Ouabain decreases sarco(endo)plasmic reticulum calcium ATPase activity in rat hearts by a process involving protein oxidation

The effect of cardiac glycosides to increase cardiac inotropy by altering Ca(2+) cycling is well known but still poorly understood. The studies described in this report focus on defining the effects of ouabain signaling on sarcoplasmic reticulum Ca(2+)-ATPase function. Rat cardiac myocytes treated with 50 microM ouabain demonstrated substantial increases in systolic and diastolic Ca(2+) concentrations. The recovery time constant for the Ca(2+) transient, tau(Ca(2+)), was significantly prolonged by ouabain. Exposure to 10 microM H(2)O(2), which causes an increase in intracellular reactive oxygen species similar to that of 50 microM ouabain, caused a similar increase in tau(Ca(2+)). Concurrent exposure to 10 mM N-acetylcysteine or an aqueous extract from green tea (50 mg/ml) both prevented the increases in tau(Ca(2+)) as well as the changes in systolic or diastolic Ca(2+) concentrations. We also observed that 50 microM ouabain induced increases in developed pressure in addition to diastolic dysfunction in the isolated perfused rat heart. Coadministration of ouabain with N-acetylcysteine prevented these increases. Analysis of sarcoplasmic reticulum Ca(2+)-ATPase protein revealed increases in both the oxidation and nitrotyrosine content in the ouabain-treated hearts. Liquid chromatography-mass spectrometric analysis confirmed that the sarcoplasmic reticulum Ca(2+)-ATPase protein from ouabain-treated hearts had modifications consistent with oxidative and nitrosative stress. These data suggest that ouabain induces oxidative changes of the sarcoplasmic reticulum Ca(2+)-ATPase structure and function that may, in turn, produce some of the associated changes in Ca(2+) cycling and physiological function.     

Yeast genome analysis identifies chromosomal translocation,gene conversion events and several sites of Ty element insertion

Paired end mapping of chromosomal fragments has been used in human cells to identify numerous structural variations in chromosomes of individuals and of cancer cell lines; however, the molecular, biological and bioinformatics methods for this technology are still in development. Here, we present a parallel bioinformatics approach to analyze chromosomal paired-end tag (ChromPET) sequence data and demonstrate its application in identifying gene rearrangements in the model organism Saccharomyces cerevisiae. We detected several expected events, including a chromosomal rearrangement of the nonessential arm of chromosome V induced by selective pressure, rearrangements introduced during strain construction and gene conversion at the MAT locus. In addition, we discovered several unannotated Ty element insertions that are present in the reference yeast strain, but not in the reference genome sequence, suggesting a few revisions are necessary in the latter. These data demonstrate that application of the chromPET technique to a genetically tractable organism like yeast provides an easy screen for studying the mechanisms of chromosomal rearrangements during the propagation of a species.     

A spectroelectrochemical and chemical study on oxidation of 7,8-dihydroxy-4-methylcoumarin (DHMC) and some related compounds in aprotic medium

Electrochemical and chemical oxidation of 7,8-hydroxy-4-methylcoumarin (DHMC 1) and 7,8-diacetoxy-4-methylcoumarin (DAMC 4) were studied to investigate the mechanisms occurring in their antioxidant activities in acetonitrile, under electron transfer and H-atom transfer conditions. Electrolysis and chemical reactions were followed on-line by monitoring the UV spectral changes with time.     

From bacterial genomics to metagenomics: concept,tools and recent advances

In the last 20 years, the applications of genomics tools have completely transformed the field of microbial research. This has primarily happened due to revolution in sequencing technologies that have become available today. This review therefore, first describes the discoveries, upgradation and automation of sequencing techniques in a chronological order, followed by a brief discussion on microbial genomics. Some of the recently sequenced bacterial genomes are described to explain how complete genome data is now being used to derive interesting findings. Apart from the genomics of individual microbes, the study of unculturable microbiota from different environments is increasingly gaining importance. The second section is thus dedicated to the concept of metagenomics describing environmental DNA isolation, metagenomic library construction and screening methods to look for novel and potentially important genes, enzymes and biomolecules. It also deals with the pioneering studies in the area of metagenomics that are offering new insights into the previously unappreciated microbial world. The authors have contributed equally to the work     

Cloning, expression, purification, and characterization of the acid alpha-mannosidase from Trypanosoma cruzi

The acid alpha-mannosidase of Trypanosoma cruzi is a broad-specificity hydrolase involved in the catabolism of glycoconjugates, presumably in the digestive vacuole. We have cloned the alpha-mannosidase gene from a T.cruzi epimastigote genomic library. The alpha-mannosidase gene was determined to be single copy by Southern analysis, and similar sequences were not detected in genomic digests of either Trypanosoma brucei or Leishmania donovani. The coding region was subcloned into the Pichia pastoris expression vector pPICZ, and alpha-mannosidase activity was detected in the medium of induced cultures. The recombinant alpha- mannosidase demonstrated a pH optimum, inhibition by swainsonine, Km, and substrate specificity consistent with the characteristics of the alpha-mannosidase previously purified from T.cruzi epimastigotes. The recombinant enzyme was purified 103-fold from the culture medium of Pichia pastoris and had a native molecular mass of 359 kDa by gel filtration. A combination of SDS-PAGE, deglycosylation with endo H, and NH2-terminal sequencing indicates that the enzyme is originally synthesized as a homodimeric polypeptide that is subsequently cleaved to form a heterotetramer composed of 57 and 46 kDa subunits. A polyclonal antibody raised to the recombinant enzyme was shown to immunoprecipitate the alpha-mannosidase from T.cruzi cell extracts and will be used in future immunolocalization studies.      

The genus Amycolatopsis: Indigenous plasmids, cloning vectors and gene transfer systems

The genus Amycolatopsis is a member of the phylogenetic group nocardioform actinomycetes. Most of the members of the genus Amycolatopsis are known to produce antibiotics. Additionally, members of this genus have been reported to metabolize aromatic compounds as the sole sources of carbon and energy. Development of genetic manipulation in Amycolatopsis has progressed slowly due to paucity of genetic tools and methods. The occurrence of indigenous plasmids in different species of Amycolatopsis is not very common. Till date, only three indigenous plasmids viz., pMEA100, pMEA300 and pA387 have been reported in Amycolatopsis species. Various vectors based on the indigenous plasmids, pMEA100, pMEA300 and pA387, have been constructed. These vectors have proved useful for molecular genetics studies of actinomycetes. Molecular genetic work with Amycolatopsis strains is not easy, since transformation methods have to be developed, or at least optimized, for each particular strain. Nonetheless, methods for efficient transformation (polyethyleneglycol (PEG) induced protoplast transformation, transformation by electroporation and direct transformation) have been developed and used successfully for the introduction of DNA into several Amycolatopsis species. The construction of plasmid cloning vectors and the development of gene transfer systems has opened up possibilities for studying the molecular genetics of these bacteria.     

Dicoumarol-induced 9-gamma-carboxyglutamic acid prothrombin: isolation and comparison with the 6-, 7-, 8-, and 10-gamma-carboxyglutamic acid isomers.

The role of gamma-carboxyglutamic acid (Gla) in prothrombin function can be effectively evaluated by characterizing dicoumarol-induced, Gla-deficient prothrombin structural isomers. In addition to the isolation of 8-, 7-, 6-, 5-, 3-, 2-, 1-, and 0-Gla isomers, we have now purified a variant prothrombin containing 9(8.80) Gla residues by barium citrate adsorption, elution, and finally by DEAE-cellulose and immunoaffinity chromatographies. Agar gel electrophoretic mobilities of the 9-Gla isomer and its fragment 1 were slower than those of the respective 10-Gla (normal) prothrombin and fragment 1, both in the absence and presence of Ca(II). In the presence of Ca(II), both 9- and 10-Gla fragments 1 moved slower than 8- and 7-Gla fragments 1. However, in the absence of metal ions, 9- and 7-Gla fragments 1 migrated at the same rate, but slower than 10- and 8-Gla fragments. Similarly, the 9-Gla fragment 1 electrofocused cathodically to 10- and 8-Gla, but comparably with 7-Gla fragment 1. The 9-Gla fragment 1 exhibited a Ca(II)-induced 44% decrease in the intrinsic fluorescence, compared with a 40% decrease in that of 10-Gla; 8-Gla fragment 1 revealed only 23% quenching. Ca(II)-dependent anti-normal prothrombin antibodies are not specific for 10-Gla prothrombin, since only a twofold molar excess of the 9-Gla isomer was required to displace equal amounts of labeled normal prothrombin. The most critical Gla residue for influencing the functional, thrombin-generating properties of prothrombin appears to be the one present in the 9-Gla isomer but absent in the 8-Gla variant, since 9-Gla prothrombin possesses four times the normal coagulant activity (78 versus 20%) of the 8-Gla isomer.