Binding of nucleotides to purified coupling factor-latent ATPase from Mycobacterium phlei.

Binding studies of various nucleotides to the purified coupling factor-latent ATPase from Mycobacterium phlei have been carried out using gel filtration, equilibrium dialysis, and ultrafiltration methods. The purified latent ATPase binds 3 mol of ADP per mol of the enzyme with an apparent dissociation constant of 68 muM. Binding of nucleotides occurred in the decreasing order: ADP, epsilon-ATP, epsilon-ADP, UDP, adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), IDP, and adenosine 5'-(alpha,beta-methylene)diphosphate (AdoP(CH2)P). AMP-P(NH)P inhibits both soluble (Ki = 77 muM) and membrane-bound latent ATPase activity. However, AMP-P(NH)P does not affect oxidative phosphorylation in membrane vesicles of M. phlei. AMP-P(NH)P exhibits one binding site per molecule of the enzyme with a dissociation constant of 71 muM. After trypsin treatment of the enzyme, the binding of ADP decreases 35%, while AMP-P(NH)P binding remains unchanged. Moreover, AMP-P(NH)P binding was not displaced by ADP. Studies with sulfhydryl agents showed that, in contrast to AMP-P(NH)P, binding of at least 1 mol of ADP requires the participation of sulfhydryl groups. The results indicate that AMP-P(NH)P and ADP do not share a common binding site and that the latent ATPase enzyme has separate sites for ATP hydrolysis and ATP synthesis.     

Application of HPLC to study the kinetics of a branched bi-enzyme system consisting of hypoxanthine-guanine phosphoribosyltransferase and xanthine oxidase--an important biochemical system to evaluate the efficiency of the anticancer drug 6-mercaptopurine in ALL cell line

The thiopurine antimetabolite 6-mercaptopurine (6MP) is an important chemotherapeutic drug in the conventional treatment of childhood acute lymphoblastic leukemia (ALL). 6MP is mainly catabolized by both hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine oxidase (XOD) to form thioinosinic monophosphate (TIMP) (therapeutically active metabolite) and 6-thiouric acid (6TUA) (inactive metabolite), respectively. The activity of both the enzymes varies among ALL patients governing the active and the inactive metabolite profile within the immature lymphocytes. Therefore, an attempt was made to study the kinetic nature of the branched bi-enzyme system acting on 6MP and to quantitate TIMP and 6TUA formed when the two enzymes are present in equal and variable ratios. The quantification of the branched kinetics using spectrophotometric method presents problem due to the closely apposed lambda(max) of the substrates and products. Hence, employing an HPLC method, the quantification of the products was done with the progress of time. The limit of quantification (LOQ) of substrate was found to be 10nM and for products as 50 nM. The limit of detection (LOD) was found to be 1 nM for the substrate and the products. The method exhibited linearity in the range of 0.01-100 microM for 6MP and 0.05-100 microM for both 6TUA and TIMP. The amount of TIMP formed was higher than that of 6TUA in the bi-enzyme system when both the enzymes were present in equivalent enzymatic ratio. It was further found that enzymatic ratios play an important role in determining the amounts of TIMP and 6TUA. This method was further validated using actively growing T-ALL cell line (Jurkat) to study the branched kinetics, wherein it was observed that treatment of 50 microM 6MP led to the generation of 12 microM TIMP and 0.8 microM 6TUA in 6 h at 37 degrees C.     

Novel selective biocatalytic deacylation studies on key precursors for bicyclonucleosides

Immobilized Candida antarctica lipase and Thermomyces lanuginosus lipase catalyze the deacylation of precursors of LNA analogs, 4'-C-acyloxymethyl-2',3',5'-tri-O-acyl-beta-L-threo-pentofuranosylthymine and 4-C-acyloxymethyl-3,5-di-O-acyl-1,2-O-(1-methylethylidene)-beta-L-threo-pentofuranose, respectively in a highly selective and efficient manner.     

Responses of Cajanus cajan and rhizospheric N-cycling communities to bioinoculants

Background and aims

Bioinoculants are commonly used for enhancing crop productivity but little information is available on their effect on key microbial communities such as those involved in the cycling of nitrogen, a major plant nutrient. Here we developed a formulation combining different bioinoculants (Bacillus megaterium, Pseudomonas fluorescens and Trichoderma harzianum) and examined their effects on both Cajanus cajan growth and N-cycling microorganisms.

Methods

Seven bioinoculant combinations were evaluated in pots under field conditions, and their effects on plant growth were measured using various biometric parameters. The abundances of the total bacterial and crenarchaeal communities along with those involved in N-cycling were monitored by qPCR at vegetative, pre-flowering, flowering and maturity stages of the crop.

Results

A significant increase in growth of C. cajan was observed when treated with mixture of three bioinoculants with dry biomass and grain yield increase by 330?% and 238?%, respectively. The combination of three bioinoculants also increased the abundance of nitrogen fixers and denitrifiers towards the flowering and maturity stages.

Conclusions

The consortium of three bioinoculants increased plant growth and grain yield of C. cajan. These bioinoculants also had a positive effect on the abundance of several N-cycling microbial communities stressing the importance of understanding non-target effects of bioinoculants together with their impact on plant growth.     

Coadapted changes in energy metabolites and body color phenotypes for resistance to starvation and desiccation in latitudinal populations of D. melanogaster

In D. melanogaster, resistance to starvation and desiccation vary in opposite directions across a geographical gradient in India but there is lack of such clinal variation on other continents. However, it is not clear whether these resistance traits or other correlated traits are the target of natural selection. For resistance to starvation or desiccation in D. melanogaster, we tested the hypothesis whether body color phenotypes and energy metabolites show correlated selection response. Our results are interesting in several respects. First, based on within population analysis, assorted darker and lighter flies from a given population showed that darker flies store higher amount of trehalose and confer greater desiccation resistance as compared with lighter flies. By contrast, lighter flies store higher lipids content and confer increased starvation tolerance. Thus, there is a trade-off for energy metabolites as well as body color phenotypes for starvation and desiccation stress. Further, trait associations within populations reflect similar patterns in geographical populations. Second, we found opposite clines for trehalose and body lipids. Third, coadapated phenotypes have evolved under contrasting climatic conditions i.e. drier and colder northern localities select darker flies with higher trehalose as well as desiccation resistance while hot and humid localities favor lighter flies with higher lipids level and greater starvation tolerance. Thus, the evolution of coadapated phenotypes associated with starvation and desiccation resistance might have resulted due to specific ecological conditions i.e. humidity changes on the Indian subcontinent.     

Development of a mutant of Trichoderma citrinoviride for enhanced production of cellulases

Considering importance of a microbial strain capable of increased cellulases production and insensitive to catabolite repression for industrial use, we have developed a mutant strain of Trichoderma citrinoviride by multiple exposures to EMS and ethidium bromide. The mutant produced 0.63, 3.12, 8.22 and 1.94 IU ml(-1) FPase, endoglucanase, beta-glucosidase and cellobiase, respectively. These levels were, respectively, 2.14, 2.10, 4.09 and 1.73 fold higher than those in parent strain. Glucose (upto 20 mM) did not repress enzyme production by the mutant under submerged fermentation conditions. In vitro activity assay with partially purified cellulase showed lack of inhibition by glucose. Interestingly, the partially purified endoglucanase and beta-glucosidase were activated by 2.0 fold and 2.6 fold, respectively, by 20 mM and 30 mM ethanol in the assay mixture. Genetic distinction of the mutant was revealed by the presence of two unique amplicans in comparative DNA fingerprinting performed using 20 random primers.     

Endothelin-1-induced macrophage inflammatory protein-1beta expression in monocytic cells involves hypoxia-inducible factor-1alpha and AP-1 and is negatively regulated by microRNA-195

Patients with sickle cell disease (SCD) exhibit a chronic inflammatory state manifested by leukocytosis and increased circulating levels of proinflammatory cytochemokines. Our studies show that placenta growth factor levels are high in SCD, and placental growth factor induces the release of the vasoconstrictor endothelin-1 (ET-1) from pulmonary microvascular endothelial cells. In this study, we observed that ET-1 increased the expression of the chemokines MIP-1β or CCL4. ET-1-induced MIP-1β mRNA expression in THP-1 cells and human peripheral blood monocytes occurred via the activation of PI3K, NADPH oxidase, p38 MAPK, and JNK-1 but not JNK-2. ET-1-induced MIP-1β expression involved hypoxia-inducible factor-1α (HIF-1α), independent of hypoxia, as demonstrated by silencing with HIF-1α small interfering RNA, EMSA, and chromatin immunoprecipitation analysis. ET-1-induced MIP-1β promoter luciferase activity was attenuated when any of the five hypoxia-response elements, AP-1, or NF-κB binding motifs in the proximal MIP-1β promoter (-1053/+43 bp) were mutated. Furthermore, ET-1 significantly downregulated the expression of a key microRNA, microRNA-195a, which showed a complementary binding site in the 3' untranslated region of MIP-1β mRNA. Moreover, ET-1-induced MIP-1β mRNA expression in either THP-1 cells or peripheral blood monocytes was reduced upon expression of microRNA-195a. Conversely, transfection of monocytes with anti-microRNA-195a oligonucleotide augmented several-fold ET-1-induced MIP-1β expression. Taken together, these studies showed that ET-1-mediated MIP-1β gene expression is regulated via hypoxia-response elements, AP-1, and NF-κB cis-binding elements in its promoter and negatively regulated by microRNA-195, which targets the 3' untranslated region of MIP-1β RNA. These studies provide what we believe are new avenues, based on targets of HIF-1α and microRNAs, for ameliorating inflammation in SCD.     

Interaction of Hermansky-Pudlak Syndrome genes in the regulation of lysosome-related organelles

Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disease caused by abnormalities in the synthesis and/or trafficking of lysosome-related organelles (LROs) including melanosomes, lamellar bodies of lung type II cells and platelet dense granules. At least 15 genes cause HPS in mice, with a significant number specifying novel subunits of protein complexes termed BLOCs (Biogenesis of Lysosome-related Organelles Complexes). To ascertain whether BLOC complexes functionally interact in vivo, mutant mice doubly or triply deficient in protein subunits of the various BLOC complexes and/or the AP-3 adaptor complex were constructed and tested for viability and for abnormalities of melanosomes, lung lamellar bodies and lysosomes. All mutants, including those deficient in all three BLOC complexes, were viable though the breeding efficiencies of multiple mutants involving AP-3 were severely compromised. Interactions of BLOC protein complexes with each other and with AP-3 to affect most LROs were apparent. However, these interactions were tissue and organelle dependent. These studies document novel biological interactions of BLOC and AP-3 complexes in the biosynthesis of LROs and assess the role(s) of HPS protein complexes in general health and physiology in mammals. Double and triple mutant HPS mice provide unique and practical experimental advantages in the study of LROs.     

Function and stability of human transcobalamin II: role of intramolecular disulfide bonds C98-C291 and C147-C187

The current studies have investigated the role of three disulfide bonds of human transcobalamin II (TC II), a plasma transporter of cobalamin (Cbl; vitamin B12), in its function and stability. When translated in vitro in the presence or absence of microsomal vesicles, TC II constructs with a single substitution, C3S or C249S, demonstrated synthesis of a stable functional protein. However, TC II synthesized in the presence of microsomal vesicles using constructs with a single (C98S, C147S, C187S, C291S), double (C3/147/S, C98/147/S) or triple (C3/98/147/S) substitution was unstable. In the absence of microsomal vesicles, the percentage of binding to Cbl-Sepharose matrix by TC II expressed by constructs C3S, C3/147/S, C98/147/S, or C3/98/147/S was 100, 49, 52, and 35%, respectively. Upon their reductive alkylation, the binding of TC II expressed by these constructs was reduced to approximately 25-30%. TC II constructs C3S or C249S, when expressed in TC II-deficient fibroblasts, produced a stable functional protein, but those expressed by constructs C147S, C187S, C291S, C3/147/S, C98/147/S, or C3/98/147/S were rapidly degraded. The intracellular degradation of TC II expressed by these constructs was inhibited by lactacystin or MG-132 but not by the lysosomal degradation inhibitors ammonium chloride or chloroquine. These studies suggest that optimal binding of Cbl by human TC II is supported by disulfide bonds C98-C291 and C147-C187 and that their disruption results in loss of Cbl binding and their rapid degradation by the proteasomal machinery.