Inhibition of hydrogen sulfide production by gene silencing attenuates inflammatory activity of LPS-activated RAW264.7 cells

Hydrogen sulfide is an inflammatory mediator and is produced by the activity of the enzyme cystathionine γ-lyase (CSE) in macrophages. Previously, pharmacological inhibition of CSE has been reported to have conflicting results, and this may be due to the lack of specificity of the pharmacological agents. Therefore, this study used a very specific approach of small interfering RNA (siRNA) to inhibit the production of the CSE in an in vitro setting. We found that the activation of macrophages by lipopolysaccharide (LPS) resulted in higher levels of CSE mRNA and protein as well as the increased production of proinflammatory cytokines and nitric oxide (NO). We successfully used siRNA to specifically reduce the levels of CSE mRNA and protein in activated macrophages. Furthermore, the levels of proinflammatory cytokines in LPS-activated macrophages were significantly lower in siRNA-transfected cells compared to those in untransfected controls. However, the production levels of NO by the transfected cells were higher, suggesting that CSE activity has an inhibitory effect on NO production. These findings suggest that the CSE enzyme has a crucial role in the activation of macrophages, and its activity has an inhibitory effect on NO production by these cells.     

Cystathionine-γ-lyase gene silencing with siRNA in monocytes/macrophages attenuates inflammation in cecal ligation and puncture-induced sepsis in the mouse

Hydrogen sulphide is an endogenous inflammatory mediator produced by cystathionine-γ-lyase (CSE) in macrophages. To determine the role of H₂S and macrophages in sepsis, we used small interference RNA (siRNA) to target the CSE gene and investigated its effect in a mouse model of sepsis. Cecal ligation puncture (CLP)-induced sepsis is characterized by increased levels of myeloperoxidase (MPO) activity, morphological changes in liver and pro-inflammatory cytokines and chemokines in the liver and lung. SiRNA treatment attenuated inflammation in the liver and lungs of mice following CLP-induced sepsis. Liver MPO activity increased in CLP-induced sepsis and treatment with siRNA significantly reduced this. Similarly, lung MPO activity increased following induction of sepsis with CLP while siRNA treatment significantly reduced MPO activity. Liver and lung cytokine and chemokine levels in CLP-induced sepsis reduced following treatment with siRNA. These findings show a crucial pro-inflammatory role for H₂S synthesized by CSE in macrophages in sepsis and suggest CSE gene silencing with siRNA as a potential therapeutic approach for this condition.     

Purification and characterization of an iron-containing alcohol dehydrogenase in extremely thermophilic bacterium <Emphasis Type="Italic">Thermotoga hypogea</Emphasis>

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It uses carbohydrates and peptides as carbon and energy sources to produce acetate, CO₂, H₂, l-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of 40 ± 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 ± 0.06 g-atoms/subunit. It was oxygen sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and Fe²⁺. The enzyme was thermostable with a half-life of about 10 h at 70°C, and its catalytic activity increased along with the rise of temperature up to 95°C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively. The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent K _m values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols.     

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.     

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.     

Effects of macrophage colony-stimulating factor on microglial responses to lipopolysaccharide and beta amyloid

A challenge for studies involving microglia cultures is obtaining sufficient cells for downstream experiments. Macrophage colony-stimulating factor (M-CSF) has been used to improve yield of microglia in culture. However, the effects of M-CSF on activation profiles of microglia cultures are still unclear. Microglia activation is characterised by upregulation of co-stimulatory molecules and an inflammatory phenotype. The aim of this study is to demonstrate whether M-CSF supplementation alters microglial responses in resting and activated conditions. Microglia derived from mixed glia cultures and the BV-2 microglia cell line were cultivated with/without M-CSF and activated with lipopolysaccharide (LPS) and beta amyloid (Aβ). We show M-CSF expands primary microglia without affecting microglial responses to LPS and Aβ, as shown by the comparable expression of MHC class II and CD40 to microglia grown without this growth factor. M-CSF supplementation in BV-2 cells had no effect on nitric oxide (NO) production. Therefore, M-CSF can be considered for improving microglia yield in culture without introducing activation artefacts.     

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.