Characterization of Drosophila nitric oxide synthase: a biochemical study

The heme and flavin-binding domains of Drosophila nitric oxide synthase (DNOS) were expressed in Escherichia coli using the expression vector pCW. The denatured molecular mass of the expressed protein was 152kDa along with a proteolytically cleaved product of 121kDa. The DNOS heme protein exhibited very low Ca(2+)/calmodulin-dependent NO synthase activity. The trypsin digestion patterns were different from nNOS. The full-length DNOS protein had high degree of stability against trypsin. The activity assay of trypsin-digested protein confirmed the same result. Urea dissociation profile of DNOS full-length protein showed that the reductase domain activity was much more susceptible towards urea than the oxygenase domain activity. Urea gradient gel of DNOS full-length protein established distinct transition of dissociation and unfolding in the range 3-4M urea. Reductase domain activity of full-length DNOS protein against external electron acceptors like cytochrome c indicated slow electron transfer from FMN. The bacterial expression of DNOS full-length protein represents an important development in structure-function studies of this enzyme and comparison with other mammalian NOS enzymes which is evolutionary significant.     

pH Alters PEG-Mediated Fusion of Phosphatidylethanolamine-Containing Vesicles

Here, we examine the different mechanisms of poly(ethylene glycol)-mediated fusion of small unilamellar vesicles composed of dioleoylphosphatidylcholine/dioleoylphosphatidylethanolamine (DOPE)/sphingomyelin/cholesterol in a molar ratio of 35:30:15:20 at pH 7.4 versus pH 5. In doing so, we test the hypothesis that fusion of this lipid mixture should be influenced by differences in hydration of DOPE at these two pH values. An examination of the literature reveals that DOPE should be less hydrated at pH 5 (where influenza virus particles fuse with endosome membranes) than at pH 7.4 (where synaptic vesicles or HIV virus particles fuse with plasma membrane). Ensemble kinetic experiments revealed substantial differences in fusion of this plasma membrane mimetic system at these two pH values. The most dramatic difference was the observation of two intermediates at pH 5 but loss of one of these fusion intermediates at pH 7.4. Analysis of data collected at several temperatures also revealed that formation of the initial fusion intermediate (stalk) was favored at pH 7.4 due to increased activation entropy. Our observations support the hypothesis that the different negative intrinsic curvature of DOPE can account for different fusion paths and activation thermodynamics in steps of the fusion process at these two pH values. Finally, the effects of 2 mol % hexadecane on fusion at both pH values seemed to have similar origins for step 1 (promotion of acyl chain or hydrocarbon excursion into interbilayer space) and step 3 (reduction of interstice energy leading to expansion to a critical stalk radius). Different hexadecane effects on activation thermodynamics at these two pH values can also be related to altered DOPE hydration. The results support our kinetic model for fusion and offer insight into the critical role of phosphatidylethanolamine in fusion.     

Clinical studies on the effect of Neem (Azadirachta indica) bark extract on gastric secretion and gastroduodenal ulcer

We have shown earlier that Neem (Azadirachta indica) bark aqueous extract has potent antisecretory and antiulcer effects in animal models and has no significant adverse effect (Bandyopadhyay et al., Life Sciences, 71, 2845-2865, 2002). The objective of the present study was to investigate whether Neem bark extract had similar antisecretory and antiulcer effects in human subjects. For this purpose, a group of patients suffering from acid-related problems and gastroduodenal ulcers were orally treated with the aqueous extract of Neem bark. The lyophilised powder of the extract when administered for 10 days at the dose of 30 mg twice daily caused a significant (p < 0.002) decrease (77%) in gastric acid secretion. The volume of gastric secretion and its pepsin activity were also inhibited by 63% and 50%, respectively. Some important blood parameters for organ toxicity such as sugar, urea, creatinine, serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase, albumin, globulin, hemoglobin levels and erythrocyte sedimentation rate remained close to the control values. The bark extract when taken at the dose of 30-60 mg twice daily for 10 weeks almost completely healed the duodenal ulcers monitored by barium meal X-ray or by endoscopy. One case of esophageal ulcer (gastroesophageal reflux disease) and one case of gastric ulcer also healed completely when treated at the dose of 30 mg twice daily for 6 weeks. The levels of various blood parameters for organ toxicity after Neem treatment at the doses mentioned above remained more or less close to the normal values suggesting no significant adverse effects. Neem bark extract thus has therapeutic potential for controlling gastric hypersecretion and gastroesophageal and gastroduodenal ulcers.     

In vitro effect of lorazepam on Mg2+ and (Na+,K+) ATPases of human foetal brain

Lorazepam (LZ), a benzodiazepine group of drug, inhibits Mg2+ and (Na+,K+) ATPases (EC 3.6.1.3) activity of human foetal and adult brain. The inhibitory effect neither varied with respect to the region (i.e. cerebrum and cerebellum) nor with the age of the foetus. The inhibition of ATPases activity indicates that the neuronal transmission processes, may be affected and raises the possibility of developmental disturbances.     

Elipsa is an early determinant of ciliogenesis that links the IFT particle to membrane-associated small GTPase Rab8

The formation and function of cilia involves the movement of intraflagellar transport (IFT) particles underneath the ciliary membrane, along axonemal microtubules. Although this process has been studied extensively, its molecular basis remains incompletely understood. For example, it is unknown how the IFT particle interacts with transmembrane proteins. To study the IFT particle further, we examined elipsa, a locus characterized by mutations that cause particularly early ciliogenesis defects in zebrafish. We show here that elipsa encodes a coiled-coil polypeptide that localizes to cilia. Elipsa protein binds to Ift20, a component of IFT particles, and Elipsa homologue in Caenorhabditis elegans, DYF-11, translocates in sensory cilia, similarly to the IFT particle. This indicates that Elipsa is an IFT particle polypeptide. In the context of zebrafish embryogenesis, Elipsa interacts genetically with Rabaptin5, a well-studied regulator of endocytosis, which in turn interacts with Rab8, a small GTPase, known to localize to cilia. We show that Rabaptin5 binds to both Elipsa and Rab8, suggesting that these proteins provide a bridging mechanism between the IFT particle and protein complexes that assemble at the ciliary membrane.     

Inhibition of azoxymethane-induced aberrant crypt foci in rat by diphenylmethyl selenocyanate through downregulation of COX-2 and modulation of glutathione-S-transferase and lipid peroxidation

Preventive intervention of colorectal cancer has become essential, as a major portion of the population could develop the disease at some point during their lives. An inverse association between dietary intake of selenium, an important biological trace element, and colorectal cancer risk has been observed through epidemiological and experimental studies. Inhibitory activity of an organoselenocyanate, diphenylmethylseleno-cyanate, was tested on azoxymethane (15 mg/kg body wt) induced colon carcino genesis in Sprague-Dawley rats. Pretreatment and concomitant treatment, at a dose of 2 mg/kg body wt, was carried out and the effect was observed on aberrant crypt foci, the preneoplastic lesion. To investigate the mechanism of action of the compound, lipid peroxidation level and glutathione-S-transferase (GST) activities were assessed in the liver as well as in the colon. Expression of cyclooxygenase-2 protein, inducible during colon carcinogenesis, was also analyzed in the colon. Inhibitory activity of the compound was shown by the reduced incidences of aberrant crypt foci in the treated groups (by 63.3%, p=0.00044 in the pretreated group, and by 44%, p=0.0067 in the concomitant treatment group). Significant induction of GST activities and significant reduction in lipid peroxidation level both in the liver as well as in the colon and suppression of cyclooxygenase-2 expression in the colon of the treated groups suggest that the compound could exert its preventive effect at different levels of the carcinogenic process. The preventive effect was better in the pretreatment group than in the concomitant treatment group, suggesting some added protection to the target tissue resulting from preadministration of the compound.     

Impact of CO2 Enrichment and Variable Nitrogen Supplies on Composition and Partitioning of Essential Nutrients of Wheat

This study was conducted to determine effects of nitrogen supply (75 and 150 kg(N) ha⁻¹) and CO₂ enrichment on partitioning of macro and micro nutrients in wheat (Triticum aestivum L. cv. HD-2285). Plants were grown from seedling emergence to maturity inside open top chambers under ambient CO₂ (CA, 350 ± 50 μmol mol⁻¹) and elevated CO₂ (CE, 600 ± 50 μmol mol⁻¹). Leaves, stems and roots of the same physiological age were analyzed for carbon, nitrogen, calcium, copper, iron, zinc and manganese content at 40, 60 and 90 d after germination. C, Cu, Mn and Zn content was higher in the stem, leaves and roots on dry mass basis under CE than CA. However, N and Fe contents decreased in CE grown plants. Ca content was unaffected due to CE and variable N supplies. This revised version was published online in July 2006 with corrections to the Cover Date.     

Water deficit as a regulatory switch for legume root responses

Plant roots perceive declining soil water potential as an initial signal which further triggers an array of physiological, morphological and molecular responses in the whole plant. Understanding the root responses with parallel insights on protein level changes has always been an area of interest for stress biologists. In a recent study, we reported drought stress-induced changes among certain structural and functional root proteins involved in reactive oxygen species (ROS) detoxification, primary and secondary metabolite biosynthetic pathways as well as proteins associated with cell signaling in an economically important legume crop Vigna radiata (L.) Wilczek. We also demonstrated photosynthetic gas exchange characteristics and root physiology under varying levels of water-deficit and recovery. In this report, we depict a closer analysis of the expression patterns of the identified proteins which were categorized into five major functional groups. These proteins represent a unique coherence and networking with each other as well as with the overall physiological and metabolic machinery in the plant cell.Key words: drought, legume, plant metabolism, root proteins, signaling, root response     

A mutation in the dam gene of Vibrio cholerae: 2-aminopurine sensitivity with intact GATC methylase activity

Vibrio cholerae mutants sensitive to 2-aminopurine (2AP) but with DNA adenine methylase activity similar to parental cells have been isolated. The mutant strains were sensitive to ultraviolet light (UV), methyl methane sulphonate (MMS) and 9-aminoacridine. The spontaneous mutation frequency of the mutants were not significantly affected. Attempts to isolate dam V. cholerae cells by screening 2AP sensitive cells have not been successful. All the mutant phenotypes could be suppressed by introducing the plasmid pRB103 carrying the dam gene of Escherichia coli into the mutant cells.