Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H+-V-ATPase

Vacuolar (H+)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an alpha-helical conformation for peptide MTM7 and in DMSO three alpha-helical regions are identified by 2D 1H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an alpha-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.     

Hosts are more important than destinations: What genetic variation in Microctonus aethiopoides (Hymenoptera: Braconidae) means for foreign exploration for natural enemies

Nucleotide sequence data were generated from the gene regions COI, 16S, and arginine kinase to assess genetic variation within the Palearctic parasitoid, Microctonus aethiopoides, reared from Sitona discoideus, S. hispidulus, and Hypera postica collected from two proximate locations in Mediterranean France. Partitioned Bayesian phylogenetic analyses of the molecular data provided strong support for the presence of at least two M. aethiopoides biotypes, one associated with Hypera species and the other with Sitona species. These new results combined with previously published data from 14 countries show that M. aethiopoides genetic variation is much more strongly correlated with host taxon than with sampling location. This contrasts with earlier perceptions that M. aethiopoides exhibits significant geographic variation, and helps to explain the widely varying biological control outcomes that have been obtained following the introductions of M. aethiopoides to Australia, New Zealand, and North America. The results strongly suggest that success rates and environmental safety in biological control would both be improved by ensuring that parasitoids collected in the native range are reared from the same host species as the one being targeted for control in the region of introduction. The results also provided insights both on the evolution of M. aethiopoides' host range, and on its evolutionary transition between solitary and gregarious larval development.     

Combined molecular and morphological phylogenetic analyses of the New Zealand wolf spider genus Anoteropsis (Araneae: Lycosidae)

Datasets from the mitochondrial gene regions NADH dehydrogenase subunit I (ND1) and cytochrome c oxidase subunit I (COI) of the 20 species in the New Zealand wolf spider (Lycosidae) genus Anoteropsis were generated. Sequence data were phylogenetically analysed using parsimony and maximum likelihood analyses. The phylogenies generated from the ND1 and COI sequence data and a previously generated morphological dataset were significantly congruent (p<0.001). Sequence data were combined with morphological data and phylogenetically analysed using parsimony. The ND1 region sequenced included part of tRNA^Leu(CUN), which appears to have an unstable amino-acyl arm and no TψC arm in lycosids. Analyses supported the existence of five species groups within Anoteropsis and the monophyly of species represented by multiple samples. A radiation of Anoteropsis species within the last five million years is inferred from the ND1 and COI likelihood phylograms, habitat and geological data, which also indicates that Anoteropsis arrived in New Zealand some time after it separated from Gondwana.     

Molecular models for intrastrand DNA G-quadruplexes

Background  

Independent surveys of human gene promoter regions have demonstrated an overrepresentation of G₃X _n1G3X _n2G₃X _n3G₃ motifs which are known to be capable of forming intrastrand quadruple helix structures. In spite of the widely recognized importance of G-quadruplex structures in gene regulation and growing interest around this unusual DNA structure, there are at present only few such structures available in the Nucleic Acid Database. In the present work we generate by molecular modeling feasible G-quadruplex structures which may be useful for interpretation of experimental data.     

Endogenous and exogenous NO attenuates conduction of vasoconstrictions along arterioles in the microcirculation

Vascular coordination in the microcirculation depends on gap junctional intercellular communication (GJIC), which is reflected by the conduction of locally initiated vasomotor responses. However, little is known about the regulation of GJIC in vivo. We hypothesized that endothelial NO regulates GJIC and therefore studied whether conduction of constrictions and dilations along the vessel wall is modulated by modifying the level of microcirculatory NO. Arterioles were focally stimulated using high K(+) or acetylcholine in the cremaster muscle in situ, and diameter changes were assessed at the local and remote upstream sites by intravital microscopy. Local stimulation with K(+) initiated a constriction that conducted along the arteriole with diminishing amplitude (length constant lambda: 371 +/- 42 mum). After N(omega)-nitro-l-arginine (l-NNA), lambda increased to 507 +/- 30 mum, indicating that GJIC is attenuated by endogenous NO. Exogenous NO, but not adenosine, reduced lambda after l-NNA in a reversible, concentration-dependent, and mainly cGMP-dependent manner as assessed by inhibition of soluble guanylate cyclase. In endothelial NO synthase-deficient mice, lambda was 530 +/- 80 mum and thus similar to that in wild-type mice after l-NNA. Exogenous NO likewise reduced lambda in these mice. The effects of NO were comparable to those of wild-type animals in Cx40-deficient mice, which excludes Cx40 as a specific target of NO. In contrast to constrictions, the amplitude of conducted dilations on acetylcholine did not diminish up to 1,300 mum and were not altered by l-NNA or exogenous NO. We conclude that endogenously released NO attenuates the conduction of vasoconstrictions most likely due to a modulation of gap junctional conductivity. We suggest that this effect is specific for smooth muscle cells, which probably transmit constricting signals, and involves connexins other than Cx40. This mechanism may support the dilatory potency of NO by preventing the conduction of remote vasoconstrictions into areas with basal or activated NO release.     

Analysis of in vivo kinetics of glycolysis in aerobic Saccharomyces cerevisiae by application of glucose and ethanol pulses

This article presents the dynamic responses of several intra- and extracellular components of an aerobic, glucose-limited chemostat culture of Saccharomyces cerevisiae to glucose and ethanol pulses within a time window of 75 sec. Even though the ethanol pulse cannot perturb the glycolytic pathway directly, a distinct response of the metabolites at the lower part of glycolysis was found. We suggest that this response is an indirect effect, caused by perturbation of the NAD/NADH ratio, which is a direct consequence of the conversion of ethanol into acetaldehyde. This effect of the NAD/NADH ratio on glycolysis might serve as an additional explanation for the observed decrease of 3PG, 2PG, and PEP during a glucose pulse. The responses measured during the ethanol pulse were used to evaluate the allosteric regulation of glycolysis. Our results confirm that FBP stimulates pyruvate kinase and suggest that this effect is pronounced. Furthermore, it appears that PEP does not play an important role in the allosteric regulation of phosphofructo kinase.     

Beta-lactams resistance and presence of class 1 integron in Pseudomonas spp. isolated from untreated hospital effluents in Brazil

The aim of the present study was to investigate the resistance profile, to detect the presence of beta-lactam resistance genes, phenotypic expression of efflux pump systems and class 1 integrons in Pseudomonas spp. strains obtained from untreated hospital effluents. Effluent samples were collected from four hospitals in Porto Alegre, RS, Brazil. Pseudomonas were isolated on MacConkey agar plates and the identification was confirmed by 16S rRNA PCR and biochemical tests. Susceptibility testing was determined by disk-diffusion method using 11 different beta-lactams and MIC assays were performed on isolates resistant to imipenem and ceftazidime. The beta-lactamase genes bla _IMP, bla _VIM, bla _SPM-1, bla _OXA-23-like, bla _OXA-24-like, bla _OXA-51-like and the intl1 gene from class 1 integron were analysed by PCR. One hundred and twenty-four isolates were recovered and the most common species was Pseudomonas pseudoalcaligenes. The resistance found among the isolates was considered high, 62 (50%) isolates were multiresistant. No isolate carrying the beta-lactamase genes tested was found among the strains. Seven isolates showed reduction of MIC for imipenem and ceftazidime in the presence of cyanide m-chlorophenylhydrazone, indicating the hyper expression of efflux pumps. From the 124 isolates, 52 (41.9%) were identified as carrying the class 1 integron gene, intI1. Untreated hospital effluents could be a source of environmental contamination due to discharge of antimicrobial resistant bacteria which can carry integron class 1 and act as a reservoir of resistance genes and have efflux pump systems.     

Evaluation of bioimpedance spectroscopy for measurements of body water distribution in healthy women before, during, and after pregnancy.

Bioimpedance spectroscopy (BIS) is a technique of interest in the study of human pregnancy because it can assess extracellular (ECW), intracellular (ICW), and total body water (TBW) as ECW plus ICW. The technique requires appropriate resistivity coefficients and has not been sufficiently evaluated during the reproductive cycle. Therefore, in a methodological study, we estimated ECW, ICW, and TBW, by means of BIS, and compared the results with the corresponding estimates obtained by using reference methods. Furthermore, results obtained by means of population-specific resistivity coefficients were compared with results obtained by means of general resistivity coefficients. These comparisons were made before pregnancy, in gestational weeks 14 and 32, as well as 2 wk postpartum in 21 healthy women. The reference methods were isotope and bromide dilution. Average ICW, ECW, and TBW, estimated by means of BIS, were in agreement with reference data before pregnancy, in gestational week 14, and postpartum. The corresponding comparison in gestational week 32 showed good agreement for ICW, whereas estimates by means of BIS were significantly (P < 0.001) lower than the corresponding reference values for ECW and TBW. Thus the BIS technique, which was based on a model developed for the nonpregnant body, estimated increases in ICW accurately, whereas increases in ECW and TBW tended to be underestimated. Estimates obtained by using population-specific and general resistivity coefficients were very similar. In conclusion, the results indicated that BIS is potentially useful for studies during pregnancy but that further work is needed before it can be generally applied in such studies.     

Satellite tracking of two Montagu’s Harriers (Circus pygargus): dual pathways during autumn migration

Autumn migration routes of two Dutch female Montagu’s Harriers (Circus pygargus) were documented for the first time using satellite telemetry. Both migrated to their African wintering area—one via the Straits of Gibraltar through the Mediterranean and the other via Italy/Tunisia. The rate of travel was comparable to values reported for larger raptor species.