Localization of m-calpain and calpastatin and studies of their association in pulmonary smooth muscle endoplasmic reticulum

Calpain and calpastatin have been demonstrated to play many physiological roles in a variety of systems. It, therefore, appears important to study their localization and association in different suborganelles. Using immunoblot studies, we have identified 80 kDa m-calpain in both lumen and membrane of ER isolated from bovine pulmonary artery smooth muscle. Treatment of the ER with Na(2)CO(3) and proteinase K demonstrated that 80 kDa catalytic subunit and 28 kDa regulatory subunit (Rs) of m-calpain, and the 110-kDa and 70-kDa calpastatin (Cs) forms are localized in the cytosolic side of the ER membrane. Coimmunoprecipitation studies revealed that m-calpain is associated with calpastatin in the cytosolic face of the ER membrane. We have also identified m-calpain activity both in the ER membrane and lumen by casein-zymography. The casein-zymogram has also been utilized to demonstrate differential pattern of the effects of reversible and irreversible cysteine protease inhibitors on m-calpain activity. Thus, a potential site of Cs regulation of m-calpain activity is created by positioning Cs, 80 kDa and 28 kDa m-calpain in the cytosolic face of ER membrane. However, such is not the case for the 80-kDa m-calpain found within the lumen of the ER because of the conspicuous absence of 28 kDa Rs of m-calpain and Cs in this locale.     

Synthesis and SAR studies of 1-substituted-n-(4-alkoxycarbonylpiperidin-1-yl)alkanes as potent antiarrhythmic agents.

Synthesis and SAR studies of the title compounds have resulted in the identification of structural and physicochemical parameter (Vw) contributing for antiarrhythmic activity. Among the two most promising compounds 3a & 3b, the 3a has shown antiarrhythmic activity comparable to quinidine.     

Production of xylanase by immobilized Trichoderma reesei SAF3 in Ca-alginate beads

In the present study, the optimum conditions for the production of xylanase by immobilized spores of Trichoderma reesei SAF3 in calcium alginate beads were determined. The operational stability of the beads during xylanase production under semi-continuous fermentation was also studied. The influence of alginate concentration (1, 2, 3, and 4%) and initial cell loading (100, 200, 300, 400, and 500 beads per flask) on xylanase production was considered. The production of xylanase was found to increase significantly with increasing concentration of alginate and reached a maximum yield of 3.12 ± 0.18 U ml⁻¹ at 2% (w/v). The immobilized cells produced xylanase consistently up to 10 cycles and reached a maximum level at the forth cycle (3.36 ± 0.2 U ml⁻¹).     

Bioisosteric replacement of dihydropyrazole of 4S-(-)-3-(4-chlorophenyl)-N-methyl-N'-[(4-chlorophenyl)-sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-caboxamidine (SLV-319) a potent CB1 receptor antagonist by imidazole and oxazole

Design, synthesis and conformational analysis of few imidazole and oxazole as bioisosters of 4S-(-)-3-(4-chlorophenyl)-N-methyl-N'-[(4-chlorophenyl)-sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-caboxamidine (SLV-319) 2 is reported. Computer assisted conformational analysis gave a direct clue for the loss of CB1 antagonistic activity of the ligands without a fine docking simulation for the homology model.     

Bioactive food components and cancer risk reduction

Research over the last three decades has provided convincing evidence to support the premise that diets rich in fruits and vegetables may be protective against the risk of different types of cancers. Initial evidence for protective effect of fruits and vegetables against cancer risk came from population-based case-control studies, which prompted intense research aimed at (a) identification of bioactive component(s) responsible for the anticancer effects of fruits and vegetables, (b) elucidation of the mechanisms by which bioactive food components may prevent cancer, and (c) determination of their efficacy for prevention of cancer in animal models. The bioactive components responsible for cancer chemopreventive effects of various edible plants have now been identified. For instance, anticancer effect of Allium vegetables including garlic is attributed to organosulfur compounds (e.g., diallyl trisulfide). Interestingly, unlike cancer chemotherapy drugs, many bioactive food components selectively target cancer cells. Molecular basis for selectivity of anticancer bioactive food components towards cancer cells remains elusive, but these agents appear promiscuous and target multiple signal transduction pathways to inhibit cancer cell growth in vitro and in vivo. Despite convincing observational and experimental evidence, however, limited effort has been directed towards clinical investigations to determine efficacy of bioactive food components for prevention of human cancers. This article reviews current knowledge on cancer chemopreventive effects of a few highly promising dietary constituents, including garlic-derived organosulfides, berry compounds, and cruciferous vegetable-derived isothiocyanates, and serves to illustrate complexity of the signal transduction mechanisms in cancer chemoprevention by these promising bioactive food components.     

Prevention of chemically induced mammary tumorigenesis by daidzein in pre-pubertal rats: the role of peroxidative damage and antioxidative enzymes

Isoflavones are biologically active plant derived compounds that have several health promoting effects. In the present study hitherto unknown effects of one of the well known isoflavonoids, daidzein, has been evaluated on its chemo-preventive action against breast cancers in pre-pubertal rats. Either daidzein (500 μg/g bwt) or vehicle, dimethyl sulphoxide (DMSO), was administered at 16th, 18th, and 20th day post-partum and the chemopreventive efficacy was evaluated in dimethylbenz[a]nthracene (DMBA) induced Sprague-Dawley rats, at 50th day. To elucidate the mechanism of action, the antioxidative status was also examined in the liver and mammary gland of prebubertal rats using two different doses of daidzein (0.5 mg/kg bwt and 50 mg/kg bwt, p.o.) for 10 days. The specific activity of antioxidant enzymes as well as reduced glutathione (GSH) level and peroxidative damage were evaluated spectrophotometrically, both in liver as well as in mammary gland. Animals treated with daidzein pre-pubertally, showed a significant reduction in the tumorigenesis of mammary gland up to 37.4% as compared to animals induced for tumors with DMBA. In animals treated with 50 mg/kg of daidzein, a significant increase in the specific activities of the antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione transferase (GST), DT-diaphorase (DTD), and in GSH content were observed in both liver and mammary gland. Expectedly, the specific activity of lactate dehydrogenase (LDH) and level of peroxidative damage was decreased, as compared to that of control group of animals. Our results suggest that, daidzein can be considered as a potent chemopreventive agent against mammary carcinogenesis in pre-pubertal animals, with modulation of antioxidant enzymes being one of its mechanisms of actions.     

Circulating anti-heat-shock-protein antibodies in normal pregnancy and preeclampsia

It has been previously reported that circulating anti-heat-shock-protein (Hsp) antibody levels are elevated in cardiovascular disorders. The aim of the present study was to determine circulating antihuman Hsp60, antimycobacterial Hsp65, and antihuman Hsp70 antibody levels in healthy pregnant women and preeclamptic patients and to investigate their relationship to the clinical characteristics of the study subjects, as well as to the markers of inflammation (C-reactive protein (CRP)), endothelial activation (von Willebrand factor antigen), or endothelial injury (fibronectin), oxidative stress (malondialdehyde) and to serum Hsp70 levels. Ninety-three preeclamptic patients and 127 normotensive healthy pregnant women were involved in this case control study. Serum anti-Hsp60, anti-Hsp65, anti-Hsp70, and Hsp70 levels were measured with enzyme-linked immunosorbent assay (ELISA). Serum CRP levels were determined by an autoanalyzer using the manufacturer’s kit. Plasma von Willebrand factor antigen levels were quantified by ELISA, while plasma fibronectin concentration by nephelometry. Plasma malondialdehyde levels were measured by the thiobarbituric-acid-based colorimetric assay. For statistical analyses, nonparametric methods were applied. Anti-Hsp60, anti-Hsp65, and anti-Hsp70 antibodies were detected in all of our serum samples. There were no significant differences in serum anti-Hsp60, anti-Hsp65, and anti-Hsp70 antibody levels between the control and preeclamptic groups. Serum levels of Hsp70 and CRP, as well as plasma levels of VWF antigen, fibronectin, and malondialdehyde, were significantly higher in preeclamptic patients than in normotensive healthy pregnant women. Serum anti-Hsp60 antibody levels showed significant correlations with serum anti-Hsp65 antibody levels both in the control and the preeclamptic groups (Spearman R = 0.55 and 0.59; p < 0.001, respectively). However, no other relationship was found between clinical features (maternal age, smoking status, parity, body mass index, gestational age at blood draw, systolic and diastolic blood pressure, gestational age at delivery, and fetal birth weight) and measured laboratory parameters of the study subjects and serum anti-Hsp antibody levels in either study group. In conclusion, anti-Hsp60 and anti-Hsp70 antibodies as naturally occurring autoantibodies are present in the peripheral circulation of healthy pregnant women. Nevertheless, humoral immunity against heat shock proteins was not associated with preeclampsia. Further studies are warranted to explore the role of heat shock proteins and immune reactivity to them in the immunobiology of normal pregnancy and preeclampsia.     

Role of protein kinase C in NADPH oxidase derived O2-mediated regulation of KV-LVOCC axis under U46619 induced increase in [Ca]i in pulmonary smooth muscle cells

Treatment of bovine pulmonary smooth muscle cells with the TxA₂ mimetic, U46619 stimulated [Ca²⁺]_i, which was inhibited upon pretreatment with apocynin (NADPH oxidase inhibitor). Pretreatment with cromakalim (K_V channel opener) or nifedepine (L-VOCC inhibitor) inhibited U46619 induced increase in [Ca²⁺]_i, indicating a role of K_V-LVOCC axis in this scenario. Neither cromakalim nor nifedepine inhibited U46619 induced increase in NADPH oxidase activity, suggesting that the NADPH oxidase activation is proximal to the K_V-LVOCC axis in the cells. Pretreatment with calphostin C (PKC inhibitor) markedly reduced U46619 induced increase in NADPH oxidase activity and [Ca²⁺]_i in the cells. Calphostin C pretreatment also markedly reduced p^47phox phosphorylation and translocation to the membrane and association with p^22phox, a component of Cyt.b₅₅₈ of NADPH oxidase in the membrane. Overall, PKC plays an important role in NADPH oxidase derived O₂⁻-mediated regulation of K_V-LVOCC axis leading to an increase in [Ca²⁺]_i by U46619 in the cells.     

Statistical optimization of <Emphasis Type="Italic">α</Emphasis>-amylase production by <Emphasis Type="Italic">Bacillus brevis</Emphasis> MTCC 7521 in solid-state fermentation using cassava bagasse

Production of α-amylase under solid-state fermentation by Bacillus brevis MTCC 7521 has been investigated using cassava bagasse as the substrate, one of the major solid wastes released during extraction of starch from cassava (Manihot esculenta). Response surface methodology was used to evaluate the effect of the main variables, i.e. incubation period (36 h), moisture holding capacity (60%), pH (7.0) and temperature (60°C) on enzyme production by applying a full factorial central composite design. The maximum hydrolysis of soluble starch (85%) and cassava starch (75%) was obtained with the application of 4 mL (≈ 14,752 units) of B. brevis crude enzyme after 5 h of incubation.