Spatial distribution and seasonal variation in fluoride enrichment in groundwater and its associated human health risk assessment in Telangana State,South India

Groundwater is a vital source of drinking water in Siddipet rural and urban regions of Central Telangana, South India and it is a major cause of fluoride toxicity in humans. The intake of elevated fluoride has a significant impact on human health, especially immediate problems that are seen in children's teeth. The primary aim of the study was to identify the seasonal variation in fluoride concentration and associated health risks in the residents of the study region. To assess the fluoride contamination in groundwater, a total of 158 samples were analyzed in two seasons. The mean concentrations of fluoride 1.26 mg/L and 2.21 mg/L were 1.46 and 2.8 times higher than the acceptable limit of 1.5 mg/L, before and after monsoon respectively. To estimate the human health risks due to the ingestion of elevated fluoride through drinking water, hazard quotient fluoride (HQ_Fluoride) was calculated using the United States Environmental Protection Agency method. HQ_Fluoride values were 0.44–2.44 and 0.89–4.67 for children, 0.36–2.00 and 0.73–3.82 for females, and 0.41–2.26 and 0.82–4.31 for males in pre- and post-monsoon seasons respectively, suggesting emphatically greater risk than the acceptable limits (HQ_Fluoride > 1), which generates health risks.     

Reactive cysteine residues in the oxidative dimerization and Cu2+ induced aggregation of human γD-crystallin: Implications for age-related cataract

Cysteine (Cys) residues are major causes of crystallin disulfide formation and aggregation in aging and cataractous human lenses. We recently found that disulfide linkages are highly and partly conserved in β- and γ-crystallins, respectively, in human age-related nuclear cataract and glutathione depleted LEGSKO mouse lenses, and could be mimicked by in vitro oxidation. Here we determined which Cys residues are involved in disulfide-mediated crosslinking of recombinant human γD-crystallin (hγD). In vitro diamide oxidation revealed dimer formation by SDS-PAGE and LC-MS analysis with Cys 111-111 and C111-C19 as intermolecular disulfides and Cys 111-109 as intramolecular sites. Mutation of Cys111 to alanine completely abolished dimerization. Addition of αB-crystallin was unable to protect Cys 111 from dimerization. However, Cu²⁺-induced hγD-crystallin aggregation was suppressed up to 50% and 80% by mutants C109A and C111A, respectively, as well as by total glutathionylation. In contrast to our recently published results using ICAT-labeling method, manual mining of the same database confirmed the specific involvement of Cys111 in disulfides with no free Cys111 detectable in γD-crystallin from old and cataractous human lenses. Surface accessibility studies show that Cys111 in hγD is the most exposed Cys residue (29%), explaining thereby its high propensity toward oxidation and polymerization in the aging lens.     

The activity and stability of wheat nitrate reductase in vitro

The activity and decay characteristics of nitrate reductase from wheat (Triticum aestivum) were studied in crude, partially-purified and highly-purified preparations. The decay of nitrate reductase activity in crude extracts was due to spontaneous dissociation of the enzyme and to the effects of two decay factors, one present in the 0–30% and the other in the 50–70% saturated (NH₄)₂SO₄ fraction of a crude extract. Low rates of factor-mediated NR decay in vitro were associated with high levels of NR activity in vivo.     

A Jonah-like chymotrypsin from the therapeutic maggot Lucilia sericata plays a role in wound debridement and coagulation

Lucilia sericata larvae are used in maggot debridement therapy, a traditional wound healing approach that has recently been approved for the treatment of chronic wounds. Maggot excretion products (MEP) contain many different proteases that promote disinfection, debridement and the acceleration of wound healing, e.g. by activating the host contact phase/intrinsic pathway of coagulation. In order to characterise relevant procoagulant proteases, we analysed MEP and identified a chymotrypsin-like serine protease with similarities to Jonah proteases from Drosophila melanogaster and a chymotrypsin from Lucilia cuprina. A recombinant form of the L. sericata Jonah chymotrypsin was produced in Escherichia coli. The activated enzyme (Jonah_m) had a pH optimum of 8.0 and a temperature optimum of 37 °C, based on the cleavage of the chromogenic peptide s-7388 and casein. Jonah_m reduced the clotting time of human plasma even in the absence of the endogenous protease kallikrein, factor XI or factor XII and digested the extracellular matrix proteins fibronectin, laminin and collagen IV, suggesting a potential mechanism of wound debridement. Based on these characteristics, the novel L. sericata chymotrypsin-like serine protease appears to be an ideal candidate for the development of topical drugs for wound healing applications.     

Two conformational states of the membrane-associated Bacillus thuringiensis Cry4Ba delta-endotoxin complex revealed by electron crystallography: implications for toxin-pore formation

The insecticidal nature of Cry delta-endotoxins produced by Bacillus thuringiensis is generally believed to be caused by their ability to form lytic pores in the midgut cell membrane of susceptible insect larvae. Here we have analyzed membrane-associated structures of the 65-kDa dipteran-active Cry4Ba toxin by electron crystallography. The membrane-associated toxin complex was crystallized in the presence of DMPC via detergent dialysis. Depending upon the charge of the adsorbed surface, 2D crystals of the oligomeric toxin complex have been captured in two distinct conformations. The projection maps of those crystals have been generated at 17A resolution. Both complexes appeared to be trimeric; as in one crystal form, its projection structure revealed a symmetrical pinwheel-like shape with virtually no depression in the middle of the complex. The other form revealed a propeller-like conformation displaying an obvious hole in the center region, presumably representing the toxin-induced pore. These crystallographic data thus demonstrate for the first time that the 65-kDa activated Cry4Ba toxin in association with lipid membranes could exist in at least two different trimeric conformations, conceivably implying the closed and open states of the pore.     

Molecular architecture of leishmania EF-1alpha reveals a novel site that may modulate protein translation: a possible target for drug development

Elongation factor-1alpha plays an essential role in eukaryotic protein biosynthesis. Recently, we have shown by protein structure modeling the presence of a hairpin-loop of 12 amino acids in mammalian EF-1alpha that is absent in the leishmania homologue [D. Nandan, A. Cherkasov, R. Sabouti, T. Yi, N.E. Reiner, Molecular cloning, biochemical and structural analysis of elongation factor-1 alpha from Leishmania donovani: comparison with the mammalian homologue, Biochem. Biophys. Res. Commun. 302 (2003) 646-652]. As a consequence of this deletion, an exposed region is available on the main body of leishmania EF-1alpha. Here we report the generation of an anti-EF-1alpha antibody (DN-3) which bound selectively to the exposed region of leishmania EF-1alpha, with no reactivity with human EF-1alpha. In a leishmania cell-free protein translation system, DN-3 substantially inhibited protein translation. A similar inhibitory effect was observed when a specific peptide based on the exposed region was used in the cell-free protein translation assay. The application of structure-based in silico methods to identify potential ligands to target the exposed region identified a small molecule that selectively attenuated in vitro translation using leishmania extracts. Moreover, this small molecule showed selective suppressive effect on multiplication of leishmania in culture. Taken together, these findings identify a novel, exposed region in leishmania EF-1alpha that may be involved in protein synthesis and a potential site for drug targeting.     

Levels of DNA strand breaks and superoxide in phorbol ester-treated human granulocytes

Phorbol ester treatment of granulocytes triggers release of superoxide (O) and a concomitant burst of DNA strand breaks. The relationship between the amount of O and the number of DNA breaks has not previously been explored. To quantify the relatively large amount of O generated over a 40-min period by 1 × 10⁶ granulocytes/mL, a discontinuous “10-min pulse” method employing cytochrome c was used; 140 nmol O per 1 × 10⁶ cells was detected. DNA strand breaks were quantified by fluorimetric analysis of DNA unwinding (FADU). To vary the level of O released by cells, inhibitors of the respiratory burst were used. Sodium fluoride (1–10 mM) and staurosporine (2–10 nM) both inhibited O production. In both cases, however, inhibition of strand breakage was considerably more pronounced than inhibition of O. Zinc chloride (50–200 μM) inhibited both O and DNA breaks, approximately equally. Dinophysistoxin-1 (okadaic acid) inhibited O production more effectively than it inhibited DNA breaks. O dismutes to H₂O₂, a reactive oxygen species known to cause DNA breaks. The addition of catalase to remove extracellular H₂O₂ had no effect on DNA breakage. Using pulse field gel electrophoresis, few double-stranded breaks were detected compared to the number detected by FADU, indicating that about 95% of breaks were single-stranded. The level of DNA breaks is not directly related to the amount of extracellular O or H₂O₂ in PMA-stimulated granulocytes. We conclude that either an intracellular pool of these reactive oxygen species is involved in breakage or that the metabolic inhibitors are affecting a novel strand break pathway. J. Cell. Biochem. 66:219–228, 1997. © 1997 Wiley-Liss Inc.     

Reconstitution of the Turkey erythrocyte adenylate cyclase sensitivity to l-epinephrine upon re-insertion of the Lubrol solubilized components into phospholipid vesicles

Turkey erythrocyte adenylate cyclase was activated by GppNHp and l-epinephrine to its stable, highly active form. In this form the enzyme could be solubilized by Lubrol-PX and subsequently re-inserted into phospholipid vesicles concomitantly with the removal of up to 99.3% of the Lubrol. The ability of GTP and l-epinephrine to reverse the GppNHp/epinephrine activated state was taken as a measure for the reappearance of hormone sensitivity in the reconstituted vesicles. An incomplete but significant reappearance of hormone sensitivity in the reconstituted adenylate cyclase was achieved. This hormone sensitivity was found to be stereospecific for (?)epinephrine. The ¹²⁵I-cyanopindolol binding properties of the reconstituted β-receptor depend on the nature of the detergent and the phospholipids used in the reconstitution.