Tyrosine phosphorylation of myosin heavy chain during skeletal muscle differentiation: an integrated bioinformatics approach

Background  

Previously it has been shown that insulin-mediated tyrosine phosphorylation of myosin heavy chain is concomitant with enhanced association of C-terminal SRC kinase during skeletal muscle differentiation. We sought to identify putative site(s) for this phosphorylation event.     

Effect of Anionic Salt and Highly Fermentable Carbohydrate Supplementations on Urine pH and on Experimentally Induced Hypocalcaemia in Cows

The objective of this experiment was to determine the effect of dietary grain on calcium homeostasis. Six rumen-fistulated dairy cows with 3 or more previous lactations and no history of parturient paresis were randomly assigned to a sequence of diets in a crossover study with 4 periods of 10 days each. Dietary treatments were: A control ration consisting of wrap grass silage alone (1), the control ration supplemented with ammonium chloride and ammonium sulphate salt solution (2), control ration following a period with supplementation (3) and control ration supplemented with increasing amounts of barley from 4 to 10 kg/cow per day, expected to produce subclinical rumen acidosis (4). Daily intake of the diets was adjusted to 14 kg DM/cow per day. On day 11, the calcium-regulating mechanisms in cows were challenged until recumbency by a standardized intravenous EDTA infusion and cows were left to recover spontaneously. Anion supplementation and the feeding of highly fermentable carbohydrate lowered urine pH below 7.0 due to subclinical acidosis. During spontaneous recovery from EDTA induced hypocalcaemia, the cows more quickly regained a whole blood free calcium concentration of 1.00 mmol/L if they had most recently been supplemented with either anionic salts or with increasing amounts of barley, as compared to the basic ration. It is concluded that so-called slug-feeding or 'steaming up' with highly fermentable carbohydrates before parturition in milk fever susceptible cows enhanced calcium homeostasis similar to the effect seen in cows on anionic diets.     

Calcitonin gene-related peptide is not essential for the development of pressure overload-induced hypertrophy in vivo.

The regulatory neuropeptide calcitonin-gene related peptide (CGRP) has been shown to evoke a hypertrophic response in isolated cardiomyocytes in vitro, an effect which was attributed to PKC activation. Activation of PKC has previously been implicated in the development of cardiac hypertrophy. We therefore investigated the role of CGRP in pressure overload-induced hypertrophy in vivo, which has not previously been reported. Constriction of the ascending aorta of rats resulted in an increase in the heart weight to body weight ratio, increased myocyte diameter, re-expression of the fetal genes ANF, MHCbeta and skeletal alpha-actin, and decreased expression of the adult genes GLUT4 and SERCA2a. Treatment of neonatal rat pups (1-2 days old) with capsaicin (50 mg/kg), resulted in the permanent de-afferentation of small-diameter unmyelinated CGRP-containing sensory C-fibres. Such treatment caused a 68% decrease in the CGRP-like immunoreactivity of hearts isolated from 10 week old rats (p < 0.001). Contrary to expectations, aortic constriction of capsaicin treated rats had no effect on the development of hypertrophy at the trophic, morphometric or gene expression levels. The results suggest that the development of pressure overload-induced hypertrophy in vivo does not require the regulatory neuropeptide CGRP.     

Inhibition of coronary thrombosis and local inflammation by a noncarbohydrate selectin inhibitor

We tested the hypothesis that selectin inhibition with blocking antibodies or a small-molecular-weight inhibitor of L-, P-, and E-selectin, methoxybenzoylpropionic acid (MBPA), prevents thrombus formation in a canine coronary Folts' model. Cyclic flow variations (CFVs) were induced by crush injury and constriction of the left anterior descending coronary artery in dogs. Systemic infusion of antibodies to P- and L-selectin abolished CFVs, respectively, in 50% and 17% of treated dogs [P = not significant (NS)]. The combination of P- and L-selectin antibodies suppressed CFVs in 60% of treated dogs (P = NS). In contrast, systemic selectin blockade by intravenous infusion or local adventitial application of MBPA markedly reduced CFVs and, in addition, reduced myocardial myeloperoxidase (MPO) activity. We conclude that inhibition of L-, P-, and E-selectin binding by a small-molecular-weight, noncarbohydrate compound markedly reduces arterial thrombosis, whereas systemic administration of antibodies to L- and P-selectin fail to reproduce this antithrombotic effect. These results underscore the role of selectins in the pathogenesis of arterial thrombosis under high shear stress and suggest that inhibition of P- and L- selectin may not suffice to prevent thrombus formation in this model. The role of E-selectin in thrombus formation in this model awaits further testing.     

A THEMIS:SHP1 complex promotes T‐cell survival

THEMIS is critical for conventional T‐cell development, but its precise molecular function remains elusive. Here, we show that THEMIS constitutively associates with the phosphatases SHP1 and SHP2. This complex requires the adapter GRB2, which bridges SHP to THEMIS in a Tyr‐phosphorylation‐independent fashion. Rather, SHP1 and THEMIS engage with the N‐SH3 and C‐SH3 domains of GRB2, respectively, a configuration that allows GRB2‐SH2 to recruit the complex onto LAT. Consistent with THEMIS‐mediated recruitment of SHP to the TCR signalosome, THEMIS knock‐down increased TCR‐induced CD3‐ζ phosphorylation, Erk activation and CD69 expression, but not LCK phosphorylation. This generalized TCR signalling increase led to augmented apoptosis, a phenotype mirrored by SHP1 knock‐down. Remarkably, a KI mutation of LCK Ser59, previously suggested to be key in ERK‐mediated resistance towards SHP1 negative feedback, did not affect TCR signalling nor ligand discrimination in vivo. Thus, the THEMIS:SHP complex dampens early TCR signalling by a previously unknown molecular mechanism that favours T‐cell survival. We discuss possible implications of this mechanism in modulating TCR output signals towards conventional T‐cell development and differentiation.     

Structure of the Bacillus anthracis Sortase A Enzyme Bound to Its Sorting Signal: A FLEXIBLE AMINO-TERMINAL APPENDAGE MODULATES SUBSTRATE ACCESS*

The endospore forming bacterium Bacillus anthracis causes lethal anthrax disease in humans and animals. The ability of this pathogen to replicate within macrophages is dependent upon the display of bacterial surface proteins attached to the cell wall by the B. anthracis Sortase A (^BaSrtA) enzyme. Previously, we discovered that the class A ^BaSrtA sortase contains a unique N-terminal appendage that wraps around the body of the protein to contact the active site of the enzyme. To gain insight into its function, we determined the NMR structure of ^BaSrtA bound to a LPXTG sorting signal analog. The structure, combined with dynamics, kinetics, and whole cell protein display data suggest that the N terminus modulates substrate access to the enzyme. We propose that it may increase the efficiency of protein display by reducing the unproductive hydrolytic cleavage of enzyme-protein covalent intermediates that form during the cell wall anchoring reaction. Notably, a key active site loop (β7/β8 loop) undergoes a disordered to ordered transition upon binding the sorting signal, potentially facilitating recognition of lipid II.