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Biomedical subjects

A Bhaduri

Publications and source records attributed to A Bhaduri.

At least 19 recordsLinked to original sources
PubMed2004

iMOT: an interactive package for the selection of spatially interacting motifs.

Functional selection and three-dimensional structural constraints of proteins relate to the retention of significant sequence similarity between proteins of similar fold and function despite poor overall sequence identity and evolutionary pressures. We report the availability of 'iMOT' (interacting MOTif) server, an interactive package for the automatic identification of spatially interacting motifs among distantly related proteins sharing similar folds and possessing common ancestral lineage. Spatial interactions between conserved stretches of a protein are evaluated by calculations of pseudo-potentials that describe the strength of interactions. Such an evaluation permits the automatic identification of highly interacting conserved regions of a protein. Interacting motifs have been shown to be useful in searching for distant homologues and establishing remote homologies among the largely unassigned sequences in genome databases. Information on such motifs should also be of value in protein folding, modelling and engineering experiments. The iMOT server can be accessed from http://www.ncbs.res.in/~faculty/mini/imot/iMOTserver.html. Supplementary Material can be accessed from: http://www.ncbs.res.in/~faculty/mini/imot/supplementary.html.

Amino Acid Motifs↗
PubMed2001

Black tea is a powerful chemopreventor of reactive oxygen and nitrogen species: comparison with its individual catechin constituents and green tea.

Production of black tea [BT] results in biotransformation of catechins of green tea [GT] to theaflavins and thearubigins. BT was found to be more efficient than GT and its individual catechin constituents in proportionate amounts in abrogating production of NO and O2(-) in activated murine peritoneal macrophages. In a reconstitution system of BT that is free of all catechins, stepwise addition of catechins showed that though all the constituents contributed to the overall effect of BT, theaflavin was the most powerful in abrogating NO production. RT-PCR analysis also showed theaflavin to be the most important constituent in down-regulating synthesis of iNOS. Clearly, BT containing theaflavin is an excellent chemopreventor against reactive oxygen and nitrogen species.

Animals↗
PubMed2001

Adrenal histoplasmosis.

A 60 year old diabetic was admitted with the history of low grade fever and weight loss of six weeks duration. After investigations, he was detected to have bilateral adrenal masses which on biopsy proved to be due to histoplasmosis. He was treated with itraconazole and made complete recovery.

Adrenal Gland Diseases↗
PubMed2000

Leishmania plasma membrane Mg2+-ATPase is a H+/K+-antiporter involved in glucose symport. Studies with sealed ghosts and vesicles of opposite polarity.

Experiments from other laboratories conducted with Leishmania donovani promastigote cells had earlier indicated that the plasma membrane Mg2+-ATPase of the parasite is an extrusion pump for H+. Taking advantage of the pellicular microtubular structure of the plasma membrane of the organism, we report procedures for obtaining sealed ghost and sealed everted vesicle of defined polarity. Rapid influx of H+ into everted vesicles was found to be dependent on the simultaneous presence of ATP (1 mm) and Mg2+ (1 mm). Excellent correspondence between rate of H+ entry and the enzyme activity clearly demonstrated the Mg2+-ATPase to be a true H+ pump. H+ entry into everted vesicle was strongly inhibited by SCH28080 (IC50 = approximately 40 microm) and by omeprazole (IC50 = approximately 50 microm), both of which are characteristic inhibitors of mammalian gastric H+,K+-ATPase. H+ influx was completely insensitive to ouabain (250 microm), the typical inhibitor of Na+,K+-ATPase. Mg2+-ATPase activity could be partially stimulated with K+ (20 mm) that was inhibitable (>85%) with SCH28080 (50 microm). ATP-dependent rapid efflux of 86Rb+ from preloaded vesicles was completely inhibited by preincubation with omeprazole (150 microm) and by 5,5'-dithiobis-(2-nitrobenzoic acid) (1 mm), an inhibitor of the enzyme. Assuming Rb+ to be a true surrogate for K+, an ATP-dependent, electroneutral stoichiometric exchange of H+ and K+(1:1) was established. Rapid and 10-fold active accumulation of [U-(14)C]2-deoxyglucose in sealed ghosts could be observed when an artificial pH gradient (interior alkaline) was imposed. Rapid efflux of [U-(14)C]d-glucose from preloaded everted vesicles could also be initiated by activating the enzyme, with ATP. Taken together, the plasma membrane Mg2+-ATPase has been identified as an electroneutral H+/K+ antiporter with some properties reminiscent of the gastric H+,K+-ATPase. This enzyme is possibly involved in active accumulation of glucose via a H+-glucose symport system and in K+ accumulation.

Adenosine Triphosphate↗
PubMed1999

Synthesis and characterization of stacked and quenched uridine nucleotide fluorophores.

Intramolecular aromatic interactions in aqueous solution often lead to stacked conformation for model organic molecules. This designing principle was used to develop stacked and folded uridine nucleotide analogs that showed highly quenched fluoroscence in aqueous solution by attaching the fluorophore 1-aminonaphthalene-5-sulfonate (AmNS) to the terminal phosphate via a phosphoramidate bond. Severalfold enhancement of fluorescence could be observed by destacking the molecules in organic solvents, such as isopropanol and dimethylsulfoxide or by enzymatic cleavage of the pyrophosphate bond. Stacking and destacking were confirmed by 1-H NMR spectroscopy. The extent of quenching of the uridine derivatives correlated very well with the extent of stacking. Taking 5-H as the monitor, temperature-variable NMR studies demonstrated the presence of a rapid interconversionary equilibrium between the stacked and open forms for uridine-5'-diphosphoro-beta-1-(5-sulfonic acid) naphthylamidate (UDPAmNS) in aqueous solution. DeltaH was calculated to be -2.3 Kcal/mol, with 43-50% of the population in stacked conformation. Fluorescence lifetime for UDPAmNS in water was determined to be 2.5 ns as against 11 ns in dimethyl sulfoxide or 15 ns for the pyrophosphate adduct of AmNS in water. Such a greatly reduced lifetime for UDPAmNS in water suggests collisional interaction between the pyrimidine and thefluorophore moieties to be responsible for quenching. The potential usefulness of such stacked and quenched nucleotide fluorophores as probes for protein-ligand interaction studies has been briefly discussed.

Fluorescence↗
PubMed1999

An arginine residue is essential for stretching and binding of the substrate on UDP-glucose-4-epimerase from Escherichia coli. Use of a stacked and quenched uridine nucleotide fluorophore as probe.

In the previous paper we demonstrated that uridine-5'-beta-1-(5-sulfonic acid) naphthylamidate (UDPAmNS) is a stacked and quenched fluorophore that shows severalfold enhancement of fluorescence in a stretched conformation. UDPAmNS was found to be a powerful competitive inhibitor (Ki = 0.2 mM) for UDP-glucose-4-epimerase from Escherichia coli. This active site-directed fluorophore assumed a stretched conformation on the enzyme surface, as was evidenced by full enhancement of fluorescence in saturating enzyme concentration. Complete displacement of the fluorophore by UDP suggested it to bind to the substrate binding site of the active site. Analysis of inactivation kinetics in presence of alpha,beta-diones such as phenylglyoxal, cyclohaxanedione, and 2,3-butadione suggested involvement of the essential arginine residue in the overall catalytic process. From spectral analysis, loss of activity could also be directly correlated with modification of only one arginine residue. Protection experiments with UDP showed the arginine residue to be located in the uridyl phosphate binding subsite. Unlike the native enzyme, the modified enzyme failed to show any enhancement of fluorescence with UDPAmNS clearly demonstrating the role of the essential arginine residue in stretching and binding of the substrate. The potential usefulness of such stacked and quenched nucleotide fluorophores has been discussed.

Arginine↗
PubMed1999

Ca2+ and calmodulin-dependent protein phosphatase from Leishmania donovani.

A protein phosphatase exclusively dependent upon micromolar amounts of Ca2+ and calmodulin has been identified and partially purified from Leishmania spp. Complete obliteration of its activity is observed in the presence of calmodulin antagonists such as trifluoperazine, fluphenazine and calmidazolium. Relative insensitivity to okadaic acid and lack of activation in the absence of Ca2+ and calmodulin distinguishes this enzyme from PP1, PP2A and PP2C-type protein phosphatases. Cross-reactivity of the enzyme was observed with antibodies that recognize both the A and B chains of calcineurin, a PP2B type Ca2+ and calmodulin-dependent phosphatase from brain. FK506, an immunosuppresive drug that inhibits the enzyme from other sources inhibited the enzyme only in the presence of exogenous FK binding protein, whereas Cyclosporin A inhibited the enzyme in crude preparations. Taken together these results reveal the presence of a Ca2+ and calmodulin-dependent phosphatase from Leishmania. This is the first report of the presence of a PP2B-type protein phosphatase from a pathogenic protozoa.

Animals↗
PubMed1998

UDP-galactose 4-epimerase from Kluyveromyces fragilis: reconstitution of holoenzyme structure after dissociation with parachloromercuribenzoate.

UDP-galactose 4-epimerase from yeast Kluyveromyces fragilis (Kluyveromyces marxianus var. marxianus) is a homodimer of molecular mass 75 kDa/subunit and has one mol NAD firmly bound/dimer. The pathway for the assembly of the holoenzyme structure has been studied after dissociating the native epimerase with p-chloromercuribenzoate into inactive mercurated monomers. The process of dissociation was not associated with unfolding of the molecules. Reconstitution of the functional holoenzyme was done by reduction with dithiothreitol and addition of extra NAD. The reaction was thus followed to monitor maturation of the enzyme from the folded monomeric state. The reconstituted enzyme was similar to the native enzyme in terms of a number of physiochemical properties such as secondary, tertiary and quarternary structures, Km for the substrate UDP-galactose, reductive inhibition, interaction with the fluorophore 1-anilino 8-naphthalene sulphonic acid (ANS), etc. Reconstitution under low ionic strength buffer (I = 0.011) shows that the presence of NAD is essential for the formation of a dimeric structure. However, dimeric apoenzyme could also be stabilized under high ionic strength buffer (I = 0.1). Reactivation was strongly dependent on pH, being most effective at pH 8.1. Kinetic evidence suggested that, at low ionic strength, assembly of NAD over dimeric apoenzyme is the rate-limiting step in expressing catalytic activity. This process has a low energy of activation of 27.2 kJ/mol.

Anilino Naphthalenesulfonates↗
PubMed1998

Oxygen therapy for children. Need-based preparation and evaluation of a self-instructional module for staff nurses on care of a child receiving oxygen therapy.

The present study was carried out with the objectives of determining the learning needs of Staff Nurses regarding care of children receiving Oxygen Therapy; finding association between learning needs and selected variables: age, total years of experience, experience in Paediatric Ward, married with or without children; determining validity of self-instructional module, on "care of child receiving Oxygen Therapy", and evaluating the effectiveness of the self-instructional module or SIM. The study was conducted in two phases. A survey approach was used for Phase-I and one group pre-test post-test design was adopted for Phase-II. The total sample of the study was 30 Staff Nurses, of 6 months experience in Paediatric Ward. The findings of the study showed high learning need status in most of the areas and the Staff Nurses also expressed the desirable need for learning in detail. It was found that age, total years of experience, experience in Paediatric Ward and married with or without children were independent of their learning need. SIM was effective in terms of gain in knowledge score as well as acceptability and utility scores of Staff Nurses.

Adult↗
PubMed1997

The plasma-membrane Ca2+-ATPase of Leishmania donovani is an extrusion pump for Ca2+.

Controlled exposure of Leishmania donovani promastigotes to hypotonic shock results in the formation of deflagellated unsealed ghosts of original polarity that largely retain the pellicular microtubular structure associated with plasma membrane of the parasite. Gentle shearing followed by suspension of the purified membrane in appropriate isotonic buffer containing Mg2+ (4mM) results in the formation of sealed everted vesicles. The presence of Mg2+ (4 mM) appears to be essential for efficient sealing and also to prevent leakiness. ATP-dependent Ca2+ accumulation can be demonstrated in these vesicles Km values for Ca2+ and ATP were 125 nM and 0.8 mM respectively. The accumulated Ca2+ reaches a concentration of 1.1 mM. Ca2+ uptake is completely inhibited by vanadate (40 microM) and several thiol-modifying agents. Using 5,5'-dithiobis-(2-nitrobenzoic acid) as the modifying agent, an excellent correlation between loss of enzyme activity and transport capability and their parallel regeneration in the presence of 2 mM dithiothreitol was demonstrated. Using 2'.7-bis(carboxyethyl)-5(6)-carboxyfluorescein as the fluorescent pH probe, it was observed that Ca2+ entry into the vesicles is accompanied by an outward movement of H+ from the vesicles. Taken together, this paper establishes that the high-affinity transmembrane Ca2+-ATPase [Ghosh, Ray, Sarkar and Bhaduri (1990) J. Biol. Chem. 265, 11345-11351; Majumdar, Mukherjee, Ray and Bhaduri (1992) J. Biol. Chem. 267, 18440-18446] is an extrusion pump for Ca2+ in this human pathogen.

Adenosine Triphosphate↗
PubMed1996

Effect of acetylation of ovalbumin on its adsorption behavior at solid/liquid interface.

This paper reports the effect of modification of lysine residues on the adsorption of ovalbumin at alumina/water interface. It has been shown that the pH dependence of the adsorption changes on acetylation of lysine. Thus at pH 7.6 acetylated ovalbumin does not show any affinity for alumina surface although unmodified protein does. It seems that although electrostatic interactions are operative, surface unfolding of proteins and surface hydrophobicity of protein also control the adsorption of ovalbumin onto alumina.

Acetylation↗
PubMed1995

Temperature-induced rapid increase in cytoplasmic free Ca2+ in pathogenic Leishmania donovani promastigotes.

We demonstrate that the cytoplasmic free Ca2+ concentration in three virulent Leishmania donovani promastigote strains is maintained at 20-30 nM level whereas the avirulent promastigotes maintain [Ca2+]i at 80-100 nM. Rapid exposure of virulent promastigotes to higher physiological temperature increases [Ca2+]i many-fold. Use of CaCl2, EGTA and Mn2+ confirmed that both release of Ca2+ from internal pool(s) and influx from outside milieu are responsible for this increase in [Ca2+]i. Application of oligomycin, CN-, and nigericin indicated the non-mitochondrial pHi sensitive pool as the possible intracellular origin for internal Ca2+ release. Exposure of these cells to acidic environment had no influence on [Ca2+]i. In contrast, the avirulent promastigotes or freshly isolated amastigotes had shown no effect of heat-shock or pH shock on [Ca2+]i of these cells. Our results indicate that the upward shift in temperature may play a role in signal transduction events in morphogenetic transformation of L. donovani promastigotes that involves mobilization of Ca2+ in cytoplasm.

Animals↗
PubMed1995

Two tryptophans at the active site of UDP-glucose 4-epimerase from Kluyveromyces fragilis.

Efficient fluorescence energy transfer from aromatic residues to the pyridine moiety of the bound coenzyme (NAD) of UDP-glucose 4-epimerase from Kluyveromyces fragilis had been reported earlier (Mukherji, S., and Bhaduri, A. (1992) J. Biol. Chem. 267, 11709-11713). We have employed N-bromosuccinimide (NBS) to identify tryptophan as the exclusive aromatic donor in the energy transfer. The characteristic UV absorption spectrum associated with Trp oxidation is observed during NBS modification of two of the four Trp residues of native epimerase along with concomitant inactivation of the enzyme. Excellent correlation between the observed inactivation and abolition of fluorescence energy transfer to coenzyme from Trp in epimerase upon treatment with NBS implicates the involvement of the same two tryptophans in both catalytic activity and fluorescence energy transfer. SDS-polyacrylamide gel electrophoresis and fluorescence data preclude gross structural/conformational changes in epimerase due to NBS oxidation. The susceptible tryptophans do not reside at the substrate binding site as substrates and UMP fail to protect against NBS modification. However, failure of sodium borohydride to reduce the bound NAD in the NBS-inactivated epimerase suggests that the reactive tryptophans are close to the coenzyme. Tryptophan fluorescence lifetime values of 1.9 and 3.9 ns for the native and 3.5 ns for the NBS-modified epimerase, complemented by a linear Stern-Volmer plot (effective Stern-Volmer constant = 2.85 M-1) of acrylamide quenching, suggest that the two key tryptophans are buried close to an intrinsic quencher, presumably NAD.

Binding Sites↗
PubMed1995

Characterization of the respiratory chain of Leishmania donovani promastigotes.

Inhibition analysis of respiration of Leishmania donovani promastigotes in resting, starved and permeabilized cells in the presence of classical electron transfer complex inhibitors such as rotenone, thenoyltrifluoroacetone and antimycin demonstrated the absence of complex I component of the respiratory chain in this organism. Cyanide failed to completely block the oxygen uptake (residual 25-30%) even at high concentrations. The alternative oxidase inhibitor for Trypanosoma brucei, salicylhydroxamic acid (SHAM) had no effect on respiration while the cytochrome o inhibitor orthohydroxydiphenyl (OHD) could block cyanide-insensitive respiration at low concentrations. Succinate-dependent O2 uptake in permeabilized cells follows the classical pathway. Oxidation of NADH by a membrane-rich fraction produced H2O2 as the end product and was insensitive to respiratory chain inhibitors. The presence of NADH-fumarate reductase was demonstrated in membrane-rich fraction and fumarate could reduce H2O2 production from NADH indicating fumarate to be an endogenous substrate for accepting electrons from NADH. A differential route for NADH oxidation was further confirmed by NADH cytochrome c reductase insensitivity to antimycin. A tentative scheme for electron transfer pathway in this organism is proposed in which a reversal of Krebs cycle enzymes occur producing succinate that can be excreted or oxidized depending upon the energy demands of the cell. Inhibition studies also suggest bifurcations of the respiratory chain that can be of minor importance for the organism.

Animals↗