Use of structure-directed DNA ligands to probe the binding of recA protein to narrow and wide grooves of DNA and on its ability to promote homologous pairing.

We have used circular dichroism and structure-directed drugs to identify the role of structural features, wide and narrow grooves in particular, required for the cooperative polymerization, recognition of homologous sequences, and the formation of joint molecules promoted by recA protein. The path of cooperative polymerization of recA protein was deduced by its ability to cause quantitative displacement of distamycin from the narrow groove of duplex DNA. By contrast, methyl green bound to the wide groove was retained by the nucleoprotein filaments comprised of recA protein-DNA. Further, the mode of binding of these ligands and recA protein to DNA was confirmed by DNaseI digestion. More importantly, the formation of joint molecules was prevented by distamycin in the narrow groove while methyl green in the wide groove had no adverse effect. Intriguingly, distamycin interfered with the production of coaggregates between nucleoprotein filaments of recA protein-M13 ssDNA and naked linear M13 duplex DNA, but not with linear phi X174 duplex DNA. Thus, these data, in conjunction with molecular modeling, suggest that the narrow grooves of duplex DNA provide the fundamental framework required for the cooperative polymerization of recA protein and alignment of homologous sequences. These findings and their significance are discussed in relation to models of homologous pairing between two intertwined DNA molecules.     

1,25 Dihydroxyvitamin D3-dependent inhibition of growth or killing of Mycobacterium avium complex in human macrophages is mediated by TNF and GM-CSF

Vitamin D3 (D3) has been shown to activate several macrophage functions. To determine whether D3 could activate macrophages to kill or inhibit intracellular growth of Mycobacterium avium complex (MAC), human monocyte-derived macrophages were treated with D3 (10(-7), 10(-8), and 10(-9) M) 24 hr before or for 48 hr after MAC infection. All three concentrations were associated with inhibition of growth or killing of MAC in a dose-dependent fashion (28 +/- 4% and 22 +/- 3% of killing and inhibition of growth, respectively, at pharmacological concentrations) when added to the monolayer before injection or 60.4 +/- 6%, 50.4 +/- 3%, and 41.4 +/- 6%, respectively, when added to the monolayers after infection. We found that D3-treated macrophages produced increased concentrations of tumor necrosis factor (TNF) and granulocyte-monocyte colony stimulating factor (GM-CSF). Subsequently, macrophages were activated by D3 in the presence of anti-TNF or anti-GM-CSF antibody: At 10(-9) M of D3 there was no inhibition of D3-dependent macrophage activation by anti-TNF antibody, whereas anti-GM-CSF antibody was associated with 100% inhibition. At 10(-8) M of D3, anti-TNF antibody inhibited 35 +/- 6% of killing, and anti-GM-CSF antibody was associated with 100% inhibition. At 10(-7) M of D3, anti-TNF antibody inhibited 58 +/- 4% and anti-GM-CSF antibody 89 +/- 3% of killing. D3 treatment is associated with anti-MAC activity in human macrophages, and this activity appears to be mediated by both TNF and GM-CSF.     

Disulfiram administration affects substance P-like immunoreactive and monoaminergic neural systems in rodent brain.

The biosynthetic enzyme peptidylglycine alpha-amidating monooxygenase catalyzes the formation of a variety of biologically active alpha-amidated peptides from respective COOH-terminal glycine-extended peptide precursors. Peptidylglycine alpha-amidating monooxygenase activity is dependent on copper, ascorbate, and molecular oxygen and is inhibited by the relatively selective copper chelator N,N-diethyldithiocarbamate or its disulfide dimer disulfiram (Antabuse). In the present study, chronic disulfiram treatment (100 mg/kg/day, for 12-25 days) resulted in significant changes in several neurochemical parameters in the mouse central nervous system, including levels of substance P-like, unamidated substance P-Gly-like, and protease-generated substance P-Gly-Lys-like immunoreactivities (SP-LI, SP-G-LI, and SP-G-K-LI, respectively). Combined high performance liquid chromatography/radioimmunoassay analyses of the extracted SP-LI, SP-G-LI, and SP-G-K-LI species indicated very similar chromatographic and immunochemical behavior as demonstrated for chemically authentic peptide standards. Additionally, changes in levels of monoamines and their metabolites were observed after drug administration. Complementary immunohistochemical analyses using affinity-purified anti-SP-G sera localized these drug-induced changes in levels of immunoreactive unamidated precursor to neural elements that normally express SP. As a functional corollary to alterations in neurochemical parameters, we observed significant disulfiram-induced increases in pain thresholds, potentiated by capsaicin treatment. Overall, our results indicate that the observed changes in steady state levels of immunoreactive SP and of the immature COOH-terminal extended forms of SP may reflect compensatory biosynthetic and posttranslational processing events in SP-containing neural systems after pharmacological challenge.     

Evaluating effect of arbuscular mycorrhizal fungal consortia and Azotobacter chroococcum in improving biomass yield of Jatropha curcas

This study was conducted to study the long-term impact of bioinoculants, Azotobacter chroococcum and arbuscular mycorrhizal fungi (AMF) on growth and biomass yield of Jatropha curcas grown in nursery and in field conditions. The experiment was set up in a randomized block design, and the following treatments was designed (T₁ = control, T₂ = Azotobacter, T₃ = inoculation with AMF, and T₄ = inoculation with Azotobacter + AMF). Data on various growth attributes (shoot height and shoot diameter) and biochemical parameters [leaf relative water content (LRWC), sugars, protein, and photosynthetic pigments] were recorded up to 6 months in the nursery and in the field (18 months). Results pertaining to morpho-physiological traits showed Azotobacter and AMF consortia increase shoot height, shoot diameter, LRWC, sugars, proteins, and photosynthetic pigments over control under nursery conditions. Besides enhancing the plant growth, these bioinoculants helped in better establishment of Jatropha plants under field conditions. A significant improvement in the shoot height, shoot diameter, fruit yield/plant, and seed yield (g)/plant was evident in 18-month-old Jatropha plants under field conditions when Azotobacter and AMF were co-inoculated. This work supports the application of bioinoculants for establishment of Jatropha curcas in semi-arid regions.     

A purine permease in Candida glabrata

Abstract Competition experiments revealed that adenine and guanine were transported by a purine permease in both Candida glabrata 4 and a C. glabrata 4 cytosine permease negative mutant. The C. glabrata 4 cytosine permease negative mutant was isolated using 5-fluorocytosine selection. This mutant no longer transported cytosine, but transported adenine and guanine. A transport system for hypoxanthine was not detected. Hence, in addition to the cytosine permease, a purine permease exists in C. glabrata . This differs from the purine cytosine permeases in Saccharomyces cereuisiae and Candida albicans which transport adenine, cytosine, guanine and hypoxanthine.     

A recognition component of the ubiquitin system is required for peptide transport in Saccharomyces cerevisiae

Peptide transport in Saccharomyces cerevisiae is controlled by three genes: PTR1, PTR2, and PTR3. PTR1 was cloned and sequenced and found to be identical to UBR1, a gene previously described as encoding the recognition component of the N-end-rule pathway of the ubiquitin-dependent proteolytic system. Independently derived ubr1 mutants, like ptr1 mutants, were unable to transport small peptides into ceils. Concomitantly, ptr1 mutants, like ubr1 mutants, were unable to degrade an engineered substrate of the N-end-rule pathway. Further, ptr1 mutants did not express PTR2, a gene encoding the integral membrane component required for peptide transport in S. cerevisiae. These results establish a physiological role for a protein previously known to be required for the degradation of N-end-rule substrates. Our findings show that peptide transport and the ubiquitin pathway—two dynamic phenomena universal to eukaryotic cells—share a common component, namely UBR1/PTR1.     

Isolation,identification and characterization of Staphylococcus sp. from Indian ethnic fermented fish product

Staphylococci from Sheedal of Northeast India was isolated, identified and characterized. All the isolated staphylococci were found to be coagulase negative. Based on the rpoB gene sequences followed by analysis using NCBI-BLAST software, seven species of Staphylococcus namely, S. piscifermentans, S. condimenti, S. arlettae, S. sciuri, S. warneri, S. nepalensis and S. hominis were recognized. Phylogenetic analyses revealed three major cluster groups. All the seven Staphylococcus showed their NaCl tolerance from 2 to 8%. No species was able to grow at 55°C. Except S. arlettae and S. sciuri, all the isolated staphylococcal species exhibited growth at pH 4–8. No isolated species was able to ferment mannitol, sucrose and arabinose. All the species exhibited moderate to maximum proteolytic and lipolytic activities. All the seven species were found to be sensitive to the antibiotics, namely, erythromycin, norfloxacin, ampicillin, streptomycin and vancomycin, whereas all were resistant to co-trimoxazole. Only S. piscifermentans was found antagonist to Salmonella enterica, Escherichia coli and Bacillus subtilis, although the clear zone was minimal. All the staphylococcal species except S. arlettae and S. sciuri exhibited hydrophobicity ranging from 25 to 66%. The observed characteristics of isolated Staphylococci from Sheedal revealed their role in fish fermentation.