Tyrosine residues in phospholipase Cgamma 2 essential for the enzyme function in B-cell signaling

Phospholipase Cgamma (PLCgamma) isoforms are regulated through activation of tyrosine kinase-linked receptors. The importance of growth factor-stimulated phosphorylation of specific tyrosine residues has been documented for PLCgamma1; however, despite the critical importance of PLCgamma2 in B-cell signal transduction, neither the tyrosine kinase(s) that directly phosphorylate PLCgamma2 nor the sites in PLCgamma2 that become phosphorylated after stimulation are known. By measuring the ability of human PLCgamma2 to restore calcium responses to the B-cell receptor stimulation or oxidative stress in a B-cell line (DT40) deficient in PLCgamma2, we have demonstrated that two tyrosine residues, Tyr(753) and Tyr(759), were important for the PLCgamma2 signaling function. Furthermore, the double mutation Y753F/Y759F in PLCgamma2 resulted in a loss of tyrosine phosphorylation in stimulated DT40 cells. Of the two kinases that previously have been proposed to phosphorylate PLCgamma2, Btk, and Syk, purified Btk had much greater ability to phosphorylate recombinant PLCgamma2 in vitro, whereas Syk efficiently phosphorylated adapter protein BLNK. Using purified proteins to analyze the formation of complexes, we suggest that function of Syk is to phosphorylate BLNK, providing binding sites for PLCgamma2. Further analysis of PLCgamma2 tyrosine residues phosphorylated by Btk and several kinases from the Src family has suggested multiple sites of phosphorylation and, in the context of a peptide incorporating residues Tyr(753) and Tyr(759), shown preferential phosphorylation of Tyr(753).     

Linear Discrete Population Models with Two Time Scales in Fast Changing Environments I: Autonomous Case

In this work we consider a structured population with groups and subgroups of individuals. The intra-group dynamics is assumed to be fast in comparison with the inter-group dynamics. We study linear discrete models where the slow dynamics is represented by a single matrix and the fast dynamics is described by means of the first k terms of a converging sequence of different matrices. The number k can be interpreted as the ratio between the two time scales.The aim of this work is to extend aggregation techniques to the case of fast changing environments. The main idea of aggregation is to build up a new system, with lower dimension, that summarizes the information concerning the fast process. This "aggregated" system provides essential information on the original one. It is shown that the asymptotic behavior of the original system can be approximated by the asymptotic behavior of the aggregated system when the ratio between the two time scales is large enough.We present an example of an age structured population in a patchy environment. The migration process is assumed to be fast in comparison with the demographic process. Numerical simulations illustrate that the asymptotic growth rate and the stable age distribution of the population in the original and the aggregated systems are getting closer as the ratio k increases.     

The influence of estrogen on hepatic cholesterol metabolism and biliary lipid secretion in rats fed fish oil

Both estrogen and dietary n-3 polyunsaturated fatty acids are known to be hypocholesterolemic, but appear to exert their effects by different mechanisms. In this study, the interaction between dietary fish oil (rich in n-3 polyunsaturated fatty acids) and estrogen in the regulation of hepatic cholesterol metabolism and biliary lipid secretion in rats was studied. Rats fed a low fat or a fish oil-supplemented diet for 21 days were injected with 17alpha-ethinyl estradiol (5 mg/kg body weight) or the vehicle only (control rats) once per day for 3 consecutive days. Estrogen-treatment led to a marked reduction in plasma cholesterol levels in fish oil-fed rats, which was greater than that observed with either estrogen or dietary fish oil alone. The expression of mRNA for cholesterol 7alpha-hydroxylase was decreased by estrogen in rats fed a low fat or a fish oil-supplemented diet, while the output of cholesterol (micromol/h/kg b.wt.) in the bile was unchanged in both groups. Cholesterol levels in the liver were increased by estrogen in rats given either diet, but there was a significant shift from cholesterol esterification to cholesteryl ester hydrolysis only in the fish oil-fed animals. Estrogen increased the concentration of cholesterol (micromol/ml) in the bile in rats fed the fish oil, but not the low fat diet. However, the cholesterol saturation index was unaffected. The output and concentration of total bile acid was also unaffected, but changes in the distribution of the individual bile acids were observed with estrogen treatment in both low fat and fish oil-fed groups. These results show that interaction between estrogen-treatment and dietary n-3 polyunsaturated fatty acids causes changes in hepatic cholesterol metabolism and biliary lipid secretion in rats, but does not increase the excretion of cholesterol from the body.     

Redox regulation of RyR-mediated Ca2+ release in muscle and neurons

Changes in the redox state of the intracellular ryanodine receptor/Ca2+ release channels of skeletal and cardiac muscle or brain cortex neurons affect their activity. In particular, agents that oxidize or alkylate free SH residues of the channel protein strongly enhance Ca(2+)-induced Ca2+ release, whereas reducing agents have the opposite effects. We will discuss here how modifications of highly reactive cysteine residues by endogenous redox agents or cellular redox state influence RyR channel activation by Ca2+ and ATP or inhibition by Mg2+. Possible physiological and pathological implications of these results on cellular Ca2+ signaling will be addressed as well.     

The acetyl xylan esterase II gene from Penicillium purpurogenum is differentially expressed in several carbon sources, and tightly regulated by pH

The expression of the acetyl xylan esterase II (axeII) gene from Penicillium purpurogenum is repressed by glucose and induced by xylan, as well as to a small degree by xylose and xylitol. This gene is expressed at neutral pH, but not under alkaline or acidic conditions, in agreement with previous findings for other xylanolytic genes of this organism. This is the first report showing pH regulation of an axe gene.     

Calcium interacts with antifreeze proteins and chitinase from cold-acclimated winter rye

During cold acclimation, winter rye (Secale cereale) plants accumulate pathogenesis-related proteins that are also antifreeze proteins (AFPs) because they adsorb onto ice and inhibit its growth. Although they promote winter survival in planta, these dual-function AFPs proteins lose activity when stored at subzero temperatures in vitro, so we examined their stability in solutions containing CaCl2, MgCl2, or NaCl. Antifreeze activity was unaffected by salts before freezing, but decreased after freezing and thawing in CaCl2 and was recovered by adding a chelator. Ca2+ enhanced chitinase activity 3- to 5-fold in unfrozen samples, although hydrolytic activity also decreased after freezing and thawing in CaCl2. Native PAGE, circular dichroism, and Trp fluorescence experiments showed that the AFPs partially unfold after freezing and thawing, but they fold more compactly or aggregate in CaCl2. Ruthenium red, which binds to Ca(2+)-binding sites, readily stained AFPs in the absence of Ca2+, but less stain was visible after freezing and thawing AFPs in CaCl2. We conclude that the structure of AFPs changes during freezing and thawing, creating new Ca(2+)-binding sites. Once Ca2+ binds to those sites, antifreeze activity, chitinase activity and ruthenium red binding are all inhibited. Because free Ca2+ concentrations are typically low in the apoplast, antifreeze activity is probably stable to freezing and thawing in planta. Ca2+ may regulate chitinase activity if concentrations are increased locally by release from pectin or interaction with Ca(2+)-binding proteins. Furthermore, antifreeze activity can be easily maintained in vitro by including a chelator during frozen storage.     

siRNAs and miRNAs: small RNA molecules for big tasks

Small RNAs have been recently discovered as important regulators of gene expression in Eukaryota. This review compares two categories of small RNAs existing in plants: short interfering RNAs (siRNAs) and microRNAs (miRNAs) and reveals similarities and differences between two intriguing processes: RNA degradation and translational repression directed by small RNAs. The disruption of miRNA-mediated regulation causes developmental abnormalities in plants, proving a fundamental role of miRNAs.     

Benzodiazepine-based selective inhibitors of mitochondrial F1F0 ATP hydrolase

A series of benzodiazepine-based inhibitors of mitochondrial F(1)F(0) ATP hydrolase were prepared and evaluated for their ability to selectively inhibit the enzyme in the forward direction. Compounds from this series showed excellent potency and selectivity for ATP hydrolase versus ATP synthase, suggesting a potentially beneficial profile useful for the treatment of ischemic heart disease.     

N-[1-Aryl-2-(1-imidazolo)ethyl]-guanidine derivatives as potent inhibitors of the bovine mitochondrial F1F0 ATP hydrolase

A series of substituted guanidine derivatives were prepared and evaluated as potent and selective inhibitors of mitochondrial F(1)F(0) ATP hydrolase. The initial thiourethane derived lead molecules possessed intriguing in vitro pharmacological profiles, though contained moieties considered non-drug-like. Analogue synthesis efforts led to compounds with maintained potency and superior physical properties. Small molecules in this series which potently and selectivity inhibit ATP hydrolase and not ATP synthase may have utility as cardioprotective agents.