NDUFS4 gene deletion. KO cells exhibited a moderately reduced maximal and routine O(2) consumption, which was fully inhibited by acute application of the CI inhibitor rotenone. The aberrant CI assembly and reduced O(2) consumption in KO cells were fully normalized by NDUFS4 gene complementation. Cellular [NAD(+)]/[NADH] ratio, lactate production and mitochondrial tetramethyl rhodamine methyl ester (TMRM) accumulation were slightly increased in KO cells. In contrast, NDUFS4 gene deletion did not detectably alter [NADP(+)]/[NADPH] ratio, cellular glucose consumption, the protein levels of hexokinases (I and II) and phosphorylated pyruvate dehydrogenase (P-PDH), total cellular adenosine triphosphate (ATP) level, free cytosolic [ATP], cell growth rate, and reactive oxygen species (ROS) levels. We conclude that the NDUFS4 subunit is of key importance in CI stabilization and that, due to the metabolic properties of the immortalized MEFs, NDUFS4 gene deletion has only modest effects at the live cell level. This article is part of a special issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions ¹. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage ^{2, 3}. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle ^4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection ⁷. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization ⁸. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.     

Influence of mitochondrial DNA level on cellular energy metabolism: implications for mitochondrial diseases

The total amount of cellular mitochondrial DNA (mtDNA) varies widely and seems to be related to the nature and metabolic state of tissues and cells in culture. It is not known, however, whether this variation has any significance in vivo, and to which extent it regulates energy production. To better understand the importance of the cellular mtDNA level, we studied the influence of a gradual reduction of mtDNA copy number on oxidative phosphorylation in two models: (a) a control human cell line treated with different concentrations of 2′, 3′-dideoxycytidine, a nucleoside analogue that inhibits mtDNA replication by interfering with mitochondrial DNA polymerase γ, and (b) a cell line derived from a patient presenting mtDNA depletion. The two models were used to construct biochemical and phenotypic threshold curves. Our results show that oxidative phosphorylation activities are under a tight control by the amount of mtDNA in the cell, and that the full complement of mtDNA molecules are necessary to maintain a normal energy production level.