Flavivirus recruits the valosin-containing protein–NPL4 complex to induce stress granule disassembly for efficient viral genome replication

Flaviviruses are human pathogens that can cause severe diseases, such as dengue fever and Japanese encephalitis, which can lead to death. Valosin-containing protein (VCP)/p97, a cellular ATPase associated with diverse cellular activities (AAA-ATPase), is reported to have multiple roles in flavivirus replication. Nevertheless, the importance of each role still has not been addressed. In this study, the functions of 17 VCP mutants that are reportedly unable to interact with the VCP cofactors were validated using the short-interfering RNA rescue experiments. Our findings of this study suggested that VCP exerts its functions in replication of the Japanese encephalitis virus by interacting with the VCP cofactor nuclear protein localization 4 (NPL4). We show that the depletion of NPL4 impaired the early stage of viral genome replication. In addition, we demonstrate that the direct interaction between NPL4 and viral nonstructural protein (NS4B) is critical for the translocation of NS4B to the sites of viral replication. Finally, we found that Japanese encephalitis virus and dengue virus promoted stress granule formation only in VCP inhibitor-treated cells and the expression of NS4B or VCP attenuated stress granule formation mediated by protein kinase R, which is generally known to be activated by type I interferon and viral genome RNA. These results suggest that the NS4B-mediated recruitment of VCP to the virus replication site inhibits cellular stress responses and consequently facilitates viral protein synthesis in the flavivirus-infected cells.     

Associations between cardiorespiratory fitness and lifestyle-related factors with DNA methylation-based ageing clocks in older men: WASEDA'S Health Study

DNA methylation-based age estimators (DNAm ageing clocks) are currently one of the most promising biomarkers for predicting biological age. However, the relationships between cardiorespiratory fitness (CRF), measured directly by expiratory gas analysis, and DNAm ageing clocks are largely unknown. We investigated the relationships between CRF and the age-adjusted value from the residuals of the regression of DNAm ageing clock to chronological age (DNAmAgeAcceleration: DNAmAgeAccel) and attempted to determine the relative contribution of CRF to DNAmAgeAccel in the presence of other lifestyle factors. DNA samples from 144 Japanese men aged 65–72 years were used to appraise first- (i.e., DNAmHorvath and DNAmHannum) and second- (i.e., DNAmPhenoAge, DNAmGrimAge, and DNAmFitAge) generation DNAm ageing clocks. Various surveys and measurements were conducted, including physical fitness, body composition, blood biochemical parameters, nutrient intake, smoking, alcohol consumption, disease status, sleep status, and chronotype. Both oxygen uptake at ventilatory threshold (VO₂/kg at VT) and peak oxygen uptake (VO₂/kg at Peak) showed a significant negative correlation with GrimAgeAccel, even after adjustments for chronological age and smoking and drinking status. Notably, VO₂/kg at VT and VO₂/kg at Peak above the reference value were also associated with delayed GrimAgeAccel. Multiple regression analysis showed that calf circumference, serum triglyceride, carbohydrate intake, and smoking status, rather than CRF, contributed more to GrimAgeAccel and FitAgeAccel. In conclusion, although the contribution of CRF to GrimAgeAccel and FitAgeAccel is relatively low compared to lifestyle-related factors such as smoking, the results suggest that the maintenance of CRF is associated with delayed biological ageing in older men.     

The oxygen delivery response to acute hypoxia during incremental knee extension exercise differs in active and trained males

Background

It is well known that hypoxic exercise in healthy individuals increases limb blood flow, leg oxygen extraction and limb vascular conductance during knee extension exercise. However, the effect of hypoxia on cardiac output, and total vascular conductance is less clear. Furthermore, the oxygen delivery response to hypoxic exercise in well trained individuals is not well known. Therefore our aim was to determine the cardiac output (Doppler echocardiography), vascular conductance, limb blood flow (Doppler echocardiography) and muscle oxygenation response during hypoxic knee extension in normally active and endurance-trained males.

Methods

Ten normally active and nine endurance-trained males (VO_2max = 46.1 and 65.5 mL/kg/min, respectively) performed 2 leg knee extension at 25, 50, 75 and 100% of their maximum intensity in both normoxic and hypoxic conditions (FIO₂ = 15%; randomized order). Results were analyzed with a 2-way mixed model ANOVA (group × intensity).

Results

The main finding was that in normally active individuals hypoxic sub-maximal exercise (25 – 75% of maximum intensity) brought about a 3 fold increase in limb blood flow but decreased stroke volume compared to normoxia. In the trained group there were no significant changes in stroke volume, cardiac output and limb blood flow at sub-maximal intensities (compared to normoxia). During maximal intensity hypoxic exercise limb blood flow increased approximately 300 mL/min compared to maximal intensity normoxic exercise.

Conclusion

Cardiorespiratory fitness likely influences the oxygen delivery response to hypoxic exercise both at a systemic and limb level. The increase in limb blood flow during maximal exercise in hypoxia (both active and trained individuals) suggests a hypoxic stimulus that is not present in normoxic conditions.

     

Changes in [Ca2+]i induced by several glucose transport-enhancing stimuli in rat epitrochlearis muscle.

The purpose of the present investigation was to establish a method for estimating intracellular Ca(2+) concentrations ([Ca(2+)](i)) in isolated rat epitrochlearis muscles. Epitrochlearis muscles excised from 4-wk-old male Sprague-Dawley rats were loaded with a fluorescent Ca(2+) indicator, fura 2-AM, for 60-90 min at 35 degrees C in oxygenated Krebs-Henseleit buffer. After fura 2 loading and subsequent 20-min incubation, the intensities of 500-nm fluorescence, induced by 340- and 380-nm excitation lights (F(total)340 and F(total)380), were measured. The fluorescences specific to fura-2 (F(fura 2)340 and F(fura 2)380) were calculated by subtracting the non-fura 2-specific component from F(total)340 and F(total)380, respectively. The ratio of F(fura 2)340 to F(fura 2)380 was calculated as R, and the change in the ratio from the baseline value (DeltaR) was used as an index of the change in [Ca(2+)](i). In resting muscle, DeltaR was stable for 60 min. Incubation for 20 min with caffeine (3-10 mM) significantly increased DeltaR in a concentration-dependent manner. Incubation with hypoxic Krebs-Henseleit buffer for 10-60 min significantly elevated DeltaR, depending on the duration of the incubation. Incubation with 50 microM N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide for 20 min significantly elevated DeltaR (P < 0.05). No significant increases in DeltaR were observed during incubation for 20 min with 2 mM 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside or with 2 mU/ml insulin. These results demonstrated that, by using the fura 2-AM fluorescence method, the changes in [Ca(2+)](i) can be monitored in the rat epitrochlearis muscle and suggest that the method can be utilized to observe quantitative information regarding [Ca(2+)](i) that may be involved in contraction- and hypoxia-stimulated glucose transport activity in skeletal muscle.     

不同退化红砂荒漠草地的水分分配格局

研究了内蒙古阿拉善盟不同过牧退化红砂草地的土壤 植物 大气系统的水分分配格局、不同退化草地和主要植物种的水分利用效率 .2 0 0 1年降雨量 12 4 .3mm ,其中试验期 119.4mm .1m深土壤水分结果表明 ,10～ 4 0cm土层受蒸散影响最大 ;由于主要共存种红砂和无芒隐子草根系分布和蒸腾强度不同等 ,含水量在 10～ 2 0cm土层以中度退化区显著低于其它样区 (P <0 .0 5 ) ,而 2 0～ 4 0cm土层以轻度退化区较低 .样地年均蒸发量为 30 .6mm ,红砂种群的年均蒸腾量为 11.9mm .随着草地退化加剧 ,裸地的蒸发量和退化指示种匍根骆驼蓬种群的蒸腾量增加 ,而红砂种群的蒸腾量降低 .与较轻度退化区比 ,中度和重度退化区的水分利用率分别下降了 14 .6 %和 4 6 .1% ,红砂水分利用率分别下降了 37.8%和 73.8% .     

Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120.

The nucleotide sequence of the entire genome of a filamentous cyanobacterium, Anabaena sp. strain PCC 7120, was determined. The genome of Anabaena consisted of a single chromosome (6,413,771 bp) and six plasmids, designated pCC7120alpha (408,101 bp), pCC7120beta (186,614 bp), pCC7120gamma (101,965 bp), pCC7120delta (55,414 bp), pCC7120epsilon (40,340 bp), and pCC7120zeta (5,584 bp). The chromosome bears 5368 potential protein-encoding genes, four sets of rRNA genes, 48 tRNA genes representing 42 tRNA species, and 4 genes for small structural RNAs. The predicted products of 45% of the potential protein-encoding genes showed sequence similarity to known and predicted proteins of known function, and 27% to translated products of hypothetical genes. The remaining 28% lacked significant similarity to genes for known and predicted proteins in the public DNA databases. More than 60 genes involved in various processes of heterocyst formation and nitrogen fixation were assigned to the chromosome based on their similarity to the reported genes. One hundred and ninety-five genes coding for components of two-component signal transduction systems, nearly 2.5 times as many as those in Synechocystis sp. PCC 6803, were identified on the chromosome. Only 37% of the Anabaena genes showed significant sequence similarity to those of Synechocystis, indicating a high degree of divergence of the gene information between the two cyanobacterial strains.     

Generation of oxidative stress contributes to the development of pulmonary hypertension induced by hypoxia.

Chronic hypoxia causes pulmonary hypertension and right ventricular hypertrophy associated with pulmonary vascular remodeling. Because hypoxia might promote generation of oxidative stress in vivo, we hypothesized that oxidative stress may play a role in the hypoxia-induced cardiopulmonary changes and examined the effect of treatment with the antioxidant N-acetylcysteine (NAC) in rats. NAC reduced hypoxia-induced cardiopulmonary alterations at 3 wk of hypoxia. Lung phosphatidylcholine hydroperoxide (PCOOH) increased at days 1 and 7 of the hypoxic exposure, and NAC attenuated the increase in lung PCOOH. Lung xanthine oxidase (XO) activity was elevated from day 1 through day 21, especially during the initial 3 days of the hypoxic exposure. The XO inhibitor allopurinol significantly inhibited the hypoxia-induced increase in lung PCOOH and pulmonary hypertension, and allopurinol treatment only for the initial 3 days also reduced the hypoxia-induced right ventricular hypertrophy and pulmonary vascular thickening. These results suggest that oxidative stress produced by activated XO in the induction phase of hypoxic exposure contributes to the development of chronic hypoxic pulmonary hypertension.     

Biotransformation of 18 beta-glycyrrhetinic acid by cell suspension cultures of Glycyrrhiza glabra.

Two biotransformation products formed from 18 beta-glycyrrhetinic acid by cell suspension cultures of Glycyrrhiza glabra were isolated and their structures determined by chemical and spectral data as 3-O-[alhpa-L-arabinopyranosyl-(1----2)-beta-D-Glucuronopy ranosyl]-24- hydroxy-18 beta-glycyrrhetinic acid and 30-O-beta-D-glycopyrano-syl-18 beta-glycyrrhetinic acid. The formation of glycyrrhizin, the main triterpene glucuronide of the licorice root, was not detected among the biotransformation products. This is the first report of the glucuronylation of an exogenous triterpene in plant cell cultures.