Functional activity of endotoxin binding factors in chronic viral hepatitis B and C

The functional state of cell-mediated and humoral antiendotoxin factors in patients with chronic viral hepatitides B and C has been studied. A decreased content of antibodies to glycolipid of chemotype Re and to Escherichia coli O14 with common enterobacterial antigen was shown to occur in these diseases. In addition, a decreased number of neutrophils, eosinophils and thrombocytes has been noted. The conclusion has been made that patients with chronic hepatitides are not protected from the pathogenic action of endotoxin penetrating from the intestines into the systemic blood stream.     

The location of a temperature-sensitive trans-dominant mutation and its effect on restriction and modification in Escherichia coli K12

An Escherichia coli K12 chromosomal EcoRI-BamHI fragment containing a mutant hsdS locus was cloned into plasmid pBR322. The mcrB gene, closely linked to hsdS, was used for selection of clones with the inserted fragment using T4 alpha gt57 beta gt14 and lambda vir. PvuII phages; the phage DNAs contain methylated cytosines and hence can be used to demonstrate McrB restriction. For the efficient expression of the hsdS gene, a BglII fragment of phage lambda carrying the pR promoter was inserted into the BamHI site of the hybrid plasmid. Under these conditions a trans-dominant effect of the hsdXts+d mutation on restriction and modification was detected. Inactivation of the hsdS gene by the insertion of the lambda phage BglII fragment into the BglII site within this gene resulted in the disappearance of the trans-dominant effect. When the cloned BamHI-EcoRI fragment was shortened by HpaI and EcoRI restriction enzymes, the trans-dominant effect was fully expressed. The results indicate that the Xts+d mutation is located in the hsdS gene. The effect of gene dosage of the HsdS subunit on the expression of Xts+d mutation was studied. The results of complementation experiments, using F'-merodiploids or plasmid pBR322 with an inserted Xts+d mutation, support the idea that the HsdSts+d product competes with the wild-type HsdS product, and has a quantitatively different effect on restriction and modification.     

Regulation of tetanus toxin-inhibited transmitter secretion in the neuromuscular junction in a calcium-free medium

The effect of Ca2+ removal from the external medium on regulation of the release of the synaptic transmitter in the tetanus toxin (TT)-inhibited neuromuscular junctions was studied on a rat phrenicodiaphragmal preparation with the aid of the conventional microelectrode technique of recording synaptic activity. As the external concentration of calcium was decreased from 2 to 0 mM, the frequency of miniature end plate potentials remained unchanged in the preparations isolated 3 to 3.5 h after intramuscular injection of TT (10(5) MLD for mouse). TT considerably reduced activation of the transmitter release, caused in intact synapses by ouabain (0.1 mM) and repetitive stimulation of the diaphragmatic nerve (50 imp/s). The data obtained indicate that in the TT-inhibited motor nerve terminals, the level of the transmitter release does not depend on the external concentration of calcium and that TT damages some of the intracellular sources of calcium.     

The relation of the X-ray B-factor to protein dynamics: insights from recent dynamic solid-state NMR data

In addressing the potential use of B-factors derived from X-ray scattering data of proteins for the understanding the (functional) dynamics of proteins, we present a comparison of B-factors of five different proteins (SH3 domain, Crh, GB1, ubiquitin and thioredoxin) with data from recent solid-state nuclear magnetic resonance experiments reflecting true (rotational) dynamics on well-defined timescales. Apart from trivial correlations involving mobile loop regions and chain termini, we find no significant correlation of B-factors with the dynamic data on any of the investigated timescales, concluding that there is no unique and general correlation of B-factors with the internal reorientational dynamics of proteins.     

Characterisation of conditioning layers formed by exopolymeric substances of Pseudomonas NCIMB 2021 on surfaces of AISI 316 stainless steel

This investigation aimed to characterise conditioning layers formed on AISI 316 stainless steel by different types of extracellular polymeric substances (EPS), i.e. biofilm, planktonic and capsular exopolymers, isolated from continuous cultures of marine Pseudomonas received from the National Collection of Industrial and Marine Bacteria (strain NCIMB 2021). Colorimetric assays and gas chromatography‐mass spectrometry analysis confirmed previously obtained results based on a FTIR and SDS‐PAGE study of Pseudomonas NCIMB 2021 EPS demonstrating the presence of protein, neutral and amino sugars and uronic acids. The content and the ratio of these macromolecules differed depending on the type of EPS. X‐ray photoelectron spectroscopy revealed that conditioning layers formed upon exposure of steel to EPS solutions were chemically dissimilar. It is proposed that the observed difference in the chemistry of conditioning layers is the likely reason for reported differences in attachment of Pseudomonas cells to EPS‐conditioned steel surfaces.     

Bottlenecks drive temporal and spatial genetic changes in alpine caddisfly metapopulations

Background

Extinction and re-colonisation of local populations is common in ephemeral habitats such as temporary streams. In most cases, such population turnover leads to reduced genetic diversity within populations and increased genetic differentiation among populations due to stochastic founder events, genetic drift, and bottlenecks associated with re-colonisation. Here, we examined the spatio-temporal genetic structure of 8 alpine caddisfly populations inhabiting permanent and temporary streams from four valleys in two regions of the Swiss Alps in years before and after a major stream drying event, the European heat wave in summer 2003.

Results

We found that population turnover after 2003 led to a loss of allelic richness and gene diversity but not to significant changes in observed heterozygosity. Within all valleys, permanent and temporary streams in any given year were not differentiated, suggesting considerable gene flow and admixture between streams with differing hydroperiods. Large changes in allele frequencies after 2003 resulted in a substantial increase in genetic differentiation among valleys within one to two years (1-2 generations) driven primarily by drift and immigration. Signatures of genetic bottlenecks were detected in all 8 populations after 2003 using the M-ratio method, but in no populations when using a heterozygosity excess method, indicating differential sensitivity of bottleneck detection methods.

Conclusions

We conclude that genetic differentiation among A. uncatus populations changed markedly both temporally and spatially in response to the extreme climate event in 2003. Our results highlight the magnitude of temporal population genetic changes in response to extreme events. More specifically, our results show that extreme events can cause rapid genetic divergence in metapopulations. Further studies are needed to determine if recovery from this perturbation through gradual mixing of diverged populations by migration and gene flow leads to the pre-climate event state, or whether the observed changes represent a new genetic equilibrium.     

Activation of endothelial TRPV4 channels mediates flow-induced dilation in human coronary arterioles: role of Ca2+ entry and mitochondrial ROS signaling

In human coronary arterioles (HCAs) from patients with coronary artery disease, flow-induced dilation is mediated by a unique mechanism involving the release of H(2)O(2) from the mitochondria of endothelial cells (ECs). How flow activates ECs to elicit the mitochondrial release of H(2)O(2) remains unclear. Here, we examined the role of the transient receptor potential vanilloid type 4 (TRPV4) channel, a mechanosensitive Ca(2+)-permeable cation channel, in mediating ROS formation and flow-induced dilation in HCAs. Using RT-PCR, Western blot analysis, and immunohistochemical analysis, we detected the mRNA and protein expression of TRPV4 channels in ECs of HCAs and cultured human coronary artery ECs (HCAECs). In HCAECs, 4α-phorbol-12,13-didecanoate (4α-PDD), a selective TRPV4 agonist, markedly increased (via Ca(2+) influx) intracellular Ca(2+) concentration. In isolated HCAs, activation of TRPV4 channels by 4α-PDD resulted in a potent concentration-dependent dilation, and the dilation was inhibited by removal of the endothelium and by catalase, a H(2)O(2)-metabolizing enzyme. Fluorescence ROS assays showed that 4α-PDD increased the production of mitochondrial superoxide in HCAECs. 4α-PDD also enhanced the production of H(2)O(2) and superoxide in HCAs. Finally, we found that flow-induced dilation of HCAs was markedly inhibited by different TRPV4 antagonists and TRPV4-specific small interfering RNA. In conclusion, the endothelial TRPV4 channel is critically involved in flow-mediated dilation of HCAs. TRPV4-mediated Ca(2+) entry may be an important signaling event leading to the flow-induced release of mitochondrial ROS in HCAs. Elucidation of this novel TRPV4-ROS pathway may improve our understanding of the pathogenesis of coronary artery disease and/or other cardiovascular disorders.