Subgenomic promoter recognition by the norovirus RNA-dependent RNA polymerases

The replication enzyme of RNA viruses must preferentially recognize their RNAs in an environment that contains an abundance of cellular RNAs. The factors responsible for specific RNA recognition are not well understood, in part because viral RNA synthesis takes place within enzyme complexes associated with modified cellular membrane compartments. Recombinant RNA-dependent RNA polymerases (RdRps) from the human norovirus and the murine norovirus (MNV) were found to preferentially recognize RNA segments that contain the promoter and a short template sequence for subgenomic RNA synthesis. Both the promoter and template sequence contribute to stable RdRp binding, accurate initiation of the subgenomic RNAs and efficient RNA synthesis. Using a method that combines RNA crosslinking and mass spectrometry, residues near the template channel of the MNV RdRp were found to contact the hairpin RNA motif. Mutations in the hairpin contact site in the MNV RdRp reduced MNV replication and virus production in cells. This work demonstrates that the specific recognition of the norovirus subgenomic promoter is through binding by the viral RdRp.     

Nucleocytoplasmic shuttling of bovine herpesvirus 1 UL47 protein in infected cells

Previous studies with transfected cells have shown that the herpes simplex virus type 1 (HSV-1) and bovine herpesvirus 1 (BHV-1) UL47 proteins shuttle between the nucleus and the cytoplasm. HSV-1 UL47 has also been shown to bind RNA. Here we examine the BHV-1 UL47 protein in infected cells using a green fluorescent protein-UL47-expressing virus. We show that UL47 is detected in the nucleus early in infection. We use fluorescence loss in photobleaching to show that nuclear UL47 undergoes rapid nucleocytoplasmic shuttling. Furthermore, we demonstrate that actinomycin D inhibits the reaccumulation of UL47 in the nuclei of infected cells. These results suggest that UL47 exhibits behavior similar to that of previously characterized RNA-transporting proteins.     

A model for integrative study of human gastric acid secretion.

We have developed a unique virtual human model of gastric acid secretion and its regulation in which food provides a driving force. Food stimulus triggers neural activity in central and enteric nervous systems and G cells to release gastrin, a critical stimulatory hormone. Gastrin stimulates enterochromaffin-like cells to release histamine, which, together with acetylcholine, stimulates acid secretion from parietal cells. Secretion of somatostatin from antral and corpus D cells comprises a negative-feedback loop. We demonstrate that although acid levels are most sensitive to food and nervous system inputs, somatostatin-associated interactions are also important in governing acidity. The importance of gastrin in acid secretion is greatest at the level of transport between the antral and corpus regions. Our model can be applied to study conditions that are not yet experimentally reproducible. For example, we are able to preferentially deplete antral or corpus somatostatin. Depletion of antral somatostatin exhibits a more significant elevation of acid release than depletion of corpus somatostatin. This increase in acid release is likely due to elevated gastrin levels. Prolonged hypergastrinemia has significant effects in the long term (5 days) by promoting enterochromaffin-like cell overgrowth. Our results may be useful in the design of therapeutic strategies for acid secretory dysfunctions such as hyper- and hypochlorhydria.     

Fine structure and metabolism of multiply innervated fast muscle fibres in teleost fish

Summary Both the fast and slow muscle fibres of advanced teleost fish are multiply innervated. The fraction of slow-fibre volume occupied by mitochondria is 31.3%, 25.5% and 24.6%, respectively, for the myotomal muscles of brook trout (Salvelinus fontinalis), crucian carp (Carassius carassius), and plaice (Pleuronectes platessa), respectively. The corresponding figures for the fast muscles of these species are 9.3%, 4.6% and 2.0%, respectively. Cytochrome-oxidase and citrate-synthetase activities in the fast muscles of 9 species of teleost range from 0.20–0.93 moles substrate utilised, g wet weight muscle^-1 min^-1 (at 15° C) or around 4–17% of that of the corresponding slow fibres. Ultrastructural analyses reveal a marked heterogeneity within the fast-fibre population. For example, the fraction of fibres with <1% or >10% mitochondria is 0,4,42% and 36, 12 and 0%, respectively, for trout, carp and plaice. In general, small fibres (<500 m²) have the highest and large fibres (>1,500 m²) the lowest mitochondrial densities. The complexity of mitochondrial cristae is reduced in fast compared to slow fibres.Hexokinase activities range from 0.4–2.5 in slow and from 0.08–0.7 moles, g wet weight^-1 min^-1 in fast muscles, indicating a wide variation in their capacity for aerobic glucose utilisation. Phosphofructokinase activities are 1.2 to 3.6 times higher in fast than slow muscles indicating a greater glycolytic potential. Lactate dehydrogenase activities are not correlated with either the predicted anaerobic scopes for activity or the anoxic tolerances of the species studied. The results indicate a considerable variation in the aerobic capacities and principal fuels supporting activity among the fast muscles of different species. Brook trout and crucian carp are known to recruit fast fibres at low swimming speeds. For these species the aerobic potential of the fast muscle is probably sufficient to meet the energy requirements of slow swimming.     

Reduced Glomerular Filtration Rate and Its Association with Clinical Outcome in Older Patients at Risk of Vascular Events: Secondary Analysis

Background

Reduced glomerular filtration rate (GFR) is associated with increased cardiovascular risk in young and middle aged individuals. Associations with cardiovascular disease and mortality in older people are less clearly established. We aimed to determine the predictive value of the GFR for mortality and morbidity using data from the 5,804 participants randomized in the Prospective Study of Pravastatin in the Elderly at Risk (PROSPER).

Methods and Findings

Glomerular filtration rate was estimated (eGFR) using the Modification of Diet in Renal Disease equation and was categorized in the ranges ([20–40], [40–50], [50–60]) ≥ 60 ml/min/1.73 m². Baseline risk factors were analysed by category of eGFR, with and without adjustment for other risk factors. The associations between baseline eGFR and morbidity and mortality outcomes, accrued after an average of 3.2 y, were investigated using Cox proportional hazard models adjusting for traditional risk factors. We tested for evidence of an interaction between the benefit of statin treatment and baseline eGFR status. Age, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol, C-reactive protein (CRP), body mass index, fasting glucose, female sex, histories of hypertension and vascular disease were associated with eGFR (p = 0.001 or less) after adjustment for other risk factors. Low eGFR was independently associated with risk of all cause mortality, vascular mortality, and other noncancer mortality and with fatal and nonfatal coronary and heart failure events (hazard ratios adjusted for CRP and other risk factors (95% confidence intervals [CIs]) for eGFR < 40 ml/min/1.73m² relative to eGFR ≥ 60 ml/min/1.73m² respectively 2.04 (1.48–2.80), 2.37 (1.53–3.67), 3.52 (1.78–6.96), 1.64 (1.18–2.27), 3.31 (2.03–5.41). There were no nominally statistically significant interactions (p < 0.05) between randomized treatment allocation and eGFR for clinical outcomes, with the exception of the outcome of coronary heart disease death or nonfatal myocardial infarction (p = 0.021), with the interaction suggesting increased benefit of statin treatment in subjects with impaired GFRs.

Conclusions

We have established that, in an elderly population over the age of 70 y, impaired GFR is associated with female sex, with presence of vascular disease, and with levels of other risk factors that would be associated with increased risk of vascular disease. Further, impaired GFR is independently associated with significant levels of increased risk of all cause mortality and fatal vascular events and with composite fatal and nonfatal coronary and heart failure outcomes. Our analyses of the benefits of statin treatment in relation to baseline GFR suggest that there is no reason to exclude elderly patients with impaired renal function from treatment with a statin.     

Diel variation in chironomid (Diptera: Insecta) exuviae abundance and taxonomic richness in near-pristine upland streams of the Greater Blue Mountains World Heritage Area,South-Eastern Australia

We assessed diurnal variation of Chironomidae exuviae in two small upland streams in temperate Australia, during summer. Understanding the diel periodicity of exuviae can be an important consideration for biomonitoring purposes or to investigate adult emergence patterns. We collected 1,813 floating exuviae, comprising 54 taxa from four subfamilies, from flowing water using a drift net. Unlike many northern hemisphere temperate studies, we observed that peak exuviae abundance (7.3 exuviae per m3) and taxon richness (1.7 taxa per m3) occurred in the dusk and night hours, with the lowest numbers (0.9 exuviae per m3) and taxon richness (0.6 taxa per m3) recorded in the late morning to early afternoon. We suggest that this could be an adaptation to avoid stressful weather during the heat of summer days, or it could be to avoid visual predators. The numerically dominant taxa exhibited peak abundance in the dusk/night samples which indicates predominant crepuscular/nocturnal patterns of adult emergence. This pattern was consistent across both streams surveyed. Our taxon inventory rose steeply during the first 24-h occasion, then at a reduced rate during the second and third days of sampling. For flowing water collections of exuviae that utilise drift-netting, we suggest that sampling at all sites includes at least three 24-h cycles and avoids periods of heavy rainfall and increased stream flow.     

Gating and inward rectifying properties of the MthK K+ channel with and without the gating ring

In MthK, a Ca2+-gated K+ channel from Methanobacterium thermoautotrophicum, eight cytoplasmic RCK domains form an octameric gating ring that controls the intracellular gate of the ion conduction pore. The binding of Ca2+ ions to the RCK domains alters the conformation of the gating ring, thereby opening the gate. In the present study, we examined the Ca2+- and pH-regulated gating and the rectifying conduction properties of MthK at the single-channel level. The open probability (Po) of MthK exhibits a sigmoidal relationship with intracellular [Ca2+], and a Hill coefficient >1 is required to describe the dependence of Po on [Ca2+], suggesting cooperative Ca2+ activation of the channel. Additionally, intracellular Ca2+ also blocks the MthK pore in a voltage-dependent manner, rendering an apparently inwardly rectifying I-V relation. Intracellular pH has a dual effect on MthK gating. Below pH 7.5, the channel becomes insensitive to Ca2+. This occurs because the gating ring is structurally unstable at this pH and tends to disassemble (Ye, S., Y. Li, L. Chen, and Y. Jiang. 2006. Cell. 126:1161-1173). In contrast, above pH 7.5, a further increase in pH shifts the Po-[Ca2+] relation towards a lower Ca2+ concentration, augments Po at saturating [Ca2+], and activates the channel even in the absence of Ca2+. Channel activity is marked by bursts of rapid openings and closings separated by relatively longer interburst closings. The duration of interburst closing and the burst length are highly Ca2+ and pH dependent, whereas the kinetics of intraburst events is Ca2+ and pH independent. The rapid intraburst openings and closings are also observed with the isolated MthK pore lacking the attached intracellular gating ring. The fast kinetic events, independent of both Ca2+ and pH, therefore appear to be determined by processes occurring within the ion conduction pore, whereas the slow events reflect the gating process controlled by Ca2+ and pH through the gating ring.     

Extrahepatic cells contribute to the progenitor/stem cell response following reduced-size liver transplantation in mice

The extent to which extrahepatic cells participate in liver regeneration following transplantation is not known. Either full-size or reduced-size livers from wild-type mice were implanted into green fluorescent protein-positive (GFP(+)) transgenic recipient mice to determine whether regenerated liver contained host-derived GFP(+) hepatic cells. After reduced-size liver transplantation, GFP(+) cells were localized to the portal zone of the liver lobule. Interestingly, GFP(+) cells stained for CD117, a marker for progenitor cells, beginning 2 days after transplantation. A significant number of GFP(+) CD117(+) cells were identified in donor livers after 28 days. GFP(+) cells comprised nearly 9% of the donor liver 28 days after reduced-size liver transplant. Moreover, GFP(+) cells also expressed the hepatic progenitor cell marker A6 and novel marker hepatic-specific antigen (HSA), as well as stem cell antigen-1 (Sca-1). Interestingly, some GFP(-) cells also were stained for CD117 and A6, suggesting that both extrahepatic and intrahepatic stem cells were present and may have contributed to the regenerative response under these conditions. Reduced-size liver transplantation using GFP(+) transgenic mice supports the hypothesis that recipient-derived progenitor cells are present and may contribute to liver regeneration following transplantation.