Results:The daily dietary calcium intake of both the urban and rural populations was low compared with the recommended dietary allowances issued by the Indian Council of Medical Research. Dietary calcium and phosphorous were significantly lower in rural adults than in urban adults (P 0.0001). The dietary phytate-to-calcium ratio was higher in rural subjects than in urban subjects (P 0.0001). The 25(OH)D concentrations of the rural subjects were higher than those of urban subjects (P 0.001), both men and women. In the rural subjects, 25(OH)D-deficient (20 ng/mL), -insufficient (20 -30 ng/mL), and -sufficient (30 ng/mL) states were observed in 44%, 39.5%, and 16.5% of the men and 70%, 29%, and 1% of the women, respectively. In the urban subjects, 25(OH)Ddeficient, -insufficient, and -sufficient states were observed in 62%, 26%, and 12% of the men and 75%, 19%, and 6% of the women, respectively. Conclusions: Low dietary calcium intake and 25(OH)D concentrations were associated with deleterious effects on bone mineral homeostasis. Prospective longitudinal studies are required to assess the effect on bone mineral density, a surrogate marker for fracture risk and fracture rates.Am J Clin Nutr 2007;85:1062-7.

Background: Little if any cutaneous production of vitamin D3 occurs at latitudes above and below 35° N and 35° S during the winter months. It was postulated that those residing in tropics synthesize enough vitamin D3 year round. Several studies have documented the effect of latitude, season and time of the day on the cutaneous production of vitamin D3 in an ampoule model. Studies from India have shown high prevalence of vitamin D deficiency despite abundant sunshine. Methods: We studied the influence of season and time of the day on synthesis of previtamin D3 in an ampoule model in Tirupati, (latitude 13.40° N and longitude 77.2° E) south India, between May 2007 to August 2008. Sealed borosilicate glass ampoules containing 50 μg of 7-DHC in 1 ml of methanol were exposed to sunlight hourly from 8 a.m. until 4 p.m. The percent conversion of 7-DHC to previtamin D3 and its photoproducts and the percent of previtamin D3 and vitamin D3 formed was estimated and related to solar zenith angle. Results: The percent conversion of 7-DHC to previtamin D3 and its photoproducts and formation of previtamin D3 and vitamin D3 was maximal between 11 a.m. to 2 p.m. of the day during the entire year (median 11.5% and 10.2% respectively at 12.30 p.m.). Conclusions: Therefore at this latitude exposure to sunlight between the hours of 11 a.m. and 2 p.m. will promote vitamin D production in the skin year round.

Yeast strains are commonly associated with sugar rich environments. Various fruit samples were selected as source for isolating yeast cells. The isolated cultures were identified at Genus level by colony morphology, biochemical characteristics and cell morphological characters. An attempt has been made to check the viability of yeast cells under different concentrations of ethanol. Ethanol tolerance of each strain was studied by allowing the yeast to grow in liquid YEPD (Yeast Extract Peptone Dextrose) medium having different concentrations of ethanol. A total of fifteen yeast strains isolated from different samples were used for the study. Seven strains of Saccharomyces cerevisiae obtained from different fruit sources were screened for ethanol tolerance. The results obtained in this study show a range of tolerance levels between 7%-12% in all the stains. Further, the cluster analysis based on 22 RAPD (Random Amplified polymorphic DNA) bands revealed polymorphisms in these seven Saccharomyces strains.

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The Krebs cycle dictates oxidative and reductive conditions in Staphylococcus aureus and is mainly regulated by isocitrate dehydrogenase (IDH) which plays pivotal role in the growth and pathogenesis of the bacteria. In the present study, IDH gene from S. aureus ATCC12600 was cloned in the Sma I site of pQE 30 vector; the resultant clone was named as UVIDH1. The insert in the clone was sequenced (accession number HM067707), and the sequence showed complete homology with IDH sequence of other S. aureus strains reported in the database indicating presence of single enzyme in S. aureus, and considerable sequence homology with other bacteria was observed; however, only 24% homology was found with NADP-dependent human IDH. Phylogenetically, the S. aureus IDH showed close identity with Bacillus subtilis and high degree of variability with other bacteria and human IDH. The expression of IDH in the clone UVIDH1 was induced with 1 mM IPTG, and the recombinant IDH was purified by passing through nickel metal chelate column; the purified recombinant IDH showed a single band in SDS-PAGE with a molecular weight of 40 kDa; K(m) and V(max) for isocitrate are 8.2 ± 0.28 and 525 ± 25 μM NADPH/mg/min, respectively, and for cofactor NADP 67.5 ± 2.82 μM and V(max) 50.5 ± 2.12 μM NADPH/mg/min.

Isocitrate dehydrogenase (IDH) gene from Staphylococcus aureus ATCC12600 was cloned, sequenced and characterized (HM067707). PknB site was observed in the active site of IDH; thus, it was predicted as IDH may be regulated by phosphorylation. Therefore, in this study, PknB, alkaline phosphatase III (SAOV 2675) and IDH genes (JN695616, JN645811 and HM067707) of S. aureus ATCC12600 were over expressed from clones PV 1, UVPALP-3 and UVIDH 1. On passing the cytosloic fractions through nickel metal chelate column, pure enzymes were obtained. Phosphorylation of pure IDH by PknB resulted in the complete loss of activity and was restored upon dephosphorylation with SAOV 2675 which indicated that phosphorylation and dephosphorylation regulate IDH activity in S. aureus. Further, when S. aureus ATCC12600 was grown in BHI broth, decreased IDH activity and increased biofilm units were observed; therefore, this regulation of IDH alters redox status in this pathogen favouring biofilm formation.

Staphylococcus aureus, a natural inhabitant of nasopharyngeal tract, survives mainly as biofilms. Previously we have observed that S. aureus ATCC 12600 grown under anaerobic conditions exhibited high rate of biofilm formation and l-lactate dehydrogenase activity. Thus, the concentration of pyruvate plays a critical role in S. aureus, which is primarily catalyzed by pyruvate kinase (PK). Analyses of the PK gene sequence (JN645815) revealed presence of PknB site in PK gene indicating that phosphorylation may be influencing the functioning of PK. To establish this hypothesis the pure enzymes of S. aureus ATCC 12600 were obtained by expressing these genes in PK 1 and PV 1 (JN695616) clones and passing the cytosolic fractions through nickel metal chelate column. The molecular weights of pure recombinant PK and PknB are 63 and 73 kDa, respectively. The enzyme kinetics of pure PK showed KM of 0.69 ± 0.02 µM, while the KM of PknB for stpks (stpks = NLCNIPCSALLSSDITASVNCAK) substrate was 0.720 ± 0.08 mM and 0.380 ± 0.07 mM for autophosphorylation. The phosphorylated PK exhibited 40 % reduced activity (PK = 0.2 ± 0.015 μM NADH/min/ml to P-PK = 0.12 ± 0.01 μM NADH/min/ml). Elevated synthesis of pyruvate kinase was observed in S. aureus ATCC 12600 grown in anaerobic conditions suggesting that the formed pyruvate is more utilized in the synthesis phase, supporting increased rate of biofilm formation.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-014-0248-3) contains supplementary material, which is available to authorized users.

Hexokinases (HKs) are the enzymes that catalyses the ATP dependent phosphorylation of Hexose sugars to Hexose-6-Phosphate (Hex-6-P). There exist four different forms of HKs namely HK-I, HK-II, HK-III and HK-IV and all of them share a common ATP binding site core surrounded by more variable sequence that determine substrate affinities. Although they share a common binding site but they differ in their kinetic functions, hence the present study is aimed to analyze the binding mode of ATP. The analysis revealed that the four ATP binding domains are showing 13 identical, 7 similar and 6 dissimilar residues with similar structural conformation. Molecular docking of ATP into the kinase domains using Molecular Operating Environment (MOE) soft ware tool clearly showed the variation in the binding mode of ATP with variable docking scores. This probably explains the variable phosphorylation rates among hexokinases family.