Effects on protease inhibition by modifying of helicase residues in hepatitis C virus nonstructural protein 3

This study of the full-length bifunctional nonstructural protein 3 from hepatitis C virus (HCV) has revealed that residues in the helicase domain affect the inhibition of the protease. Two residues (Q526 and H528), apparently located in the interface between the S2 and S4 binding pockets of the substrate binding site of the protease, were selected for modification, and three enzyme variants (Q526A, H528A and H528S) were expressed, purified and characterized. The substitutions resulted in indistinguishable K(m) values and slightly lower k(cat) values compared to the wild-type. The K(i) values for a series of structurally diverse protease inhibitors were affected by the substitutions, with increases or decreases up to 10-fold. The inhibition profiles for H528A and H528S were different, confirming that not only did the removal of the imidazole side chain have an effect, but also that minor differences in the nature of the introduced side chain influenced the characteristics of the enzyme. These results indicate that residues in the helicase domain of nonstructural protein 3 can influence the protease, supporting our hypothesis that full-length hepatitis C virus nonstructural protein 3 should be used for protease inhibitor optimization and characterization. Furthermore, the data suggest that inhibitors can be designed to interact with residues in the helicase domain, potentially leading to more potent and selective compounds.     

Exercise affects the gene expression profiles of human white blood cells.

White blood cells (WBCs) express tens of thousands of genes, whose expression levels are modified by genetic and external factors. The purpose of the present study was to investigate the effects of acute exercise on gene expression profiles (GEPs) of WBCs and to identify suitable genes that may serve as surrogate markers for monitoring exercise and training load. Five male participants performed an exhaustive treadmill test (ET) at 80% of their maximal O(2) uptake (Vo(2 max)) and a moderate treadmill test (MT) at 60% Vo(2 max) for exactly the same time approximately 2 wk later. WBCs were isolated by the erythrocyte lysis method. GEPs were measured using the Affymetrix GeneChip technology. After scaling, normalization, and filtering, groupwise comparisons of gene expression intensities were performed, and several measurements were validated by real-time PCR. We found 450 genes upregulated and 150 downregulated (>1.5-fold change; ANOVA with Benjamini-Hochberg correction, P < 0.05) after ET that were closely associated with the gene ontology lists "response to stress" and "inflammatory response". Analysis of mean expression levels after MT showed that the extent of up- and downregulation was workload dependent. The genes for the stress (heat shock) proteins HSPA1A and HSPH1 and for the matrix metalloproteinase MMP-9 showed the most prominent increases, whereas the YES1 oncogene (YES1) and CD160 (BY55) were most strongly reduced. Despite different methodological approaches used, the consistency of our results with the expression data of another study (Connolly PH, Caiozzo VJ, Zaldivar F, Nemet D, Larson J, Hung SP, Heck JD, Hatfield GW, Cooper DM. J Appl Physiol 97: 1461-1469, 2004) suggests that expression fingerprints are useful tools for monitoring exercise and training loads and thereby help to avoid training-associated health risks.     

Autonomic cardiovascular and respiratory control during prolonged spaceflights aboard the International Space Station.

Impaired autonomic control represents a cardiovascular risk factor during long-term spaceflight. Little has been reported on blood pressure (BP), heart rate (HR), and heart rate variability (HRV) during and after prolonged spaceflight. We tested the hypothesis that cardiovascular control remains stable during prolonged spaceflight. Electrocardiography, photoplethysmography, and respiratory frequency (RF) were assessed in eight male cosmonauts (age 41-50 yr, body-mass index of 22-28 kg/m2) during long-term missions (flight lengths of 162-196 days). Recordings were made 60 and 30 days before the flight, every 4 wk during flight, and on days 3 and 6 postflight during spontaneous and controlled respiration. Orthostatic testing was performed pre- and postflight. RF and BP decreased during spaceflight (P < 0.05). Mean HR and HRV in the low- and high-frequency bands did not change during spaceflight. However, the individual responses were different and correlated with preflight values. Pulse-wave transit time decreased during spaceflight (P < 0.05). HRV reached during controlled respiration (6 breaths/min) decreased in six and increased in one cosmonaut during flight. The most pronounced changes in HR, BP, and HRV occurred after landing. The decreases in BP and RF combined with stable HR and HRV during flight suggest functional adaptation rather than pathological changes. Pulse-wave transit time shortening in our study is surprising and may reflect cardiac output redistribution in space. The decrease in HRV during controlled respiration (6 breaths/min) indicates reduced parasympathetic reserve, which may contribute to postflight disturbances.     

A critical review on natural compounds interacting with the plant plasma membrane H+-ATPase and their potential as biologicals in agriculture

The plant plasma membrane (PM) H⁺-ATPase is an essential enzyme controlling plant growth and development. It is an important factor in response to abiotic and biotic stresses and is subject to tight regulation. We are in demand for new sustainable natural growth regulators and as a key enzyme for regulation of transport into the plant cell the PM H⁺-ATPase is a potential target for these. In this review, we have evaluated the known non-protein natural compounds with regulatory effects on the PM H⁺-ATPase, focusing on their mechanism of action and their potential as biologicals/growth regulators in plant production of future sustainable agriculture.     

School performance in Danish children exposed to maternal type 1 diabetes in utero: A nationwide retrospective cohort study

BackgroundConflicting results have been reported concerning possible adverse effects on the cognitive function of offspring of mothers with type 1 diabetes (O-mT1D). Previous studies have included offspring of parents from the background population (O-BP), but not offspring of fathers with type 1 diabetes (O-fT1D) as the unexposed reference group.Methods and findingsThis is a population-based retrospective cohort study from 2010 to 2016. Nationally standardized school test scores (range, 1 to 100) were obtained for public school grades 2, 3, 4, 6, and 8 in O-mT1D and compared with those in O-fT1D and O-BP. Of the 622,073 included children, 2,144 were O-mT1D, and 3,474 were O-fT1D. Multiple linear regression models were used to compare outcomes, including the covariates offspring with type 1 diabetes, parity, number of siblings, offspring sex, smoking during pregnancy, parental age, and socioeconomic factors. Mean test scores were 54.2 (standard deviation, SD 24.8) in O-mT1D, 54.4 (SD 24.8) in O-fT1D, and 56.4 (SD 24.7) in O-BP. In adjusted analyses, the mean differences in test scores were −1.59 (95% CI −2.48 to −0.71, p < 0.001) between O-mT1D and O-BP and −0.78 (95% CI −1.48 to −0.08, p = 0.03) between O-fT1D and O-BP. No significant difference in the adjusted mean test scores was found between O-mT1D and O-fT1D (p = 0.16). The study’s limitation was no access to measures of glycemic control during pregnancy.ConclusionsO-mT1D achieved lower test scores than O-BP but similar test scores compared with O-fT1D. Glycemic control during pregnancy is essential to prevent various adverse pregnancy outcomes in women with type 1 diabetes. However, the present study reduces previous concerns regarding adverse effects of in utero hyperglycemia on offspring cognitive function.

Anne Lærke Spangmose and colleagues examine the association between school performance and exposure to maternal or paternal type 1 diabetes in utero in Denmark.     

Biogeography of Aegagropila linnaei (Cladophorophyceae,Chlorophyta): a widespread freshwater alga with low effective dispersal potential shows a glacial imprint in its distribution

Aim Aegagropila linnaei is a freshwater macroalga that is generally regarded as a rare species. It is apparently absent from large but seemingly suitable areas of the Northern Hemisphere, implying a limited dispersal potential and an imprint of Pleistocene glaciations in its biogeography. However, despite the popularity of its enigmatic lake ball‐form, detailed biogeographical studies of A. linnaei have never been conducted. The main means of reproduction of A. linnaei is fragmentation and akinetes are not formed, supporting the assumption of limited dispersal capacity. The aim of this study was to reconstruct the biogeography of A. linnaei, and to identify possible refugia during glaciations, as well as to evaluate dispersal potential by quantitative desiccation experiments. Location Palaearctic. Methods The current distribution of A. linnaei was inferred from herbarium specimens, literature data and recent field observations. All herbarium specimens were morphologically re‐examined. Desiccation experiments were performed with vegetative filaments of three isolates of A. linnaei, as no specialized resistant stages are known. For comparison, the widespread freshwater algae Cladophora glomerata and Rhizoclonium sp. were included. Internal transcribed spacer (ITS) ribosomal DNA sequences were generated and a ribotype network was constructed. Results Aegagropila linnaei was recorded from 283 locations in freshwater and brackish environments. The majority of locations were in central and northern Europe in previously glaciated areas. Desiccation experiments showed that A. linnaei is very susceptible to desiccation. Based on ITS sequences of 34 samples, five different ribotypes were identified. Four of these ribotypes had a restricted distribution. Aegagropila linnaei represents a single species with little genetic variation (0.1–0.5%). Main conclusions This is the most comprehensive study of this species so far, reporting many new locations and tackling several taxonomic problems. Few additional finds were made from North America, and the origin of A. linnaei is inferred to be in Asia. The highest density of its present‐day locations is in previously glaciated areas in Europe, where glacial ice‐dammed lakes might have functioned as refugia. Low effective long‐distance dispersal capacity is inferred, based on high susceptibility to desiccation and its modes of dispersal.     

Analyzing protein-protein interactions in cell membranes

Interactions among membrane proteins regulate numerous cellular processes, including cell growth, cell differentiation and apoptosis. We need to understand which proteins interact, where they interact and to which extent they interact. This article describes a set of novel approaches to measure, on the surface of living cells, the number of clusters of proteins, the number of proteins per cluster, the number of clusters or membrane domains that contain pairs of interacting proteins and the fraction of one protein species that interacts with another protein within these domains. These data can then be interpreted in terms of the function of the protein-protein interactions.     

Definition of a short region of XPG necessary for TFIIH interaction and stable recruitment to sites of UV damage

XPG is the human endonuclease that cuts 3' to DNA lesions during nucleotide excision repair. Missense mutations in XPG can lead to xeroderma pigmentosum (XP), whereas truncated or unstable XPG proteins cause Cockayne syndrome (CS), normally yielding life spans of <7 years. One XP-G individual who had advanced XP/CS symptoms at 28 years has been identified. The genetic, biochemical, and cellular defects in this remarkable case provide insight into the onset of XP and CS, and they reveal a previously unrecognized property of XPG. Both of this individual's XPG alleles produce a severely truncated protein, but an infrequent alternative splice generates an XPG protein lacking seven internal amino acids, which can account for his very slight cellular UV resistance. Deletion of XPG amino acids 225 to 231 does not abolish structure-specific endonuclease activity. Instead, this region is essential for interaction with TFIIH and for the stable recruitment of XPG to sites of local UV damage after the prior recruitment of TFIIH. These results define a new functional domain of XPG, and they demonstrate that recruitment of DNA repair proteins to sites of damage does not necessarily lead to productive repair reactions. This observation has potential implications that extend beyond nucleotide excision repair.     

Rap2B-dependent stimulation of phospholipase C-epsilon by epidermal growth factor receptor mediated by c-Src phosphorylation of RasGRP3

Receptor tyrosine kinase regulation of phospholipase C-epsilon (PLC-epsilon), which is under the control of Ras-like and Rho GTPases, was studied with HEK-293 cells endogenously expressing PLC-coupled epidermal growth factor (EGF) receptors. PLC and Ca(2+) signaling by the EGF receptor, which activated both PLC-gamma1 and PLC-epsilon, was specifically suppressed by inactivation of Ras-related GTPases with clostridial toxins and expression of dominant-negative Rap2B. EGF induced rapid and sustained GTP loading of Rap2B, binding of Rap2B to PLC-epsilon, and Rap2B-dependent translocation of PLC-epsilon to the plasma membrane. GTP loading of Rap2B by EGF was inhibited by chelation of intracellular Ca(2+) and expression of lipase-inactive PLC-gamma1 but not of PLC-epsilon. Expression of RasGRP3, a Ca(2+)/diacylglycerol-regulated guanine nucleotide exchange factor for Ras-like GTPases, but not expression of various other exchange factors enhanced GTP loading of Rap2B and PLC/Ca(2+) signaling by the EGF receptor. EGF induced tyrosine phosphorylation of RasGRP3, but not RasGRP1, apparently caused by c-Src; inhibition of c-Src interfered with EGF-induced Rap2B activation and PLC stimulation. Collectively, these data suggest that the EGF receptor triggers activation of Rap2B via PLC-gamma1 activation and tyrosine phosphorylation of RasGRP3 by c-Src, finally resulting in stimulation of PLC-epsilon.     

Sexual harassment in a live-bearing fish (<Emphasis Type="Italic">Poecilia mexicana</Emphasis>): influence of population-specific male mating behaviour

We investigated male sexual behaviour and the cost of sexual harassment, as measured by the reduction of female feeding time in the presence of a male, in a cave-dwelling population of Poecilia mexicana, in which sexual harassment does not occur naturally. We asked whether the lack of sexual harassment in this population is due to low sexual activity of the males, or low feeding motivation of the females. We experimentally increased the sexual activity of males or the females feeding motivation, or we used a combination of both treatments. Female feeding time was not lower in the presence of a male than in the presence of a female after sexual deprivation of the males or food deprivation of the females. Only in the combined experiment was female feeding time lower in the presence of a large male than in the presence of a small male, indicating a weak effect of sexual harassment by large males. Virgin females did not suffer a cost of sexual harassment, indicating that sexual experience does not cause the lack of sexual harassment in cave mollies. Males from a surface population, where sexual harassment occurs, significantly reduced the feeding time of cave-dwelling females even though these males exhibited surprisingly little sexual behaviour. The sexual activity of cave mollies did not correlate with male body size in any experiment, indicating that even after sexual deprivation, small cave molly males do not switch to the alternative mating behaviour known in surface-dwelling P. mexicana, where sexual activity is correlated negatively with male body size.