Amino acid residues involved in autophosphorylation and phosphotransfer activities are distinct in nucleoside diphosphate kinase from Mycobacterium tuberculosis

Nucleoside diphosphate kinase (NdK) is a ubiquitous enzyme in both prokaryotes and eukaryotes and is primarily involved in the maintenance of cellular nucleotide pools. We have cloned ndk from Mycobacterium tuberculosis strain H37Ra and expressed it in Escherichia coli as a fusion protein with glutathione S-transferase. The purified protein, following thrombin cleavage and gel permeation chromatography, was found to be hexameric with a monomeric unit molecular mass of approximately 16.5 kDa. The protein exhibited nucleotide binding, divalent cation-dependent autophosphorylation, and phosphate transfer ability from nucleoside triphosphate to nucleoside diphosphate. Although UDP inhibited the catalytic activity of the recombinant protein, the classic inhibitors, like cromoglycate, 5'-adenosine 3'-phosphate, and adenosine 3'-phosphate 5'-phosphosulfate, had no effect on the activity. Among three histidine residues in the protein, His-117 was found to be essential for autophosphorylation. However, in subsequent phosphate transfer, we observed that His-53 had a significant contribution. Consistent with this observation, substitution of His-53 with either Ala or Gln affected the ability of the recombinant protein to complement NdK function in Pseudomonas aeruginosa. Furthermore, mutational analysis established critical roles for Tyr-50 and Arg-86 of the M. tuberculosis protein in maintaining phosphotransfer ability.     

Phylogeny,phenotypic and nutritional characteristics of estuarine soil actinomycetes having broad-spectrum antimicrobial activity derived from an ecologically guided bioprospecting programme

No investigation has been done to identify ecological factors selecting for broad-spectrum antimicrobial activity of actinomycetes in the estuaries. Previously, we established that, in the Sundarbans (the world’s largest tidal mangrove forest), high antagonistic potential (AP) sampling sites were influenced by tides, while the low AP sites were not. We now report molecular phylogenetic analysis, morphological, physiological and biochemical characteristics of actinomycetes of high AP sites. The effects of soil organic carbon, nitrogen and ionic content (which strongly influenced AP) on the strength and spectrum of activity of the isolates were also studied in shake flasks. Molecular phylogenetic analysis showed sequences of our strains to be 96–99% similar to the 16S rDNA sequences of Streptomyces. Results showed variation among sporophore sizes, ornamentation and number of spores. Jaccard’s similarity coefficients of the isolates varied from 0.512 to 0.884 indicating disparity in the biochemical and physiological characteristics, possibly due to spatial separation of the sampling sites. Top soil in the intertidal zone of estuaries is generated from settling and consolidation of fluid mud and Streptomyces would be expected to produce broad-spectrum antimicrobials in such virgin soil. Isolates should be collected from the narrow band between the mean high and low tide marks to maximize chances of finding broad-spectrum activity. Considering the study on nutritional requirements vis-à-vis the field studies, it was concluded that results of this investigation corroborated the field observations where soil nitrogen, rather than organic carbon or ionic concentration, played a major role in determining the antimicrobial spectrum.     

Catechin and Catechin Fractions as Biochemical Markers to Study the Diversity of Indian Tea (Camellia sinensis (L.) O. Kuntze) Germplasm

The heterogeneous Indian tea germplasm includes ‘China’, ‘Assam’, ‘Cambod’, and their hybrids which were evaluated using biochemical markers viz., total catechin and their fractions, for varietal identification and characterization. Principal component analysis (PCA) of biochemical characters showed that the total catechin and trihydroxylated catechin has higher eigenvalues. The first two principal components (PCs) could differentiate more than 90% of the clones studied. This grouping based on first two principal component matrices differentiated ‘China’, and their hybrids with ‘Assam’ and ‘Cambod’ variety. Morphologically indistinct large‐leaved ‘Cambod’ variety and ‘Assam’ varieties could not be differentiated using biochemical markers, since both varietal types taxonomically belong to a single species. Clones of ‘China’ type showed low total catechin content and catechin ratio which are distinctly grouped. The ‘China–Assam’ and ‘China–Cambod’ hybrids formed intermediate groups between ‘China’ PC group and ‘Cambod’/‘Assam’ PC groups, providing evidence for genetic control of catechin ratio variation. Tea clones which are differentially positioned in the PC group could be explained based on the genetic contribution by other varietal type as parents. This biochemical characterization will be a useful tool in the development of quality‐tea clones with different proportion of total catechin and their fractions.     

Structural characterization of a water-soluble beta-(1-->6)-linked D-glucan isolated from the hot water extract of an edible mushroom, Agaricus bitorquis

A water-soluble polysaccharide, isolated from the hot aqueous extract of an edible mushroom, Agaricus bitorquis, was found to consist of d-glucose only. On the basis of total hydrolysis, methylation analysis, and NMR studies (¹H, ¹³C, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit was established as→6)-β-d-Glcp-(1→     

Haematological and histological changes in fish Heteropneustes fossilis exposed to pesticides from industrial waste water

The present study investigated the potential changes in hematologic parameters and histology of fish Heteropneustes fossilis when exposed to industrial waste water of different concentrations. The toxicant enter into the fish body from different rout, inhalation, dermal oral, and other several rout for entering in the fish body. The blood parameters of fish H. fossilis control group and treatment were investigated on 1st, 5th, 10th, and 20th days of experiment, hematologic variation was observed in RBC, WBC, MCV, MCH, and MCHC count. Histopathologic changes in liver, intestine, gill, muscle, and heart showed increasing degrees of damage in the tissues in correlation with the accumulation pattern of pesticides, while liver, intestine, gill, muscle, and heart of control group exhibited a normal architecture. Toxic effects of pesticides vary in different organs of the fish H. fossilis significant. The fish exposed to higher concentration showed uncoordinated alterations in behavioral responses, especially erratic and jerky swimming, physiologic, malformation, histologic, hematologic and biochemical changes, frequent surfacing and in gulping, mucus secretion, an increase in opercular movement, and copious secretion of mucus of all over the body.     

Leishmania donovani affects antigen presentation of macrophage by disrupting lipid rafts

Leishmania donovani-infected splenic macrophages and P388D1 (P388D1(I)) failed to activate T cells in response to low dose of exogenous peptide. The membrane fluidity of P388D1(I) was greater than that of the normal counterpart P388D1(N), but could be reduced either by exposing the cell below phase transition point or by loading cholesterol into membrane (L-P388D1(I)), and this was associated with enhanced Ag-presenting ability of P388D1(I). Presentation of endogenous leishmanial Ag, kinetoplastid membrane protein-11, was also defective, but could be corrected by loading cholesterol into membrane. Because membrane rafts are important for Ag presentation at a low peptide dose, raft architecture of P388D1(I) was studied using raft (CD48 and cholera toxin-B) and non-raft (CD71) markers in terms of their colocalization with I-A(d). Binding of anti-CD48 mAb and cholera toxin B subunit decreased significantly in P388D1(I), and consequently, colocalization with I-A(d) was not seen, but this could be restored in L-P388D1(I). Conversely, colocalization between I-A(d) and CD71 remained unaffected regardless of the presence or the absence of intracellular parasites. P388D1(N) and L-P388D1(I), but not P388D1(I), formed peptide-dependent synapse with T cells quite efficiently and this was found to be corroborated with both intracellular Ca2+ mobilization in T cells and IL-2 production. This indicated that intracellular parasites disrupt the membrane rafts, possibly by increasing the membrane fluidity, which could be corrected by making the membrane rigid. This may be a strategy that intracellular L. donovani adopts to evade host immune system.     

Light-induced inhibition of papain by a {Mn-NO}6 nitrosyl: identification of papain-SNO adduct by mass spectrometry

Modification of Cys25 at the active site of the cysteine protease papain by S-nitrosylation inhibits its hydrolytic ability. Previous studies have demonstrated that NO donors N-nitrosoanilines inhibit papain activity via formation of S-NO bond formation at the active site while NO donors such as S-nitroso-N-acetyl-penicillamine (SNAP), N-nitrosoaniline derivatives, and S-nitroso-glutathione (GSNO) inhibit the enzyme via S-thiolation by thiyl radicals generated from the S-nitrosothiols. In this study, we report papain inactivation by a photosensitive {Mn-NO}(6) nitrosyl [(PaPy(3))Mn(NO)](ClO(4)) (1) where PaPy(3)(-) is the anion of the designed ligand N,N-bis(2-pyridylmethyl)amine-N-ethyl-2-pyridine-2-carboxamide. This nitrosyl releases NO upon exposure to visible light of low intensity (50W tungsten lamp). With N(alpha)-benzoyl-l-arginine-p-nitroanilide (l-BApNA) as the substrate, the dissociation constant for the breakdown of the enzyme-inactivator complex (K(I)) and the overall inactivation rate constant (k(i)) were calculated to be 2.46mM and 64.8min(-1), respectively. The papainS-NO adduct has been identified using electrospray mass spectrometry (ESI-MS). The results demonstrate that controlled inactivation of papain can be achieved with the {Mn-NO}(6) nitrosyl 1 and light. The reaction is clean and the extent of inactivation is directly proportional to the exposure time.     

Neue diterpene und sesquiterpene aus südafrikanischen Helichrysum-arten

The investigation of several South African Helichrysum species afforded in addition to known compounds two new kaurenic acid derivatives, three new labdane derivatives, three aromadendrene derivatives and geranylterpinene. The structures are elucidated mainly by extensive ¹H NMR-studies. The chemotaxonomic importance of these findings is discussed briefly.