Conformation of the closed channel state of colicin A in proteoliposomes: an umbrella model

Colicin A (ColA) is a water-soluble toxin that forms a voltage-gated channel in the cytoplasmic membrane of Escherichia coli. Until now, two models were proposed for the closed channel state: the umbrella model and the penknife model. Mutants of ColA, each containing a single cysteine, were labeled with a nitroxide spin label, reconstituted into liposomes, and studied by electron paramagnetic resonance (EPR) spectroscopy to study the membrane-bound closed channel state. The spin-labeled ColA variants in solution and in liposomes of native E. coli lipid composition were analyzed in terms of the mobility of the nitroxide, its accessibility to paramagnetic reagents, and the polarity of its microenvironment. The EPR data determined for the soluble ColA pore-forming domain are in agreement with its crystal structure. Moreover, the EPR results show that ColA has a conformation in liposomes different from its water-soluble conformation. Residues that belong to helices H8 and H9 are significantly accessible for O₂ but not for nickel-ethylene diamine diacetic acid, indicating their location inside the membrane. In addition, the polarity values determined from the hyperfine tensor component A_zz of residues 176, 181, and 183 (H9) indicate the location of these residues close to the center of the lipid bilayer, supporting a transmembrane orientation of the hydrophobic hairpin. Furthermore, the accessibility and polarity data suggest that the spin-labeled side chains of the amphipathic helices (H1-H7 and H10) are located at the membrane-water interface. Evidence that the conformation of the closed channel state in artificial liposomes depends on lipid composition is given. The EPR results for ColA reconstituted into liposomes of E. coli lipids support the umbrella model for the closed channel state.     

Importance of the cytochrome pathway of mitochondrial electron transport over the alternative pathway during the Kok effect in leaf discs of pea (Pisum sativum)

The Kok effect refers to the progressive light-induced inhibition of dark respiration at low light intensities, which saturates around the light compensation point. This appears as a sudden break around the light compensation point in the plot of photosynthesis versus light intensity. The magnitude of the break can be considered as a measure of the Kok effect. In the present work, the importance of different components of dark respiration during the Kok effect was investigated by using low concentrations of mitochondrial inhibitors in leaf discs of pea ( Pisum sativum L. cv. Azad P1). The effects of glucose (stimulates respiration) and 0.8 M sorbitol (imposes osmotic stress and inhibits photosynthesis) were also studied for comparison. The magnitude of the break decreased significantly in the presence of antimycin A or oligomycin (inhibitors of cytochrome pathway of mitochondrial electron transport and ATP synthase, respectively). In contrast, there was no significant change with salicylhydroxamic acid (SHAM; an inhibitor of alternative pathway of mitochondrial electron transport). The magnitude of the break increased significantly with glucose, and decreased on exposure to osmotic stress. Our results suggest that the Kok effect (inhibition of dark respiration in light) is modulated by inhibitors of cytochrome pathway and ATP synthesis, but not that of the alternative pathway.     

Cytogenetic effects in workers occupationally exposed to tobacco dust

Tobacco dust mainly contains nitrosamines, which are readily absorbed by the body tissues like skin, respiratory epithelium, and mucous membrane of mouth, nose and intestines. Exposure to tobacco dust is known to affect the respiratory tracts in humans. In the present study, cytogenetic effects of exposure to tobacco dust are evaluated in 154 male tobacco factory workers and 138 age and sex matched controls by analysing chromosomal aberrations in their peripheral blood lymphocytes. The workers were in the age group of 20-55 years and were employed in the tobacco processing factory for 1-32 years. Heparinised blood samples were collected from workers and control subjects and lymphocyte cultures were carried out by using standard technique. Slides were prepared and 150 metaphases were screened for each sample for various structural and numerical types of abnormalities. A statistically significant increase was observed in the frequencies of chromosomal aberrations in non-smoking and smoking exposed groups when compared to the respective controls. An increase in the frequencies of chromosomal aberrations was also observed with increase in years of service in the exposed subjects.     

Synthesis,Molecular Properties Prediction and Antimicrobial Activity of Imidazolyl Schiff Bases,Triazoles and Azetidinones

Benzylidenehydrazinyl imidazoles ( 3 ) are prepared from 2‐hydrazinyl imidazoles ( 2 ) on treatment with hydrazine. The imine functionality in 3 is utilized to develop 5′‐aryl‐N‐(4‐aryl‐1H‐imidazol‐2‐yl)‐1H‐1,2,3‐triazol‐1‐amines ( 5 ) by 1,3‐dipolar cycloaddition of diazomethane followed by aromatization with I₂ in DMSO. Compounds 3 are also explored to prepare 4′‐aryl‐1‐(4‐aryl‐1H‐imidazol‐2‐ylamino)‐3‐chloroazetidin‐2‐ones ( 6 ) on treatment with chloroacetyl chloride. The Molinspiration calculations predicted that 3 , 5 and 6 have molecular hydrophobicity, conformational flexibility, good intestinal absorption and bioactivity scores. The chloro, bromo and nitro substituted imidazolyl azetidinones ( 6c , 6d , 6f ) and nitro substituted imidazolyl triazole ( 5f ) exhibited excellent antibacterial activity on B. subtilis, whereas chloro and nitro substituted imidazolyl triazoles ( 5c , 5f ) showed prominent antifungal activity on A. niger.     

Biosynthesis of polymyxin by Bacillus polymyxa. II. On the nature and interaction of the multienzyme complex with the end product polymyxin

The interaction of polymyxin with the producer organism Bacillus polymyxa has been shown to be at the level of membranes, resulting in an enhancement of the activities of its own biosynthetic enzymes. This enhancement has been shown to be due to the solubilization of the membrane-associated multienzyme complex by polymyxin in a specific manner. The relevance of this physiological feature was also indicated by the appearance of the soluble multienzyme complex activity only in cells, which synthesize maximal amounts of polymyxin. Purification of the polymyxin released multienzyme complex from the membranes and the soluble form of the complex from the stationary phase cells has revealed several similarities between them. Both contain two major fractions of the molecular weights of 300,000 and 170,000, harboring all the polymyxin component amino acid-activating enzymes. Their multisubunit nature was established by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Using mutants blocked in sporulation and/or antibiotic synthesis, it was shown that the interaction of polymyxin with the producer organism was inoperative when antibiotic production was curtailed. This interaction has been suggested as one of the early sporulation-specific phenemenon.     

Chemopreventive effect of piperine on mitochondrial TCA cycle and phase-I and glutathione-metabolizing enzymes in benzo(a)pyrene induced lung carcinogenesis in Swiss albino mice

Piperine is a major component of black (Piper nigrum Linn) and long pepper (Piper longum Linn) used widely in various systems of traditional medicine. We have evaluated the effect of piperine on mitochondrial tricarboxylic acid cycle and phase I and glutathione-metabolizing enzymes in Benzo(a)pyrene induced experimental lung carcinogenesis in swiss albino mice. Lung cancer bearing mice showed a significant decrease in the activities of mitochondrial enzymes-isocitrate dehydrogenase (ICDH), -ketoglutarate dehydrogenase (KDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and significantly increased NADPH-Cytochorome reductase (NADPH-C reductase), cytochrome P450 (cyt-p450) and cytochrome b5(cyt-b5). The activities of glutathione-metabolizing enzymes glutathione peroxidase(GPx), glutathione reductase (GR) and glucose-6-phospho dehydrogenase(G6PDH) were significantly lowered in lung-cancer bearing mice when compared with control mice. Piperine supplementation to tumour-induced animals significantly lowered the phase-I enzymes (NADPH-C reductase, cyt-p450 and cyt-b5)) and there was a rise in glutathione-metabolizing enzymes (GPx, GR and G6PDH), which indicated an antitumour and anti-cancer effect. Comparison of normal control mice and mice administered piperine only as drug control showed no significant variations in enzyme activities. Piprine administration to benzo(a)pyrene induced animals significantly increased the activities of mitochondrial enzymes, thereby suggesting its role in mitochondrial energy production.     

Maternal dietary folate and/or vitamin B12 restrictions alter body composition (adiposity) and lipid metabolism in Wistar rat offspring

Maternal vitamin deficiencies are associated with low birth weight and increased perinatal morbidity and mortality. We hypothesize that maternal folate and/or vitamin B₁₂ restrictions alter body composition and fat metabolism in the offspring. Female weaning Wistar rats received ad libitum for 12 weeks a control diet (American Institute of Nutrition-76A) or the same with restriction of folate, vitamin B₁₂ or both (dual deficient) and, after confirming vitamin deficiency, were mated with control males. The pregnant/lactating mothers and their offspring received their respective diets throughout. Biochemical and body composition parameters were determined in mothers before mating and in offspring at 3, 6, 9 and 12 months of age. Vitamin restriction increased body weight and fat and altered lipid profile in female Wistar rats, albeit differences were significant with only B₁₂ restriction. Offspring born to vitamin-B₁₂-restricted dams had lower birth weight, while offspring of all vitamin-restricted dams weighed higher at/from weaning. They had higher body fat (specially visceral fat) from 3 months and were dyslipidemic at 12 months, when they had high circulating and adipose tissue levels of tumor necrosis factor α, leptin and interleukin 6 and low levels of adiponectin and interleukin 1β. Vitamin-restricted offspring had higher activities of hepatic fatty acid synthase and acetyl-CoA-carboxylase and higher plasma cortisol levels. In conclusion, maternal and peri-/postnatal folate and/or vitamin B₁₂ restriction increased visceral adiposity (due to increased corticosteroid stress), altered lipid metabolism in rat offspring perhaps by modulating adipocyte function and may thus predispose them to high morbidity later.     

Influence of monosaccharides on the fibrillogenesis of type I collagen

The in vitro self-assembly of Type I rat tail tendon collagen in the presence of monosaccharides was studied turbidimetrically. D(+)glucose and D(+)mannose delayed fibril formation significantly while D(-)fructose showed no variation. The effect of glucose was concentration dependent. Binding of glucose during fibrillogenesis was shown by the incorporation of [14C]glucose and also by SDS-polyacrylamide slab gel electrophoresis of the product. The results indicate that in vitro nonenzymatic glycosylation can take place in the short duration required for fibril formation.     

Therapeutic effect of paclitaxel and propolis on lipid peroxidation and antioxidant system in 7,12 dimethyl benz(a)anthracene-induced breast cancer in female Sprague Dawley rats

Breast cancer is one of the most common cancers in women of developed and developing countries. The optimum management of which requires a multidisciplinary approach including the use of certain biochemical and molecular markers. The effect of propolis along with paclitaxel on 7,12 dimethyl benz(a)anthracene (DMBA) induced experimental breast cancer was investigated in female Sprague Dawley rats. Female Sprague Dawley rats were divided into five groups of six animals each. Group I served as normal control animal. Group II animals received DMBA (20 mg in 0.5 ml sunflower oil and 0.5 ml of saline) i.p. to develop mammary tumor by the end of 90 days. Group III were breast cancer animals treated with 33 mg paclitaxel/kg body weight (bw) weekly once for 4 weeks. Group IV were breast cancer-bearing animals treated with 50 mg propolis/kg bw for 30 days. Group V were breast cancer-bearing animals treated with both paclitaxel and propolis as mentioned above. Administration of paclitaxel and propolis effectively suppressed breast cancer, which is revealed by the decrease in the extent of lipid peroxidation (LPO) with concomitant increase in the activities of enzymic antioxidants (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)) and non-enzymic antioxidants (reduced glutathione (GSH), Vitamin C and Vitamin E) levels when compared to breast cancer-bearing animals treated with either paclitaxel or propolis alone. From our results, we conclude that propolis is a potent antioxidant and, when given in combination with paclitaxel, offers maximum protection against DMBA induced mammary carcinogenesis.