Conformational change of syntaxin linker region induced by Munc13s initiates SNARE complex formation in synaptic exocytosis

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin-1 adopts a closed conformation when bound to Munc18-1, preventing binding to synaptobrevin-2 and SNAP-25 to form the ternary SNARE complex. Although it is known that the MUN domain of Munc13-1 catalyzes the transition from the Munc18-1/syntaxin-1 complex to the SNARE complex, the molecular mechanism is unclear. Here, we identified two conserved residues (R151, I155) in the syntaxin-1 linker region as key sites for the MUN domain interaction. This interaction is essential for SNARE complex formation in vitro and synaptic vesicle priming in neuronal cultures. Moreover, this interaction is important for a tripartite Munc18-1/syntaxin-1/MUN complex, in which syntaxin-1 still adopts a closed conformation tightly bound to Munc18-1, whereas the syntaxin-1 linker region changes its conformation, similar to that of the LE mutant of syntaxin-1 when bound to Munc18-1. We suggest that the conformational change of the syntaxin-1 linker region induced by Munc13-1 initiates ternary SNARE complex formation in the neuronal system.     

Role of extracellular charged amino acids in the yeast alpha-factor receptor

The yeast pheromone receptor, Ste2p, is a G protein coupled receptor that initiates cellular responses to alpha-mating pheromone, a 13 residue peptide that carries a net positive charge at physiological pH. We have examined the role of extracellular charged groups on the receptor in response to the pheromone. Substitutions of Asn or Ala for one extracellular residue, Asp275, affected both pheromone binding and signaling, suggesting that this position interacts directly with ligand. The other seven extracellular acidic residues could be individually replaced by polar residues with no detectable effects on receptor function. However, substitution of Ala for each of these seven residues resulted in impairment of signaling without affecting pheromone binding, implying that the polar nature of these residues promotes receptor activation. In contrast, substitution of Ala for each of the six positively charged residues at the extracellular surface of Ste2p did not affect signaling.     

Docosahexaenoic acid is selectively enriched in plasma phospholipids during pregnancy in Trinidadian women--results of a pilot study

The fetal demand for docosahexaenoic acid (DHA) has to be satisfied by the mother. We determined the fatty acids in maternal plasma non-esterified fatty acid (NEFA), triacylglycerol (TAG) and phosphatidylcholine (PC), in a cross-sectional study of non-pregnant (n = 10), pregnant (n = 19), and postpartum (n = 9) women. There were lipid class-dependent differences in plasma polyunsaturated fatty acid (PUFA) concentrations between groups. During pregnancy, DHA was most highly enriched in PC, about 230%, with more modest enrichment for linoleic acid (LA) and arachidonic acid (AA), and no enrichment of alpha-linolenic acid (alpha-LNA). There was relative enrichment of LA, AA and alpha-LNA in TAG, but not of DHA. There was no specific enrichment of any PUFA in the NEFA pool. These data accord with the suggestion that the enrichment of alpha-LNA in TAG and of DHA in phospholipids reflects hepatic regulation of n-3 PUFA metabolism which potentially enhances the delivery of DHA to the placenta.     

Carolina Chickadee (Aves, Paridae, Poecile carolinensis) Vocalization Rates: Effects of Body Mass and Food Availability under Aviary Conditions

We evaluated the effect of body mass and several environmental factors on vocalization rates of Carolina chickadees ( Poecile carolinensis ) housed in an aviary. Two different nonsong vocalizations (tseet and chick-a-dee) and song (fee-bee fee-bay) were recorded. Food was delivered from a feeder and three different levels of food access were presented to each bird: 10, 40 and 55 min/d. Two scales of body mass were measured: 'dawn mass' and 'focal mass' (mass during a focal observation divided by dawn mass). Across all birds, there was a significant negative correlation between both nonsong vocalization rates and body mass (both dawn and focal mass) and the effect of mass on call rate was greatest for days when food was relatively abundant. Nonsong vocalizations were also given at higher rates when food was limited (10 min/d) than when food was more abundant (40 and 55 min/d). No changes in call rates with time of day were observed. Overall, song rates were substantially lower than nonsong rates. Unlike nonsong rates, song rates were highest in birds that had relatively high dawn mass. No significant correlation between song rates and focal mass was observed, and no significant correlation between song rates and time of day was observed. Finally, vocalizations from nonfocal birds had little effect on vocalization rates of focal birds. Our results suggest that nonsong communicative signals are more important for birds facing energetic stress, while song is more important for birds that are not energetically stressed.     

A Deletion Map of cyc1 Mutants and Its Correspondence to Mutationally Altered Iso-1-Cytochromes c of Yeast

Mutants arising spontaneously from sporulated cultures of certain strains of yeast, Saccharomyces cerevisiae, contained deletions of the CYC1 gene which controls the primary structure of iso-1-cytochrome c. At least 60 different kinds of deletions were uncovered among the 104 deletions examined and these ranged in length from those encompassing only two adjacent point mutants to those encompassing at least the entire CYC1 gene. X-ray-induced recombination rates of crosses involving these deletions and cyc1 point mutants resulted in the assignment of 211 point mutants to 47 mutational sites and made it possible to unambiguously order 40 of these 47 sites. Except for one mutant, cyc1-15, there was a strict colinear relationship between the deletion map and the positions of 13 sites that were previously determined by amino acid alterations in iso-1-cytochromes c from intragenic revertants.     

Homing endonuclease mediated gene targeting in Anopheles gambiae cells and embryos

Homing endonuclease genes (HEGs) are ‘selfish’ genetic elements that combine the capability to selectively disrupt specific gene sequences with the ability to rapidly spread from a few individuals to an entire population through homologous recombination repair events. Because of these properties, HEGs are regarded as promising candidates to transfer genetic modifications from engineered laboratory mosquitoes to wild-type populations including Anopheles gambiae the vector of human malaria. Here we show that I-SceI and I-PpoI homing endonucleases cleave their recognition sites with high efficiency in A. gambiae cells and embryos and we demonstrate HEG-induced homologous and non-homologous repair events in a variety of functional assays. We also propose a gene drive system for mosquitoes that is based on our finding that I-PpoI cuts genomic rDNA located on the X chromosome in A. gambiae, which could be used to selectively incapacitate X-carrying spermatozoa thereby imposing a severe male-biased sex ratio.     

The mutational structure of metabolism in Caenorhabditis elegans

A properly functioning organism must maintain metabolic homeostasis. Deleterious mutations degrade organismal function, presumably at least in part via effects on metabolic function. Here we present an initial investigation into the mutational structure of the Caenorhabditis elegans metabolome by means of a mutation accumulation experiment. We find that pool sizes of 29 metabolites vary greatly in their vulnerability to mutation, both in terms of the rate of accumulation of genetic variance (the mutational variance, VM) and the rate of change of the trait mean (the mutational bias, ΔM). Strikingly, some metabolites are much more vulnerable to mutation than any other trait previously studied in the same way. Although we cannot statistically assess the strength of mutational correlations between individual metabolites, principal component analysis provides strong evidence that some metabolite pools are genetically correlated, but also that there is substantial scope for independent evolution of different groups of metabolites. Averaged over mutation accumulation lines, PC3 is positively correlated with relative fitness, but a model in which metabolites are uncorrelated with fitness is nearly as good by Akaike's Information Criterion.     

CO2 elimination as a function of frequency and tidal volume in rabbits during HFO

CO2 elimination (VCO2) was monitored during high-frequency oscillation (HFO) over a frequency (f) range of 2-30 Hz in anesthetized and paralyzed rabbits to determine whether effective gas exchange could be achieved in this species, to determine the f and tidal volume (VT) dependence of gas exchange in this species, and to compare these results with those from dog and human studies. We were able to produce VCO2 levels during HFO that exceeded normal steady-state levels of CO2 production with VT's less than the total dead space volume. VCO2 was related to f in a curvilinear fashion, whereas in some rabbits VCO2 became independent of f at higher frequencies. This curvilinear relationship between f and VCO2 is similar to data from humans but contrasts with the linear relationship found in dogs. Evidence is presented indicating frequency-dependent behavior of gas exchange is correlated with a frequency-dependent decrease in respiratory system resistance. We propose that the frequency-dependent mechanical properties of the rabbit lung may also account for the species differences in HFO gas exchange.