The v-ATPase V0 subunit a1 is required for a late step in synaptic vesicle exocytosis in Drosophila

The V(0) complex forms the proteolipid pore of an ATPase that acidifies vesicles. In addition, an independent function in membrane fusion has been proposed largely based on yeast vacuolar fusion experiments. We have isolated mutations in the largest V(0) component vha100-1 in flies in an unbiased genetic screen for synaptic malfunction. The protein is only required in neurons, colocalizes with markers for synaptic vesicles as well as active zones, and interacts with t-SNAREs. Loss of vha100-1 leads to vesicle accumulation in synaptic terminals, suggesting a deficit in release. The amplitude of spontaneous release events and release with hypertonic stimulation indicate normal levels of neurotransmitter loading, yet mutant embryos display severe defects in evoked synaptic transmission and FM1-43 uptake. Our data suggest that Vha100-1 functions downstream of SNAREs in synaptic vesicle fusion.     

Angiotensin converting enzyme gene polymorphism and myocardial infarction a large association and linkage study

The DD genotype of the angiotensin converting enzyme (ACE) polymorphism has been associated with myocardial infarction (MI). However, sample sizes of many case-control studies showing positive association were small and data were inconsistent. Furthermore, no family-based study is available.In a case-control study frequencies of the ACE genotypes were compared in 1319 unrelated patients with previous MI before 60 years of age (616 from the MONICA Augsburg region and 703 from rehabilitation centers in south Germany) and in 2381 population controls from the MONICA Augsburg study region). Furthermore, linkage and association of the ACE I/D polymorphism with MI were tested in 246 informative families using the sib-transmission/disequilibrium test (S-TDT).Overall, no excess of the D allele was found in MI patients (frequency 0.53 versus 0.57 in the general population; P=0.2). The ACE DD genotype was even slightly less frequent in groups with MI compared to the general population controls (0.26 versus 0.33 in women and 0.28 versus 0.33 in men). Similar results were also obtained in 247 men with low cardiovascular risk. In the family-based study, the frequency of the D allele was not different in siblings with or without previous MI (0.53 versus 0.50, respectively; S-TDT P=0.15) indicating no linkage or association of the D allele with MI.In a case-control study of MI patients and controls from the general population as well as a family study neither association nor linkage of the ACE D allele with MI was detected despite sample sizes that were among the largest samples studied so far.     

Exploring pleiotropy using principal components

A standard multivariate principal components (PCs) method was utilized to identify clusters of variables that may be controlled by a common gene or genes (pleiotropy). Heritability estimates were obtained and linkage analyses performed on six individual traits (total cholesterol (Chol), high and low density lipoproteins, triglycerides (TG), body mass index (BMI), and systolic blood pressure (SBP)) and on each PC to compare our ability to identify major gene effects. Using the simulated data from Genetic Analysis Workshop 13 (Cohort 1 and 2 data for year 11), the quantitative traits were first adjusted for age, sex, and smoking (cigarettes per day). Adjusted variables were standardized and PCs calculated followed by orthogonal transformation (varimax rotation). Rotated PCs were then subjected to heritability and quantitative multipoint linkage analysis. The first three PCs explained 73% of the total phenotypic variance. Heritability estimates were above 0.60 for all three PCs. We performed linkage analyses on the PCs as well as the individual traits. The majority of pleiotropic and trait-specific genes were not identified. Standard PCs analysis methods did not facilitate the identification of pleiotropic genes affecting the six traits examined in the simulated data set. In addition, genes contributing 20% of the variance in traits with over 0.60 heritability estimates could not be identified in this simulated data set using traditional quantitative trait linkage analyses. Lack of identification of pleiotropic and trait-specific genes in some cases may reflect their low contribution to the traits/PCs examined or more importantly, characteristics of the sample group analyzed, and not simply a failure of the PC approach itself.     

NarE: a novel ADP-ribosyltransferase from Neisseria meningitidis

Mono ADP-ribosyltransferases (ADPRTs) are a class of functionally conserved enzymes present in prokaryotic and eukaryotic organisms. In bacteria, these enzymes often act as potent toxins and play an important role in pathogenesis. Here we report a profile-based computational approach that, assisted by secondary structure predictions, has allowed the identification of a previously undiscovered ADP-ribosyltransferase in Neisseria meningitidis (NarE). NarE shows structural homologies with E. coli heat-labile enterotoxin (LT) and cholera toxin (CT) and possesses ADP-ribosylating and NAD-glycohydrolase activities. As in the case of LT and CT, NarE catalyses the transfer of the ADP-ribose moiety to arginine residues. Despite the absence of a signal peptide, the protein is efficiently exported into the periplasm of Neisseria. The narE gene is present in 25 out of 43 strains analysed, is always present in ET-5 and Lineage 3 but absent in ET-37 and Cluster A4 hypervirulent lineages. When present, the gene is 100% conserved in sequence and is inserted upstream of and co-transcribed with the lipoamide dehydrogenase E3 gene. Possible roles in the pathogenesis of N. meningitidis are discussed.     

The role of tetrahydrobiopterin in superoxide generation from eNOS: enzymology and physiological implications

Tetrahydrobiopterin (BH₄) is a ubiquitous pteridine metabolite that serves as a NOS cofactor. Recently, we showed that BH₄ efficiently inhibits superoxide generation from the heme group at the oxygenase domain of eNOS. This role indicates that BH₄ acts as a redox switch in the catalytic mechanism of the enzyme, which may have important consequences in the physiology of the endothelium. Here the mechanism by which BH₄ inhibits superoxide release from eNOS and the "uncoupling" effects of oxidized BH₄ metabolites are presented. The implications of the disparate actions of fully reduced and oxidized BH 4 metabolites in the control of eNOS biochemistry are discussed in the light of clinical data indicating that BH 4 levels are important in the regulation of superoxide levels and of endothelial reactivity.     

The development of exoerythrocytic stages of Plasmodium inui shortti in New World monkeys

Attempts are being made to adapt Old World monkey malarial parasites to New World monkeys for vaccine and molecular studies. Several of these (Plasmodium cynomolgi Berok, Plasmodium fragile, and Plasmodium knowlesi) grow readily but have failed to produce infective gametocytes. Plasmodium gonderi and Plasmodium fieldi develop in the liver after sporozoite inoculation but have failed to establish infection in the erythrocyte. Anopheles dirus mosquitoes infected with Plasmodium inui shortti by feeding on infected macaques transmitted the infection to Saimiri boliviensis monkeys. Infective gametocytes were produced, and sporozoite transmission from Saimiri to Saimiri monkey was obtained. Exoerythrocytic stages have also been observed in the liver tissue of Saimiri monkeys. The availability of the complete transmission cycle provides an additional resource for immunologic and vaccine studies.     

Particle and prey detection by mechanoreceptive copepods: a mathematical analysis

When particles move through fluids, they produce far-field pressure differences and near-field fluid deformations. Here we evaluate if a copepod, relying on mechanoreceptive antennulary setal hairs, can detect pressure changes caused by a variety of signal sources. We first provide a correction of the copepod mechanoreception model of Legier-Visser et al. (1986), showing how an object above a minimum size should be detectable. The pressure change P created by an object of this minimum size was 385 dynes/cm², based on biomechanical relationships for a rigid seta bending with respect to the exoskeletal body and using the neurophysiological detection threshold of a 10 nm bend of the sensory seta (Yen et al., 1992). The P for: a 3 m particle = 0.01 dynes/cm², a 50 m particle = 0.16 dynes/cm², an escaping nauplius = 78 dynes/cm², a revolving prey = 10^–5 dynes/cm², a 1 mm copepod escaping at 1 m/s at a distance of 1 mm from the mechanoreceptive sensory hairs of its captor = 312 dynes/cm². Only the copepod escaping at high-speed close to the captor would create a pressure difference that could elicit a response. At this point, we conclude that pressure differences are rarely of a magnitude that is perceptible and that additional information must be derived for a copepod to detect prey. Other signals include fluid deformations as well as other types of stimuli (odor, shadows). Like most organisms, a copepod will rely on all sensory modalities to find food, avoid predators, and track mates, assuring their survival in the aquatic environment. It also is possible that the biomechanical model is insufficient for estimating pressure differences causing the cuticular deformation or that further analysis is necessary to improve our certainty of the sensitivity of the copepod seta.     

Polarographic measurement of hydrogen sulfide production and consumption by mammalian tissues

The role of nitric oxide (NO) in redox cell signaling is widely accepted. However, the biological role of another candidate small inorganic signaling molecule and the subject of this study, hydrogen sulfide (H2S), is much less known. H2S as a reductant and nucleophile has numerous potential cellular targets; however, its rapid biological oxidation suggests a fleeting cellular existence. The challenge of accurate real-time measurement of H2S at low micromolar or nanomolar concentrations in biological preparations represents a major impediment to H2S investigations. We here demonstrate the use of a novel polarographic H2S sensor (PHSS) to follow rapid changes in H2S concentration in common buffered biological solutions with a detection limit near 10 nM. The PHSS, used in combination with O2 and NO sensors in multisensor respirometry, shows stability, a high signal-to-noise ratio, and signal specificity for H2S. Preparations of rat vascular tissue exhibit H2S production on the addition of sulfhydryl-bearing amino acid substrates and H2S consumption when supplied with exogenous H2S. Taken together, these findings suggest the existence of dynamic steady-state cellular H2S levels. The PHSS should facilitate the investigation of H2S biology by providing a previously unattainable continuous record of H2S under biologically relevant conditions.