Ribavirin-Resistant Mutants of Human Enterovirus 71 Express a High Replication Fidelity Phenotype during Growth in Cell Culture

It has been shown in animal models that ribavirin-resistant poliovirus with a G64S mutation in its 3D polymerase has high replication fidelity coupled with attenuated virulence. Here, we describe the effects of mutagenesis in the human enterovirus 71 (HEV71) 3D polymerase on ribavirin resistance and replication fidelity. Seven substitutions were introduced at amino acid position 3D-G64 of a HEV71 full-length infectious cDNA clone (26M). Viable clone-derived virus populations were rescued from the G64N, G64R, and G64T mutant cDNA clones. The clone-derived G64R and G64T mutant virus populations were resistant to growth inhibition in the presence of 1,600 μM ribavirin, whereas the growth of parental 26M and the G64N mutant viruses were inhibited in the presence of 800 μM ribavirin. Nucleotide sequencing of the 2C and 3D coding regions revealed that the rate of random mutagenesis after 13 passages in the presence of 400 μM ribavirin was nearly 10 times higher in the 26M genome than in the mutant G64R virus genome. Furthermore, random mutations acquired in the 2C coding regions of 26M and G64N conferred resistance to growth inhibition in the presence of 0.5 mM guanidine, whereas the G64R and G64T mutant virus populations remained susceptible to growth inhibition by 0.5 mM guanidine. Interestingly, a S264L mutation identified in the 3D coding region of 26M after ribavirin selection was also associated with both ribavirin-resistant and high replication fidelity phenotypes. These findings are consistent with the hypothesis that the 3D-G64R, 3D-G64T, and 3D-S264L mutations confer resistance upon HEV71 to the antiviral mutagen ribavirin, coupled with a high replication fidelity phenotype during growth in cell culture.     

Differential sensitivity of oleosins to proteolysis during oil body mobilization in sunflower seedlings

Until now, there has been no conclusive demonstration of any in vivo oleosin degradation at the early stages of oil body mobilization. The present work on sunflower (Helianthus annuus L.) has demonstrated limited oleosin degradation during seed germination. Seedling cotyledon homogenization in Tris-urea buffer, followed by SDS-PAGE, revealed three oleosins (16, 17.5 and 20 kDa). Incubation of oil bodies with total soluble protein from 4-day-old seedlings resulted in oleosin degradation. In vitro and in vivo degradation of the 17.5-kDa oleosin was faster than the other two, indicating its greater susceptibility to proteolysis. Oleosin degradation by the total soluble protein resulted in a transient 14.5-kDa polypeptide, followed by an 11-kDa protease-protected fragment, which appeared post-germinatively and accumulated corresponding to increased rate of lipid mobilization. A 65-kDa protease, active at pH 7.5-9.5, was zymographically detected in the total soluble protein. Its activity increased along with in vivo accumulation of the protease-protected fragment during seed germination and accompanying lipid mobilization. Protease-treated oil bodies were more susceptible to maize lipase action. Differential proteolytic sensitivity of different oleosins in the oil body membranes could be a determinant of oil body longevity during seed germination.     

Aerobic training increases the expression of adiponectin receptor genes in the peripheral blood mononuclear cells of young men

Little is known about the effect of exercise training on the expression of adiponectin receptor genes in peripheral blood mononuclear cells (PBMCs). In this study, we investigated the effects of aerobic training on the expression of AdipoR1 and AidpoR2 mRNAs in PBMCs, whole body insulin sensitivity, and circulating adiponectins in men. Thirty young men were randomly assigned to either a control (n=15) or an exercise (n=15) group. Subjects assigned to the exercise group underwent a 12-week jogging and/or running programme on a motor-driven treadmill at an intensity of 60%-75% of the age-based maximum heart rate with duration of 40 minutes per session and a frequency of 5 days per week. Two-way mixed ANOVA with repeated measures was used to test any significant time-by-group interaction effects for the measured variables at p=0.05. We found significant time-by-group interaction effects for waist circumference (p=0.001), VO_2max (p<0.001), fasting insulin (p=0.016), homeostasis model assessment for insulin resistance (HOMA-IR) (p=0.010), area under the curve (AUC) for insulin response during the 75-g oral glucose tolerance test (p=0.002), high-molecular weight (HMW) adiponectin (p=0.016), and the PBMC mRNA levels of AdipoR1 (p<0.001) and AdipoR2 (p=0.001). The exercise group had significantly increased mRNA levels of AdipoR1 and AdipoR2 in PBMCs, along with increased whole body insulin sensitivity and HMW adiponectin, decreased waist circumference, and increased VO_2max compared with the control group. In summary, the current findings suggest that exercise training modulates the expression of AdipoR1 and AdipoR2 mRNAs in PBMCs, implying that manipulation of the expression of these genes could be a potential surrogate for lifestyle intervention-mediated improvements of whole body insulin sensitivity and glucose homeostasis.     

Genetic and environmental heterogeneity of residual variance of weight traits in Nellore beef cattle

Background

Many studies have provided evidence of the existence of genetic heterogeneity of environmental variance, suggesting that it could be exploited to improve robustness and uniformity of livestock by selection. However, little is known about the perspectives of such a selection strategy in beef cattle.

Methods

A two-step approach was applied to study the genetic heterogeneity of residual variance of weight gain from birth to weaning and long-yearling weight in a Nellore beef cattle population. First, an animal model was fitted to the data and second, the influence of additive and environmental effects on the residual variance of these traits was investigated with different models, in which the log squared estimated residuals for each phenotypic record were analyzed using the restricted maximum likelihood method. Monte Carlo simulation was performed to assess the reliability of variance component estimates from the second step and the accuracy of estimated breeding values for residual variation.

Results

The results suggest that both genetic and environmental factors have an effect on the residual variance of weight gain from birth to weaning and long-yearling in Nellore beef cattle and that uniformity of these traits could be improved by selecting for lower residual variance, when considering a large amount of information to predict genetic merit for this criterion. Simulations suggested that using the two-step approach would lead to biased estimates of variance components, such that more adequate methods are needed to study the genetic heterogeneity of residual variance in beef cattle.     

The Vascular Endothelial Growth Factor Inhibitors Ranibizumab and Aflibercept Markedly Increase Expression of Atherosclerosis-Associated Inflammatory Mediators on Vascular Endothelial Cells

Introduction

Recent studies have suggested that the VEGF inhibitors, Ranibizumab and Aflibercept may be associated with an excess of cardiovascular events, potentially driven by increasing atheroma instability, leading to plaque rupture and clinical events. Inflammation plays a key role in the progression of atherosclerotic plaque and particularly conversion to an unstable phenotype. Here, we sought to assess the in vitro effects of these drugs on the expression of key inflammatory mediators on endothelial cells.

Methods

Human coronary artery endothelial cells were co-incubated for 16h with Ranibizumab (0.11nM) or Aflibercept (0.45nM), as determined by each drug’s peak serum concentration (Cmax). Expression at protein (ELISA) and gene (RT-PCR) level of inflammatory chemokines CCL2, CCL5 and CXC3L1 as well as gene expression for the cell adhesion molecules VCAM-1, ICAM-1 and the key NF-κb protein p65 was assessed. VEGF-A protein levels were also determined.

Results

Both drugs significantly increased chemokine, cell adhesion molecule (CAM) and p65 expression, while decreasing VEGF-A protein secretion. At equivalent Cmax concentrations, Aflibercept was significantly more pro-inflammatory than Ranibizumab. Reduction of secreted VEGF-A levels significantly attenuated inflammatory effects of both drugs, whereas blockade of the VEGF-A receptor or silencing of VEGF-A gene synthesis alone had no effect, suggesting that binding of drug to secreted VEGF-A is crucial in promoting inflammation. Finally, blockade of Toll-like receptor 4 significantly reduced inflammatory effects of both drugs.

Conclusion

We demonstrated here, for the first time, that both drugs have potent pro-inflammatory effects, mediated via activation of Toll-like receptor 4 on the endothelial cell surface by drug bound to VEGF-A. Further studies are required to investigate whether these effects are also seen in vivo.     

Lipid Composition of Whole Roots and of Ca2+, Mg2+-activated Adenosine Triphosphatases from Wheat and Oat as Related to Mineral Nutrition

Lipid composition of whole roots of wheat (Triticum vulgare Vill. cv. Svenno Spring Wheat) and oat (Avena sativa L. cv. Brighton) and of cell wall fractions, mitochondrial fractions and microsomal fractions of these roots were studied. Lipid composition depended upon the level of mineral nutrition. In wheat total phospholipids, phosphatidyl choline and sulfolipid content was highest in the roots grown at the higher salt concentration, while the reverse was true for oat roots. In both species glycolipid and sterol content was lower in the high salt roots, at the same time as higher proportions of them were built into the microsomal fraction. Phosphatidyl choline content of the wheat root membrane fractions increased with the salt level, while the opposite occurred in the oat roots. The phosphatidyl choline content may be correlated with the (Ca²⁺, Mg²⁺)-stimulated ATPase activity.     

Temperature Response and Effect of Ca2+ and Mg2+ on ATPases from Roots of Oats and Wheat as Influenced by Growth Temperature and Nutritional Status

Activation by Ca²⁺, Mg²⁺, Zn²⁺, or Mn²⁺ of adenosine triphosphatases in a microsomal fraction from wheat roots depends upon the growth temperature when the plants are grown under low salt conditions, but not when the plants get a full-strength culture medium. At low ionic strength, cultivation at 25°C gives only half as high activation as cultivation at 18°C or at high ionic strength at both temperatures. Corresponding data for activation of ATPases from oats also show that low ionic strength during growth gives the highest temperature dependence. Low temperature together with low salt conditions during growth gives the highest ATPase activity after stimulation with divalent cations. High growth temperature and full-strength medium decrease the ATPase activity. Activation energies (Ea) were calculated for the two temperature intervals 35–20°C and 20–5°C. The dominating ATPase stimulation (Ca²⁺ in wheat, Mg²⁺ in oats) is characterized by high specific activity combined with a low Ea value. The differences in ATPase activity between oats and wheat can be correlated with different cultivation requirements known from agriculture.     

Relationship of nitrate supply with growth rate, plasma membrane-bound and cytosolic nitrate reductase, and tissue nitrate content in tobacco plants

cNR, cytosolic nitrate reductase
PM-NR, plasma membrane-bound nitrate reductase

Activities of plasma membrane-bound nitrate reductase (PM-NR) and cytosolic nitrate reductase (cNR) in tobacco (Nicotiana tabacum L. cv. Samsun) are regulated differently, depending upon the nitrate supply to the culture medium (in sand culture). The cNR activity of roots was higher at low nitrate concentrations with the maximum at 5 mM nitrate supply and declined to low values beyond 5 mM . In contrast, the PM-NR activity of roots increased with higher nitrate concentrations with the maximum at 25 mM nitrate and clearly decreased only at 40 mM . This high PM-NR activity correlated with a low growth rate and might be one of the responses to excess nitrate. Internal nitrate and total nitrogen content of the tissues, however, showed a relative minimum in shoots and in roots of between 15 and 25 mM external nitrate. With declining PM-NR activities beyond 25 mM external nitrate, the nitrate content in the tissue increased indicating an inverse relationship between tissue nitrate content and root PM-NR activity. In leaves both NR activities (cNR and PM-NR) correlated with the internal nitrate content, but with a different response at low nitrate.