Cloning and characterization of the <Emphasis Type="Italic">Tne</Emphasis>DI restriction–modification system of <Emphasis Type="Italic">Thermotoga neapolitana</Emphasis>

A putative Type II restriction–modification system of Thermotoga neapolitana, TneDI, was cloned into Escherichia coli XL1-Blue MRF′ and characterized. Gene CTN_0339 specifies the endonuclease R.TneDI, while CTN_0340 encodes the cognate DNA methyltransferase M.TneDI. Both enzymes were purified simply by heating the cell lysates of E. coli followed by centrifugation. The enzymes were active over a broad range of temperatures, from 42°C to at least 77°C, with the highest activities observed at 77°C. R.TneDI cleaved at the center of the recognition sequence (CG↓CG) and generated blunt-end cuts. Overexpression of R.TneDI in BL21(DE3) was confirmed by both SDS-PAGE and Western blotting.     

Pentoxifylline attenuates cardiac dysfunction and reduces TNF-alpha level in ischemic-reperfused heart

Although pentoxifylline (PTXF), a phosphodiesterase inhibitor, has been reported to exert beneficial effects in cardiac bypass surgery, its effect and mechanisms against ischemia-reperfusion (I/R) injury in heart are poorly understood. Because I/R is known to increase the level of tumor necrosis factor (TNF)-alpha in myocardium and PTXF has been shown to depress the production of TNF-alpha in failing heart, this study examined the hypothesis that PTXF may attenuate cardiac dysfunction and reduce TNF-alpha content in I/R heart. For this purpose, isolated rat hearts were subjected to global ischemia for 30 min followed by reperfusion for 2-30 min. Although cardiac dysfunction due to ischemia was not affected, the recovery of heart function upon reperfusion was markedly improved by PTXF treatment. This cardioprotective effect of PTXF was dose dependent; maximal effect was seen at a concentration of 125 microM. TNF-alpha, nuclear factor-kappaB (NF-kappaB), and phosphorylated NF-kappaB contents were decreased in ischemic heart but were markedly increased within 2 min of starting reperfusion. The ratio of cytosolic-to-homogenate NF-kappaB was decreased, whereas the ratio of particulate-to-homogenate NF-kappaB was increased in I/R hearts. These changes in TNF-alpha and NF-kappaB protein contents as well as in NF-kappaB redistribution due to I/R were significantly attenuated by PTXF treatment. The results of this study indicate that the cardioprotective effects of PTXF against I/R injury may be due to reductions in the activation of NF-kappaB and the production of TNF-alpha content.     

Sarcoplasmic reticulum Ca2+ transport and gene expression in congestive heart failure are modified by imidapril treatment

This study was designed to test the hypothesis that blockade of the renin-angiotensin system improves cardiac function in congestive heart failure by preventing changes in gene expression of sarcoplasmic reticulum (SR) proteins. We employed rats with myocardial infarction (MI) to examine effects of an angiotensin-converting enzyme inhibitor, imidapril, on SR Ca(2+) transport, protein content, and gene expression. Imidapril (1 mg.kg(-1).day(-1)) was given for 4 wk starting 3 wk after coronary artery occlusion. Infarcted rats exhibited a fourfold increase in left ventricular end-diastolic pressure, whereas rates of pressure development and decay were decreased by 60 and 55%, respectively. SR Ca(2+) uptake and Ca(2+) pump ATPase, as well as Ca(2+) release and ryanodine receptor binding activities, were depressed in the failing hearts; protein content and mRNA levels for Ca(2+) pump ATPase, phospholamban, and ryanodine receptor were also decreased by approximately 55-65%. Imidapril treatment of infarcted animals improved cardiac performance and attenuated alterations in SR Ca(2+) pump and Ca(2+) release activities. Changes in protein content and mRNA levels for SR Ca(2+) pump ATPase, phospholamban, and ryanodine receptor were also prevented by imidapril treatment. Beneficial effects of imidapril on cardiac function and SR Ca(2+) transport were not only seen at different intervals of MI but were also simulated by another angiotensin-converting enzyme inhibitor, enalapril, and an ANG II receptor antagonist, losartan. These results suggest that blockade of the renin-angiotensin system may increase the abundance of mRNA for SR proteins and, thus, may prevent the depression in SR Ca(2+) transport and improve cardiac function in congestive heart failure due to MI.     

Mechanisms of lysophosphatidic acid-induced increase in intracellular calcium in vascular smooth muscle cells

Although lysophosphatidic acid (LPA) is known to cause an increase in intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMCs), the mechanisms of [Ca2+]i mobilization by LPA are not fully understood. In the present study, the effect of LPA on [Ca2+]i mobilization in cultured A10 VSMCs was examined by Fura-2 fluorescence technique. The expression of LPA receptors was studied by immunostaining. LPA was observed to increase [Ca2+]i in a concentration-dependent manner; this increase was dependent on the concentration of extracellular Ca2+. Both sarcolemmal (SL) Na(+)-Ca2+ exchange inhibitors (amiloride, Ni2+ and KB-R7943) and Na(+)-H+ exchange inhibitor (MIA) as well as SL store-operated Ca2+ channel (SOC) antagonists (SK&F 96365, tyrphostin A9 and gadolinium), unlike SL Ca2+ channel antagonists (verapamil and diltiazem), inhibited the LPA-induced increase in [Ca2+]i. In addition, sarcoplasmic reticulum (SR) Ca2+ channel blocker (ryanodine), SR Ca2+ channel opener (caffeine), SR Ca2+ pump ATPase inhibitor (thapsigargin) and inositol 1,4,5-trisphosphate (InsP3) receptor antagonists (xestospongin and 2-aminoethoxydiphenyl borate) were found to inhibit the LPA-induced Ca2+ mobilization. Furthermore, phospholipase C (PLC) inhibitor (U 73122) and protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) attenuated the LPA-induced increase in [Ca2+]i. These results indicate that Ca2+ mobilization by LPA involves extracellular Ca2+ entry through SL Na(+)-Ca2+ exchanger, Na(+)-H+ exchanger and SL SOCs. In addition, ryanodine-sensitive and InsP(3)-sensitive intracellular Ca2+ pools may be associated with the LPA-induced increase in [Ca2+]i. Furthermore, the LPA-induced [Ca2+]i mobilization in VSMCs seems to be due to the activation of both PLC and PKC.     

High-throughput microplate phosphorylation assays based on DevR-DevS/Rv2027c 2-component signal transduction pathway to screen for novel antitubercular compounds

DevR-DevS (Rv3133c-Rv3132c) and DevR-Rv2027c have been established through their autophosphorylation and phospho-transfer properties to constitute bonafide regulatory 2-component systems of Mycobacterium tuberculosis. DevR has also been shown by others to play a key regulatory role in the expression of M. tuberculosis genes comprising the dormancy regulon. The authors describe high-throughput phosphorylation assays in a microplate format using DevS and Rv2027c histidine kinases and DevR response regulator proteins from M. tuberculosis. The assays were designed to measure [gamma-(32)P]ATP-dependent autophosphorylation of DevS/Rv2027c and also the phosphotransfer reaction to DevR. First, the optimal reaction conditions were established using the conventional method of radiolabeling the 2-component proteins by [gamma-(32)P]ATP and followed by gel electrophoresis-based analysis. Next, the assays were converted to a high-throughput format in which the radiolabeled protein retained on a filter using mixed cellulose ester-based 96-well filter plates was analyzed for radioactivity retention by scintillation counting. The utility of these assays to screen for inhibitors is illustrated using 2-mercaptobenzimidazole, ethidium bromide, and EDTA. The high quality and flexibility of these assays will enable their use in high-throughput screening for new antitubercular compounds directed against 2-component systems that comprise a novel target in dormant mycobacteria.     

Sorghum straw for xylanase hyper-production by Thermomyces lanuginosus (D2W3) under solid-state fermentation

This paper reports the production of very high levels of cellulase free xylanase and associated hemicellulases by an indigenous thermophilic isolate of Thermomyces lanuginosus (D(2)W(3)) using solid-state fermentation. Sorghum straw, an inexpensive and abundant source of carbon supported maximal xylanase activity (11,855 units/g dry substrate). Culturing T. lanuginosus D(2)W(3) on sorghum straw and optimizing other culture conditions (media types, particle size of carbon source, inoculum level, inoculum age and additives), yielded increased levels of xylanase (39,726 units/g dry substrate). Further optimization of enzyme production was carried out using Box-Behnken design of experiments with three independent variables (inoculum level, glycerol and ammonium sulphate concentrations) which resulted in very high levels of xylanase, 48,000+/-1774 units/g dry substrate, and 2.6+/-0.2, 13.4+/-0.56, 68+/-1.7, 1.4+/-0.08, 1.2+/-0.05 (units/g dry substrate) of beta-xylosidase, alpha-galactosidase, pectinase, beta-mannosidase and alpha-L-arabinofuranosidase, respectively.     

Synthesis and SAR of novel oxazolidinones: discovery of ranbezolid

Novel oxazolidinones were synthesized containing a number of substituted five-membered heterocycles attached to the 'piperazinyl-phenyl-oxazolidinone' core of eperezolid. Further, the piperazine ring of the core was replaced by other diamino-heterocycles. These modifications led to several compounds with potent activity against a spectrum of resistant and susceptible gram-positive organisms, along with the identification of ranbezolid (RBx 7644) as a clinical candidate.     

Short-term zinc deficiency in diet induces increased oxidative stress in testes and epididymis of rats

In order to determine the effects of Zinc deficient diet on oxidative stress in testis and epididymis, various parameters viz: total proteins, lipid peroxidation, hydroperoxides, antioxidant capacity and enzymatic activities are evaluated in rats fed on zinc deficient diet for 2, 4 and 6 weeks. Total proteins, water and lipid solouble antioxidant capacity decreased while lipid peroxidation (TBARS) and hydroperoxides concentration increased in testes, caput and cauda epididymis except in 2ZD (testes) where hydroperoxides revealed a significant decrease. GSH decreased in testes and caput and cauda epididymis. GPx and gamma-GT activities increased in testes and caput and cauda epididymis of zinc deficient rats. Further, GST increased in testes but exhibited decreases after 2 and 4 weeks and an increase after 6 weeks in caput and cauda epididymis of zinc deficient rats. GR activities decreased in testes but it increased in caput and cauda epididymis of zinc deficient rats. Thus, zinc deprivation results in increased sensitivity to oxidative stress. All these may have been as a consequence of increased ROS generation and/or decreased zinc dependent antioxidant processes.     

Pharmacological basis of different targets for the treatment of atherosclerosis

The development of atherosclerotic plaque is a highly regulated and complex process which occurs as a result of structural and functional alterations in endothelial cells, smooth muscle cells (SMCs), monocytes/macrophages, T-lymphocytes and platelets. The plaque formation in the coronary arteries or rupture of the plaque in the peripheral vasculature in latter stages of atherosclerosis triggers the onset of acute ischemic events involving myocardium. Although lipid lowering with statins has been established as an important therapy for the treatment of atherosclerosis, partially beneficial effects of statins beyond decreasing lipid levels has shifted the focus to develop newer drugs that can affect directly the process of atherosclerosis. Blockade of renin angiotensin system, augmentation of nitric oxide availability, reduction of Ca(2+) influx, prevention of oxidative stress as well as attenuation of inflammation, platelet activation and SMC proliferation have been recognized as targets for drug treatment to control the development, progression and management of atherosclerosis. A major challenge for future drug development is to formulate a combination therapy affecting different targets to improve the treatment of atherosclerosis.