Molecular cloning and characterization of Brugia malayi hexokinase

5' EST from filarial gene database has been subjected to 3' rapid amplification of cDNA ends (RACE), semi-nested PCR and PCR to obtain full-length cDNA of Brugia malayi. Full-length hexokinase gene was obtained from cDNA using gene specific primers. The elicited PCR product was cloned, sequenced and expressed as an active enzyme in Escherichia coli. Sequence analysis of B. malayi hexokinase (BmHk) revealed 59% identity with nematode Caenorhabditis elegans but low similarity with all other available hexokinases including human. BmHk, an apparent tetramer with subunit molecular mass of 72 kDa, was able to phosphorylate glucose, fructose, mannose, maltose and galactose. The Km values for glucose, fructose and ATP were found to be 0.035+/-0.005, 75+/-0.3 and 1.09+/-0.5 mM respectively. BmHk was strongly inhibited by ADP, glucosamine, N-acetyl glucosamine and mannoheptulose. The recombinant enzyme was found to be activated by glucose-6-phosphate. ADP exhibited noncompetitive inhibition with the substrate glucose (Ki=0.55 mM) while, mixed type of inhibition was observed with inorganic pyrophosphate (PPi) when ATP was used as substrate (Ki=9.92 microM). The enzyme activity is highly dependent on maintenance of free sulfhydryl groups. CD analysis indicated that BmHk is composed of 37% alpha-helices and 26% beta-sheets. The observed differences in kinetic properties of BmHk as compared to host enzyme may facilitate designing of specific inhibitors against BmHk.     

Finger millet (Eleucine coracana) flour as a vehicle for fortification with zinc

Millets, being less expensive compared to cereals and the staple for the poorer sections of population, could be the choice for fortification with micronutrients such as zinc. In view of this, finger millet, widely grown and commonly consumed in southern India, was explored as a vehicle for fortification with zinc in this investigation. Finger millet flour fortified with either zinc oxide or zinc stearate so as to provide 50 mg zinc per kg flour, was specifically examined for the bioaccessibility of the fortified mineral, as measured by in vitro simulated gastrointestinal digestion procedure and storage stability. Addition of the zinc salts increased the bioaccessible zinc content by 1.5–3 times that of the unfortified flour. Inclusion of EDTA along with the fortified salt significantly enhanced the bioaccessibility of zinc from the fortified flours, the increase being three-fold. Inclusion of citric acid along with the zinc salt and EDTA during fortification did not have any additional beneficial effect on zinc bioaccessiblity. Moisture and free fatty acid contents of the stored fortified flours indicated the keeping quality of the same, up to 60 days. Both zinc oxide and zinc stearate were equally effective as fortificants, when used in combination with EDTA as a co-fortificant. The preparation of either roti or dumpling from the fortified flours stored up to 60 days did not result in any significant compromise in the bioaccessible zinc content. Thus, the present study has revealed that finger millet flour can effectively be used as a vehicle for zinc fortification to derive additional amounts of bioaccessible zinc, with reasonably good storage stability, to combat zinc deficiency.     

Use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction

A general method is presented here for the determination of the Km, kcat, and kcat/Km of fluorescence resonance energy transfer (FRET) substrates using a fluorescence plate reader. A simple empirical method for correcting for the inner filter effect is shown to enable accurate and undistorted measurements of these very important kinetic parameters. Inner filter effect corrected rates of hydrolysis of a FRET peptide substrate by hepatitis C virus (HCV) NS3 protease at various substrate concentrations enabled measurement of a Km value of 4.4 +/- 0.3 microM and kcat/Km value of 96,500 +/- 5800 M-1 s-1. These values are very close to the HPLC-determined Km value of 4.6 +/- 0.7 microM and kcat/Km value of 92,600 +/- 14,000 M-1 s-1. We demonstrate that the inner filter effect correction of microtiter plate reader velocities enables rapid measurement of Ki and Ki' values and kinetic inhibition mechanisms for HCV NS3 protease inhibitors.     

Production of L-Phenylacetyl Carbinol by Immobilized Cells of Saccharomyces Cerevisiae

Conversion of benzaldehyde to L-phenylacetyl carbinol (L-PAC) was achieved with immobilized, growing cells of Saccharomyces cerevisiae in different reactors. Product formation increased (31%) with the subsequent initial reuses of the entrapped cells. Biomass production and PAC formation depleted (40 and 57%, respectively) after 4-5 continuous growth and biotransformation cycles. With the regeneration of the biocatalysts, catalytic activity of the cells was resumed. The highest yields were in a stirred tank reactor (29 g PAC) from 77 g benzeldehyde with 14 repeated uses of entrapped cells.     

Synthesis of bicyclic biaryls as glucose-6-phosphatase inhibitors

Biaryls, 7-naphthyl-5-s-amino-2,3-dihydrobenzo[b]thiophene-4-carbonitriles (3a-e), 8-(1-naphthyl)-6-s-amino-isothiochroman-5-carbonitriles (6a-d), 4-(1-naphthyl)-2-s-aminobezocycloalkene-1-carbonitriles (6e-j), 8-naphthyl-6-s-amino-2-ethyl-1,2,3,4-tetrahydro-isoquinoline-5-carbonitrle (6k-n), 1-naphthyl-3-s-amino-10H-9-thia-phenantherene-4-carbonitriles (8a-e) and 1-(1-naphthyl)-3-s-amino-9,10-dihydrophenantherene-4-carbonitriles (8f-i) have been prepared through carbanion induced ring transformation reactions of 6-naphthyl-3-cyano-4-s-amino-2H-pyran-2-ones (1) from respective ketones (2, 5, and 7). These compounds have been evaluated for their glucose-6-phosphatase inhibitory activity and only 6a, c, j, m, c, d, h displayed significant inhibition of the glucose-6-phosphatase.     

Effect of ascorbic acid on stimulatory status of activated mouse peritoneal phagocytes

Mouse peritoneal macrophages (MPM) when elicited by the antioxidant ascorbic acid have been found to be significantly stimulatory, exhibiting marked alteration at the cellular and enzyme levels. Alterations recorded were as follows--cellular yield per mouse, their protein content, lysosomal acid hydrolase levels and capability to phagocyte, all were significantly enhanced. The new stimulant was observed to produce no synergistic action on MPM when thioglycollate, BCG or endotoxin along with the same stimulated the latter. Levels of antioxidants like ascorbic acid and glutathione were found to be enhanced in elicited macrophages whereas superoxide dismutase levels varied when the three above stimulators were administered. However, the ascorbic acid elicited cells showed an increase in glutathione levels and a decrease in SOD levels but no change in total intracellular ascorbic acid levels. Further, though ascorbic acid interaction enhanced the phagocytic capability of MPM as compared to resident cells, no significant boosting of phagocytic process could be observed when each of three stimulators coupled with ascorbic acid was used for macrophage elicitation.     

Fine root biomass and tree species effects on potential N mineralization in afforested sodic soils

The study was carried out under three types of plantation forest of 40 years, growing on infertile sodic soils, poor in organic matter and N content, of Indogangetic alluvium at Lucknow (26°45 N; 80°53 E). Fine root biomass estimated under three forests did not differ much with season, or with species (106–113 g m^-2) but varied with soil depth to 0.45 m. The proportion of very fine roots (<0.5 mm) increased with soil depth. Available N in soil was greatest under mixed forest followed by Eucalyptus camaldulensis and Acacia nilotica planted soils. N was maximum in summer season and decreased with soil depth. Nitrogen mineralization during anerobic incubation of 14 days could not be differentiated by tree species, but the monsoon season favoured the process and winter season retarded it. Mineralization decreased with soil depth corresponding to fine roots. There was a reduction in bulk density of soil, pH and EC in forested soil compared to a similar but non forested soil, whereas, organic C and total N increased in forested soils. N mineralization was found to be affected significantly with the fine root biomass and available N content in the soils, whereas negative relations of mineralized N with pH and EC were noticed, though these were not significantly different in this study.     

Studying vertical dependence to improve NFS performance in wireless networks

Wireless networks experience a high level of errors and losses. These physical layer characteristics have an impact on the performance of the higher layers. In addition, the performance of each protocol layer is contingent on the behavior of the other layers. Vertical dependency is a term which describes this inter-dependence between layers. In the wireless and mobile environment, the effects of vertical dependence are particularly pronounced due to the dynamic nature of the environment and due to the fact that traditional assumptions about protocol layer interactions do not always hold. In this paper, we consider the vertical dependencies between various layers in the protocol stack, studying the performance of the Network File System under various error models and improvement techniques. Our experimental results demonstrate the dependency of the application performance on the details of the error characteristics and other protocol layers. After studying the vertical dependencies, we improve NFS performance by implementing changes to the application level reliability mechanisms. Understanding of the vertical dependencies enables development of effective methods for performance enhancement and efficient reaction to errors and changes on the wireless media. This revised version was published online in August 2006 with corrections to the Cover Date.     

Saccharomyces cerevisiae Hop1 protein zinc finger motif binds to the Holliday junction and distorts the DNA structure: implications for holliday junction migration

Saccharomyces cerevisiae HOP1, which encodes a component of synaptonemal complex, plays an important role in crossing over between homologues. Hop1p contains a zinc finger motif, and substitution of a conserved Cys371 by Ser rendered the hop1 mutant allele defective in sporulation and meiosis. However, the molecular mechanism underlying the function of Hop1 zinc finger motif (ZnF) remains obscure. Here we show that wild-type Hop1 ZnF binds significantly better to the Holliday junction compared with other recombination intermediates. Consequently, the salt titration midpoint for dissociation of the Holliday junction-ZnF complex was higher than the complexes containing flush-ended linear or tailed duplex DNA. Although DNase I footprinting showed that Hop1 ZnF binds to each of the four arms of the junction, KMnO4 probing and 2-aminopurine fluorescence emission data disclosed that it distorts the DNA structure along a pair of symmetrical arms. Molecular modeling studies show that Hop1 ZnF forms a unique zinc-binding fold, reminiscent of the basic helix-loop-helix motif. In the presence of Zn2+, docking studies show that alpha helix 1, which is replete with basic amino acid residues, makes stabilizing contacts with the sugar-phosphate backbone. Structural comparison revealed a striking similarity between RecG wedge domain and Hop1 ZnF motif. We propose that Hop1 ZnF motif plays a key role in the physical monitoring of recombination intermediates and branch migration of the Holliday junction.