Effects of quercetin on antioxidant defense in streptozotocin-induced diabetic rats.

In light of evidence that some complications of diabetes mellitus may be caused or exacerbated by oxidative damage, we investigated the effects of subacute treatment with the antioxidant quercetin on tissue antioxidant defense systems in streptozotocin-induced diabetic Sprague-Dawley rats (30 days after streptozotocin induction). Quercetin, 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one, was administered at a dose of 10mg/kg/day, ip for 14 days, after which liver, kidney, brain, and heart were assayed for degree of lipid peroxidation, reduced and oxidized glutathione content, and activities of the free-radical detoxifying enzymes catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase. Treatment of normal rats with quercetin increased serum AST and increased hepatic concentration of oxidized glutathione. All tissues from diabetic animals exhibited disturbances in antioxidant defense when compared with normal controls. Quercetin treatment of diabetic rats reversed only the diabetic effects on brain oxidized glutathione concentration and on hepatic glutathione peroxidase activity. By contrast, a 20% increase in hepatic lipid peroxidation, a 40% decline in hepatic glutathione concentration, an increase in renal (23%) and cardiac (40%) glutathione peroxidase activities, and a 65% increase in cardiac catalase activity reflect intensified diabetic effects after treatment with quercetin. These results call into question the ability of therapy with the antioxidant quercetin to reverse diabetic oxidative stress in an overall sense.     

A dynamic metabolite valve for the control of central carbon metabolism

Successful redirection of endogenous resources into heterologous pathways is a central tenet in the creation of efficient microbial cell factories. This redirection, however, may come at a price of poor biomass accumulation, reduced cofactor regeneration and low recombinant enzyme expression. In this study, we propose a metabolite valve to mitigate these issues by dynamically tuning endogenous processes to balance the demands of cell health and pathway efficiency. A control node of glucose utilization, glucokinase (Glk), was exogenously manipulated through either engineered antisense RNA or an inverting gene circuit. Using these techniques, we were able to directly control glycolytic flux, reducing the specific growth rate of engineered Escherichia coli by up to 50% without altering final biomass accumulation. This modulation was accompanied by successful redirection of glucose into a model pathway leading to an increase in the pathway yield and reduced carbon waste to acetate. This work represents one of the first examples of the dynamic redirection of glucose away from central carbon metabolism and enables the creation of novel, efficient intracellular pathways with glucose used directly as a substrate.     

Nucleotide sequence of a cDNA encoding porcine testis 17 alpha-hydroxylase cytochrome P-450.

We describe the isolation and characterization of a cDNA encoding the complete porcine neonatal testis 17 alpha-hydroxylase/C-17,20-lyase cytochrome P-450. The deduced amino acid sequence is 509 amino acids in length.     

Neurotrophic peptide aldehydes: solid phase synthesis of fellutamide B and a simplified analog

A combination of solid phase and solution phase synthetic methods have been used to complete the total synthesis of the neurotrophic lipopeptide aldehyde fellutamide B (2). The beta-hydroxy aliphatic tail was prepared by regioselective reductive opening of a cyclic sulfate, and later coupled to a solid phase resin. The synthetic compound was then examined in cytotoxicity and nerve growth factor (NGF) induction assays. A simplified analog of fellutamide B also showed activity.     

Atrial Arrhythmia in Ageing Spontaneously Hypertensive Rats: Unraveling the Substrate in Hypertension and Ageing

Background

Both ageing and hypertension are known risk factors for atrial fibrillation (AF) although the pathophysiological contribution or interaction of the individual factors remains poorly understood. Here we aim to delineate the arrhythmogenic atrial substrate in mature spontaneously hypertensive rats (SHR).

Methods

SHR were studied at 12 and 15 months of age (n = 8 per group) together with equal numbers of age-matched normotensive Wistar-Kyoto control rats (WKY). Electrophysiologic study was performed on superfused isolated right and left atrial preparations using a custom built high-density multiple-electrode array to determine effective refractory periods (ERP), atrial conduction and atrial arrhythmia inducibility. Tissue specimens were harvested for structural analysis.

Results

Compared to WKY controls, the SHR demonstrated: Higher systolic blood pressure (p<0.0001), bi-atrial enlargement (p<0.05), bi-ventricular hypertrophy (p<0.05), lower atrial ERP (p = 0.008), increased atrial conduction heterogeneity (p = 0.001) and increased atrial interstitial fibrosis (p = 0.006) & CD68-positive macrophages infiltration (p<0.0001). These changes resulted in higher atrial arrhythmia inducibility (p = 0.01) and longer induced AF episodes (p = 0.02) in 15-month old SHR. Ageing contributed to incremental bi-atrial hypertrophy (p<0.01) and atrial conduction heterogeneity (p<0.01) without affecting atrial ERP, fibrosis and arrhythmia inducibility. The limited effect of ageing on the atrial substrate may be secondary to the reduction in CD68-positive macrophages.

Conclusions

Significant atrial electrical and structural remodeling is evident in the ageing spontaneously hypertensive rat atria. Concomitant hypertension appears to play a greater pathophysiological role than ageing despite their compounding effect on the atrial substrate. Inflammation is pathophysiologically linked to the pro-fibrotic changes in the hypertensive atria.     

Beta 2-adrenoceptor stimulation increases the number of antigen-specific precursor B lymphocytes that differentiate into IgM-secreting cells without affecting burst size.

Previous studies have shown that early addition of a beta 2-adrenergic agonist to whole splenocyte cultures immunized with SRBC induced an increase in the number of cells secreting Ag-specific antibody. Because of the low frequency of Ag-specific B lymphocytes in these cultures, it has been difficult to determine the cellular mechanism by which this increase is produced. To gain insight into this cellular mechanism, the present study was designed to evaluate the responsiveness of TNP-specific B lymphocytes cultured at both high density and limiting dilution with keyhole limpet hemocyanin (KLH)-specific, IL-4-producing Th lymphocytes, TNP-KLH, and the beta 2-adrenergic agonist, terbutaline. The results showed that a maximal twofold increase in both the number of anti-TNP IgM-secreting cells and the amount of anti-TNP IgM secretion occurred in terbutaline-exposed lymphocytes after 5 days of bulk culture. This response occurred in a concentration-dependent manner and was inhibited by concomitant culture with beta-adrenoceptor antagonists. No appreciable change was measured in the level of either IgG1 secretion in terbutaline plus Ag-exposed bulk cultures or MHC class II expression on terbutaline plus Ag-exposed TNP-specific B lymphocytes as compared with Ag alone. These data raised the possibility that beta 2-adrenoceptor stimulation induced either the differentiation of a larger proportion of TNP-specific B lymphocyte precursors into anti-TNP IgM-secreting cells, or the extensive proliferation of a constant number of TNP-specific B lymphocyte precursors, or both. Limiting dilution results showed that beta 2-adrenoceptor stimulation induced a twofold increase in the number of TNP-specific B lymphocyte precursors that differentiated into anti-TNP IgM-secreting cells, without affecting the number of anti-TNP IgM-secreting cells produced by each precursor clone.     

Ionic mechanism and role of phytochrome-mediated membrane depolarisation in caulonemal side branch initial formation in the mossPhyscomitrella patens

In caulonemal filaments of the mossPhyscomitrella patens (Hedw.), red light triggers a phytochrome-mediated transient depolarisation of the plasma membrane and the formation of side branch initials. Three-electrode voltage clamp and ion flux measurements were employed to elucidate the ionic mechanism and physiological relevance of the red-light-induced changes in ion transport. Current-voltage analyses indicated that ion channels permeable to K⁺ and Ca²⁺ are activated at the peak of the depolarisation. Calcium influx evoked by red light coincided with the depolarisation in various conditions, suggesting the involvement of voltage-gated Ca²⁺ channels. Respective K⁺ fluxes showed a small initial influx followed by a dramatic transient efflux. A role of anion channels in the depolarising current is suggested by the finding that Cl^– efflux was also increased after red light irradiation. In the presence of tetraethylammonium (10 mM) or niflumic acid (1 M), which block the red-light-induced membrane depolarisation and ion fluxes, the red-light-promoted formation of side branch initials was also abolished. Lanthanum (100 M), which inhibits K⁺ fluxes and part of the initial Ca²⁺ influx activated by red light, reduced the development of side branch initials in red light by 50%. The results suggest a causal link between the red-light-induced ion fluxes and the physiological response. The sequence of events underlying the red-light-triggered membrane potential transient and the role of ion transport in stimulus-response coupling are discussed in terms of a new model for ion-channel interaction at the plasma membrane during signalling.Abbreviations [Ca²⁺]_c cytosolic free Ca²⁺ - I-V current-voltage - E equilibrium potential - Pr red-light-absorbing phytochrome form - Pr far-red-light-absorbing phytochrome form - SPQ 6-methoxy-l-(3-sulphonatopropyl)quinolinium - TEA tetraethylammonium     

Crystallization of acetate kinase from Methanosarcina thermophila and prediction of its fold.

The unique biochemical properties of acetate kinase present a classic conundrum in the study of the mechanism of enzyme-catalyzed phosphoryl transfer. Large, single crystals of acetate kinase from Methanosarcina thermophila were grown from a solution of ammonium sulfate in the presence of ATP. The crystals diffract to beyond 1.7 A resolution. Analysis of X-ray data from the crystals is consistent with a space group of C2 and unit cell dimensions a = 181 A, b = 67 A, c = 83 A, beta = 103 degrees. Diffraction data have been collected from the crystals at 110 and 277 K. Data collected at 277 K extend to lower resolution, but are more reproducible. The orientation of a noncrystallographic two-fold axis of symmetry has been determined. Based on an analysis of the predicted amino acid sequences of acetate kinase from several organisms, we hypothesize that acetate kinase is a member of the sugar kinase/actin/hsp70 structural family.