Biomedical subjects
Publications and source records attributed to A K Datta.
A bacterial strain capable of biodegradation of lantadene A (22 beta-angeloyloxy-3-oxoolean-12-en-28-oic acid) has been isolated from soil using lantadene A as the sole carbon source. The organism is rod shaped, Gram negative, motile and has been identified as Pseudomonas pickettii. This is the first report of the biodegradation of a pentacyclic triterpenoid.
Inactivation of adenosine kinase (Adk) from Leishmania donovani correlates with the modification of two conformationally vicinal cysteine residues. In contrast, Adk from hamster liver, despite being sensitive to monothiol-blocking reagents, was insensitive to dithiol modifiers. Inactivation kinetics and substrate-protection studies along with double-modification experiments successively with N-ethylmaleimide in the presence of Ado and sodium m-arsenite-2,3-dimercaptopropanol or diazenedicarboxylic acid bis-N,N'-dimethylamide supported assignment of the two thiols at the Ado-binding site. Cystine bridge formation impaired the ability of the modified enzyme to bind to the substrate. Tryptophan fluorescence of the enzyme was quenched after modification by dithiol-blocking reagents with concomitant loss of activity. However, treatment of the enzyme with methylmethanethiosulphonate (MMTS) led to complete inactivation without a marked change in protein fluorescence. Ado protected both fluorescence and catalytic activity against inactivation by both MMTS and dithiol-blocking reagents. Stern-Volmer quenching analysis of the native and Ado-complexed enzyme suggested that, of the four tryptophan residues, at least one is located at or near the active site. Furthermore quenching constants of native, Ado-complexed and dithiol-modified enzyme in the presence of either acrylamide or KI indicated spatial proximity of tryptophan and two cysteine residues within the hydrophobic domain of the Ado-binding site. Taken together the results suggest important function(s) for the cysteine residue(s). A schematic model is proposed to explain the inactivation of the enzyme by both monothiol- and dithiol-blocking reagents.
A Ni(II)-binding serpin, pNiXa, is abundant in Xenopus oocytes and embryos. Kinetic assays show that purified pNiXa strongly inhibits bovine alpha-chymotrypsin (Ki = 3 mM), weakly inhibits porcine elastase (K1 = 0.5 microM), and does not inhibit bovine trypsin. The reversible, slow-binding inhibition of alpha-chymotrypsin by pNiXa is unaffected by Ni(II). Ovochymase in egg exudates is inhibited by pNiXa, but to a limited extent, even at high pNiXa concentrations. An octadecapeptide that models the His-rich domain (-HRHRHEQQGHHDSAKHGH-) of pNiXa forms six-coordinate, octahedral Ni(II)-complexes when the N-terminus is acetylated, and a square-planar Ni(II)-complex when the N-terminus is unblocked. Spectroscopy reveals two distinct types of octahedral Ni(II)-coordination to the N-acetylated octadecapeptide, involving, respectively, 3-4 and 5-6 imidazole nitrogens; the octadecapeptide undergoes partial, reversible precipitation in pH- and Ni(II)-dependent fashion, suggesting an insoluble, Ni(II)-coupled (Hx)n-dimer. Such (Hx)n-peptide interaction is confirmed by an enzyme-linked biotinavidin assay with N-biotin-KHRHRHE-amide and N-acetyl-KHRHRHE-resin beads, which become coupled after adding Ni(II) or Zn(II). H2O2 oxidation of 2'-deoxyguanosine to mutagenic 8-hydroxy-2'-deoxyguanosine is enhanced by the octahedral Ni(II)-octadecapeptide complex, although the effect is more intense with the square-planar Ni(II)-octadecapeptide complex. Immunoperoxidase staining of whole mounts with pNiXa antibody shows that pNiXa is distributed throughout gastrula-stage embryos and is localized during organogenesis in the brain, eye, spinal cord, myotomes, craniofacial tissues, and other sites of Ni(II)-induced anomalies. Patterns of pNiXa staining are similar in controls and Ni(II)-exposed embryos. Binding of Ni(II) to pNiXa may cause embryotoxicity by enhancing oxidative reactions that produce tissue injury and genotoxicity. Although the natural target proteinases for pNiXa inhibition have not been established, pNiXa may be an important regulator of proteolysis during embryonic development.
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The toxicity of ozone, the major component of photochemical smog, is related to its powerful oxidising ability, and many of its deleterious effects are mediated through free radical reactions. As the majority of ozone oxidation events are thought to be confined to the pulmonary epithelial lining fluid, we studied the interaction of ozone with a range of small molecular weight antioxidants found within this compartment: ascorbic acid (AH2), uric acid (UA), and reduced glutathione (GSH). Epithelial lining fluid obtained as bronchoalveolar lavage (BAL) fluid, was taken from 16 male subjects and the antioxidant concentrations determined for each subject. BAL fluid samples from nine of these subjects were then exposed, using an interfacial exposure system, to a range (50-1000 ppb) of ozone concentrations. Both AH2 and UA were consumed by ozone in a time and ozone concentration dependent manner, with mean consumption rates of 1.7 +/- 0.8 and 1.0 +/- 0.5 pmol L-1 s-1 ppb-1, respectively. Considerable intersubject variation was however observed. The individual rates of consumption for each antioxidant were significantly correlated with the respective initial antioxidant concentration. In contrast, although GSH was consumed at 50 ppb ozone, the rate of consumption did not change with increasing ozone concentration. We conclude that there is differential depletion of BAL fluid antioxidants, suggesting a reactivity hierarchy toward ozone in human ELF of AH2 > UA > > GSH.
Hypo-osmotic sperm swelling (HOS) profile is found to be a better predictor of the fertilisability in those husbands possessing subnormal semen parameters. The group where husband's HOS gradings were less than 60%, no one could conceive his fertile wife even after intra-uterine insemination (IUI) for 8 times. However, in the other group where the HOS values were greater than 60%, conception were registered in 6 oligospermic and 2 asthenospermic husbands after 2 to 8 IUI amongst 22 married couples. No conception occurred in any normal coital cycle. All the pregnancies were delivered by elective caesarean section (4 females and 3 males), their birth weights were 2.705 +/- 0.425 kg. One aborted spontaneously at 10 weeks' gestation. The emerging fact in this study is where the HOS values are below 60% the male fertility is reduced, the chances of conception are trivial but fecundity is relatively fair where HOS reading is greater than 60%. It is also a no risk and high benefit method to pick and choose subfertile husbands who are likely to conceive their fertile wives by assisted reproductive techniques.
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All members of the sialyltransferase gene family cloned to date contain a conserved region, the "sialylmotif," consisting of 48-49 amino acids in the center of the coding sequence. To investigate the function of this motif, mutant constructs of the Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase were designed by site-directed mutagenesis, replacing 11 individual conserved amino acids with alanine. Each of the mutants was expressed in COS-1 cells, and eight of these retained sialyltransferase activity, allowing comparison of their enzymatic properties with that of the wild type enzyme. Kinetic analysis showed that six of eight mutants had a 3-12-fold higher Km for the donor substrate CMP-NeuAc relative to the wild type enzyme, while the Km values for the acceptor substrate were within 0.5-1.2-fold of the wild type for all eight mutants evaluated. The Ki of the donor substrate analog CDP was also evaluated for the recombinant sialyltransferase with the Val to Ala mutation at residue 220, which produced a 6-fold increase in Km of CMP-NeuAc. A corresponding increase in Ki of 3.4-fold was observed for CDP, indicating a decreased affinity for the cytidine nucleotide. Taken together, these results suggest that the conserved sialylmotif in the sialyltransferase gene family participates in the binding of the common donor substrate, CMP-NeuAc.
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Values of Km were determined for three purified sialyltransferases and the corresponding recombinant enzymes. The enzymes were Gal beta 1-4GlcNAc alpha 2-6 sialyltransferase and Gal beta 1-3(4)GlcNAc alpha 2-3 sialyltransferase from rat liver; these enzymes are responsible for the attachment of sialic acid to N-linked oligosaccharide chains; and the Gal beta 1-3GalNAc alpha 2-3 sialyltransferase from porcine submaxillary gland that is responsible for the attachment of sialic acid to O-linked glycoproteins and glycolipids. A procedure for the large scale expression of active sialyltransferases from recombinant baculovirus-infected insect cells is described. For the liver enzymes values of Km were determined using rat and human asialo alpha 1 acid glycoprotein and N-acetyllactosamine as variable substrates; lacto-N-tetraose was also used with the Gal beta 1-3(4)GlcNAc alpha 2-3 sialyltransferases. Antifreeze glycoprotein was used as the macromolecular acceptor for the porcine enzyme. Values for Km were also determined using CMP-NeuAc as the variable substrate.
Lipocortin-1 (LC-1; annexin-1) may mediate some anti-inflammatory actions of the glucocorticoids, probably after binding to specific cell surface binding sites. We have quantified LC-1 levels in bronchoalveolar lavage (BAL) fluid and cells collected from seven healthy volunteers before and after 7 days of treatment with an oral glucocorticoid, prednisolone (30 mg/day). Extracellular BAL LC-1 was higher and cellular LC-1 was lower after prednisolone than before [extracellular: before, median 98 ng/mg albumin (range 48-350 ng/mg albumin); after, 236 ng/mg albumin (19-414 ng/mg albumin); P < 0.05. Cellular: before, 23.3 ng/10(6) cells (14.6-26.9 ng/10(6) cells); after, 18.0 ng/10(6) cells (122-268 ng/10(6) cells); P < 0.05]. The distribution of LC-1 within BAL cells ex vivo (cell surface = 25%, cytosol = 50%, membrane = 25%) was unaffected by prednisolone treatment. However, in adherent cells that had been cultured for 4 h, 70-80% of the LC-1 was on the cell surface. In summary, prednisolone appears to promote cellular release of LC-1. The difference in distribution of cellular LC-1 in BAL cells ex vivo and in vitro may reflect adherence and/or activation.
The purpose of this study was to determine whether the Ni(II)-tetraglycine complex system (NiG4) that is known to disproportionate H2O2 at pH > or = 8 can catalyze oxidation of the guanine residues in 2'-deoxyguanosine (dG), calf thymus DNA, and calf thymus nucleohistone (NH) by H2O2 at physiological pH. Incubation of dG with H2O2 in the presence of NiG4 at 37 degrees C, produced two effects: (a) formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and (b) decomposition of dG to 2,6-diamino-4-hydroxy-5-formamidopyrimidine and several low molecular weight unidentified products. The magnitude of both effects depended on incubation time (1-48 h), H2O2 concentration (7.5-40 mM), NiG4 concentration (0.1 or 1 mM), and pH (6.0-8.0). The effects were not detected below pH 6 and above pH 8.0. For 0.1 mM NiG4 and 7.5 mM H2O2, production of 8-OH-dG from dG (0.75 mM) during 24 h at 37 degrees C was significantly lower than from NH (1 mg/ml) or DNA (0.5 mg/ml), indicating possible specific effects that might be related to the strength of interaction of NiG4 with dG, NH, or DNA. The results indicate production of hydroxyl radical or other oxidizing species in the reaction of H2O2 with NiG4 at pH 7-8. Reactions like this may be relevant to the mechanisms of Ni(II)-mediated oxidative damage, observed in vitro and in vivo, which may contribute to the toxic and carcinogenic effects of this metal.
The presence of arginine at the active site of Leishmania donovani adenosine kinase was studied by chemical modification, followed by the characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal (PGO), butane-2,3-dione and cyclohexane-1,2-dione all irreversibly inactivated the enzyme. In contrast, adenosine kinase from hamster liver was insensitive to these reagents. The inactivation of the enzyme by PGO followed pseudo-first-order kinetics, with a second-order rate constant of 39.2 min-1.M-1. Correlation between the stoichiometry of PGO modification and extent of inactivation indicated that modification of a single residue per molecule suffices for the loss of activity. Reactivity of the essential arginine residue towards PGO was affected by the presence of adenosine (Ado) and other competing alternative substrates, consistent with an arginine residue located proximal to the Ado-binding site. The enzyme showed an intrinsic fluorescence with an emission maximum at 340 nm when excited at 295 nm. The protein fluorescence was partially quenched on addition of Ado. PGO modification also led to significant quenching of the fluorescence. However, the fluorescence of the Ado-protected enzyme, which displayed 82% of the original activity after PGO treatment, was retained. The kinetic analyses of the partially modified enzyme showed an increase in the Km for Ado from 14 to 55 microM. Furthermore, the inability of the modified enzyme to bind to 5'-AMP-Sepharose 4B affinity column provided additional evidence that modification is attended by decrease in affinity of the enzyme for Ado. The results are consistent with the interpretation that modification of the active-site arginine residue affects activity by interfering with the binding of the substrate to the active site.
We have isolated, purified, and identified by chemical analyses and mass spectrometry, a novel 3-oxo-2-tetradecyloctadecanoate (dehydrocorynomycolate)-containing phospholipid (PL-1) from the chloroform-methanol extract of Corynebacterium diphtheriae. This phospholipid was separated from all of the other known dehydrocorynomycolate and 3-hydroxy-2-tetradecyloctadecanoate (corynomycolate)-containing lipids and found to be unstable even at -20 degrees C. It was present in trace amounts as a homologous series (molecular weights of 1400 and 1404 as the methyl esters) and composed of a dehydrocorynomycolate, a phosphate group, a diacylglycerol, and an unidentified amine-containing component. Because of the complexity of these phospholipids, their complete structural determination is yet to be completed. A cell-free extract of C. diphtheriae catalyzed the incorporation of radiolabel from [14C]palmitic acid into PL-1. This incorporation was ATP-dependent, and the rate was linear with respect to both time and protein concentration. The radiolabel was incorporated primarily into the dehydrocorynomycoloyl moiety of PL-1. While avidin did not show any significant effect, cerulenin showed a marked inhibition of this reaction. Based on these results, we suggest that this dehydrocorynomycolate-containing PL-1 may be the long-sought acyl carrier-containing product of a Claisen-type condensation.
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A potential dolichol recognition sequence (PDRS) with the 11-residue consensus F-I/V-X-F/Y-X-X-I-P-F-X-F/Y can be found in each of five microsomal enzymes that interact with dolichol or a dolichol derivative. However, no direct evidence demonstrating a role for the PDRSs has been reported. Hamster UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT) differs from the other enzymes by having two PDRSs. Stable CHO-K1 transfectants were created that expressed elevated amounts of normal GPT, GPT with a scramble mutation at the first PDRS (nearest the amino terminus), or GPT with a triple alanine-replacement mutation at the second PDRS. The mutant enzymes had no detectable catalytic activity in vivo, but were fully capable of conferring cellular resistance to the GPT inhibitor tunicamycin. In vitro studies with membrane preparations confirmed that the mutant enzymes were catalytically inactive and also showed that their recovery in microsomes was diminished compared with normal enzyme. These data demonstrate that each PDRS of hamster GPT is necessary for normal enzyme function. The implications of these data for possible roles of the PDRSs are discussed.
The oxidation of 2'-deoxyguanosine (dG) to 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in free dG and the dG residues of DNA and nucleohistone with H2O2 in the presence of Ni(II) and histidyl oligopeptides, carnosine, homocarnosine and anserine was studied at physiological pH. The oxidation of free dG with H2O2 was enhanced by the oligopeptides, but not by Ni(II) alone. Much greater enhancement was produced by equimolar mixtures of Ni(II) with any of the oligopeptides or with L-histidine. In contrast, the oxidation of dG residues in DNA and nucleohistone with H2O2 was not affected by the oligopeptides, but was enhanced by Ni(II). The latter enhancement remained practically unchanged when Ni(II) was accompanied by equimolar amounts of homocarnosine or anserine, whereas carnosine tended to attenuate that enhancement. The extent of formation of 8-OH-dG in free dG depended on time and concentration of H2O2 and was highest at pH 7.4. An electron spin resonance study of the reaction mixture containing H2O2, Ni(II), carnosine, homocarnosine and/or anserine provided evidence for the generation of .OH radical in the reaction media. Although it has previously been concluded that carnosine, anserine and homocarnosine might serve as anti-oxidants, the present study does not support that conclusion and shows that these compounds may even act as pro-oxidants, especially when they are complexed with Ni(II). The results suggest that carnosine, homocarnosine and anserine, by enhancing oxidation of the free dG pool, may potentiate the carcinogenic effects of Ni(II) since the resulting 8-OH-dG can be misincorporated into DNA and thus produce a mutagenic lesion.
Members of the phthiocerol dimycocerosate family of waxes were extracted from Mycobacterium bovis BCG, Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans and a skin biopsy from a leprosy patient. The waxes were degraded by alkaline hydrolysis and the mycocerosic acids converted to pentafluorobenzyl ester. Profiles of the esters, recorded using electron-capture gas-chromatography, gave characteristic profiles for the mycocerosates from M. leprae but those from M. bovis, M. tuberculosis and M. kansasii were superficially similar. The mycocerosate profiles from M. marinum and M. ulcerans were similar, but distinct from the others. Selected ion monitoring negative ion-chemical ionisation gas chromatography-mass spectrometry of of the pentafluorobenzyl esters allowed the analysis of mycocerosate isomers not revealed on gas chromatography alone. M. bovis and M. tuberculosis had similar profiles of C29, C30 and C32 mycocerosates; and additional C33 component was also present in M. kansasii. The mycocerosates from M. marinum and M. ulcerans were C27, C29 and C30 and those from M. leprae were distinct in having C29, C30, C32, C33 and C34 components. These methods have excellent potential for use in the detection of mycobacterial disease by direct analysis of infected tissue without prior cultivation of the causative agent.