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Biomedical subjects

A Bhaduri

Publications and source records attributed to A Bhaduri.

At least 37 records · Page 2Linked to original sources
PubMed1992

Allosteric modulation of Leishmania donovani plasma membrane Ca(2+)-ATPase by endogenous calmodulin.

The plasma membrane of the human pathogen Leishmania donovani possesses a high-affinity transmembrane Ca(2+)-ATPase that has its catalytic site oriented toward the cytoplasmic milieu (Ghosh, J., Ray, M., Sarkar, S., and Bhaduri, A. (1990) J. Biol. Chem. 265, 11345-11351). When the enzyme is studied in its more authentic, physiologically relevant, membrane-associated form, it exhibits pronounced sigmoidal kinetics with Ca2+ (K0.5 approximately 700 nM) in a trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid buffering system that effectively complexes all available Mg2+. Addition of exogenous Mg2+ (60 microM) completely abolishes sigmoidicity and establishes strictly hyperbolic kinetics, and the Km for Ca2+ reduces to 100 nM. Mg2+ can be replaced by heterologous calmodulin. The exclusive dependence of the enzyme on only Ca2+ for its activity and its positive allosteric modulation by Mg2+ distinguish this enzyme from other well-characterized plasma membrane Ca(2+)-ATPases. Employing this Ca(2+)-ATPase as the assay system, a soluble endogenous activating protein factor was purified that, by several criteria, corresponds to authentic calmodulin. The parasite calmodulin shifts the kinetics to hyperbolic kinetics, increases the Vmax 2-fold, and most important lowers the Km (approximately 100 nM) to a physiological level. The interaction with endogenous calmodulin thus converts the enzyme from a totally inactive to a fully active state.

Adenosine Triphosphate↗
PubMed1992

An essential histidine residue for the activity of UDPglucose 4-epimerase from Kluyveromyces fragilis.

UDPglucose 4-epimerase from Kluyveromyces fragilis was completely inactivated by diethylpyrocarbonate following pseudo-first order reaction kinetics. The pH profile of diethylpyrocarbonate inhibition and reversal of inhibition by hydroxylamine suggested specific modification of histidyl residues. Statistical analysis of the residual enzyme activity and the extent of modification indicated modification of 1 essential histidine residue to be responsible for loss in catalytic activity of yeast epimerase. No major structural change in the quarternary structure was observed in the modified enzyme as shown by the identical elution pattern on a calibrated Sephacryl 200 column and association of coenzyme NAD to the apoenzyme. Failure of the substrates to afford any protection against diethylpyrocarbonate inactivation indicated the absence of the essential histidyl residue at the substrate binding region of the active site. Unlike the case of native enzyme, sodium borohydride failed to reduce the pyridine moiety of the coenzyme in the diethylpyrocarbonate-modified enzyme. This indicated the presence of the essential histidyl residue in close proximity to the coenzyme binding region of the active site. The abolition of energy transfer phenomenon between the tryptophan and coenzyme fluorophore on complete inactivation by diethylpyrocarbonate without any loss of protein or coenzyme fluorescence are also added evidences in this direction.

Diethyl Pyrocarbonate↗
PubMed1992

Distinct functional roles of two active site thiols in UDPglucose 4-epimerase from Kluyveromyces fragilis.

UDPglucose 4-epimerase from Kluyveromyces fragilis was earlier shown to have two conformationally vicinal thiols at the active site. Upon treatment with diamide, these thiols form a disulfide linkage across the subunits that results in coordinated loss of catalytic activity and coenzyme fluorescence (Ray, M., and Bhaduri, A. (1980) J. Biol. Chem. 255, 10777-10786). Employing a number of thiol-specific reagents, we now suggest discriminatory and nonidentical roles for these two thiols. Kinetic and statistical analysis of 5,5'-dithiobis-(2-nitrobenzoic acid) and N-ethylmaleimide modification reaction of epimerase show that only one thiol is essential for activity. Consecutive modification experiments clearly show that the same active thiol is modified in both cases. However, significant differences are observed when the reactivity of these reagents is monitored in terms of coenzyme fluorescence. Treatment with N-ethylmaleimide leads to a form of inactive enzyme that fully retains its fluorescent properties whereas modification with 5,5'-dithiobis-(2-nitrobenzoic acid), on the other hand, results in the loss of both activity and fluorescence. The closely spaced nonessential second thiol, which is not modified by N-ethylmaleimide is therefore involved in generating and maintaining the coenzyme fluorescence. Modification studies with a series of spin-labeled maleimide shows that only 3-(maleimidomethyl)proxyl causes partial quenching of coenzyme fluorescence. This suggests that the active thiol is situated at a distance of 4.5 A approximately from the coenzyme fluorophore.

Binding Sites↗
PubMed1992

Antileishmanial activity of hamycin: a polyene antibiotic.

Hamycin, a polyene antibiotic, now in extensive use in the treatment of candidiasis and otomycosis, is found to be remarkably effective in killing Leishmania donovani promastigotes in a liquid medium at a concentration of 0.2 microgram/ml. The glucose stimulated respiration and the uptake of 2-deoxy-D[U-14C]-glucose was inhibited in cells treated with the drug at a growth inhibitory concentration. An immediate release of isotopic glucose from preloaded cells could be demonstrated after exposure to hamycin. All the above effects could be effectively prevented in the presence of ergosterol. The primary site of action of hamycin on L. donovani promastigote cells appears to be membrane sterols that result in the loss of the permeability barrier to small metabolites. The lower minimum inhibitory concentration of hamycin compared to other established drugs warrants further study in the context of increasing reports of clinical resistance to pentavalent antimonials.

Amphotericin B↗
PubMed1992

Microenvironment at the substrate binding subsite of the active site of UDPglucose 4-epimerase from Kluyveromyces fragilis using a fluorescent analog of UMP.

A chromophorics and fluorescent analog of uridine 5'-monophosphate (UMP), a known competitive inhibitor of UDPglucose 4-epimerase was synthesised. This analog, namely 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) uridine 5'-monophosphate, was found to be a powerful reversible inhibitor of UDPglucose 4-epimerase indicating its interaction with the substrate binding site of the enzyme. The extreme sensitivity of the fluorescence emission spectrum of this analog to solvent polarity makes it an excellent probe for the study of the environment at the active site of the enzyme. We report here the effective use of this UMP analog to demonstrate that the hydroxyl groups of the ribose moiety of UMP and presumably the substrates (UDPgalactose and UDPglucose) do not reside in a hydrophobic milieu.

Binding Sites↗
PubMed1990

Acivicin: a highly active potential chemotherapeutic agent against visceral leishmaniasis.

Acivicin, a chlorinated amino acid antibiotic, is found to be remarkably effective in killing both the vector and the host form of the parasitic protozoa, Leishmania donovani, the causative agent for visceral leishmaniasis or Kala-azar. The ED50 (50 nM) for the pathogenic amastigote form in in vitro screening system is significantly lower than the reported values for other drugs under trial. The drug irreversibly inactivates both in vitro and in vivo carbamyl phosphate synthetase II, the first enzyme of the pyrimidine biosynthetic pathway. The irreversible inactivation of this sensitive target enzyme and lack of effective reversal by glutamine makes acivicin a preferred candidate for potential chemotherapy against increasing number of Kala-azar cases that are reported to be unresponsive to pentavalent antimonials.

Animals↗
PubMed1990

A high affinity Ca2(+)-ATPase on the surface membrane of Leishmania donovani promastigote.

A Ca2(+)-dependent ATP-hydrolytic activity was detected in the crude membrane ghost of the promastigote or vector form of the protozoal parasite Leishmania donovani, the pathogen responsible for kala azar. The Ca2(+)-ATPase was purified to apparent homogeneity after solubilization with deoxycholate. The enzyme consists of two subunits of Mr = 51,000 and 57,000 and has an apparent molecular weight of 215,000 +/- 12,000. The enzyme activity is exclusively dependent on Ca2+, and the pure enzyme can hydrolyze 1.6 mumol of ATP/min/mg of protein. The apparent Km for Ca2+ is 35 nM, which is further reduced to 12 nM in the presence of heterologous calmodulin. The enzyme is sensitive to vanadate, but is insensitive to oligomycin and ouabain. The enzyme is strongly associated with the plasma membrane and has its catalytic site oriented toward the cytoplasmic face. The enzyme spans across the plasma membrane as surface labeling with radioiodine shows considerable radioactivity in the completely purified enzyme. The localization and orientation of this high affinity, calmodulin-sensitive Ca2(+)-ATPase suggest some role of this enzyme in Ca2+ movement in the life cycle of this protozoal parasite.

Adenosine Triphosphate↗
PubMed1990

Plasma prolactin as an indicator of disease progression in advanced breast cancer.

Serial plasma prolactin levels were measured in 144 breast cancer patients (premenopausal [PR-M], N = 64; postmenopausal [PO-M], N = 80) and compared with respective controls. Patients with breast cancer were grouped into those who (1) developed distant metastasis, (2) developed local recurrence, (3) stable disease, and (4) responded to the various therapeutic modalities at the end of 2 years. The authors' analysis showed excellent correlation between serial plasma prolactin changes and the response to therapy or progression of disease in patients with advanced breast carcinoma.

Biomarkers, Tumor↗
PubMed1988

Prognostic value of squamous cell carcinoma antigen in patients with uterine cervical carcinoma.

Squamous cell carcinoma antigen (SCC Ag) was estimated in 30 controls, in 16 patients with benign lesions of the uterine cervix, and in 51 patients with uterine cervical carcinoma. The rate of positivity of the antigen among the cancer patients was 87% (N = 49). SCC Ag estimations were of no diagnostic value, since 37% of patients with benign lesions had elevated levels compared to controls. SCC Ag was highly correlated to histologic subtype. The highest values were obtained in keratinizing tumors, followed by large cell nonkeratinizing and small cell nonkeratinizing types. Moreover, elevation of SCC Ag was statistically significant (p less than 0.001) among all the three histologic subtypes compared to controls. However, SCC Ag levels were not consistently correlated to the stage of the disease. Patients with pretherapeutic SCC Ag levels above 30 ng/ml had a faster recurrence rate and shorter survival than those who exhibited antigen values below 2.0 ng/ml. From our results, it is suggested that SCC Ag has limited use as a parameter for early diagnosis of cervical carcinoma, but it seems to reflect advancement of the disease. These findings indicate that SCC Ag elevation may prove to be a valuable marker in predicting subclinical disease.

Antigens, Neoplasm↗
PubMed1988

Aspartate transcarbamylase from Leishmania donovani. A discrete, nonregulatory enzyme as a potential chemotherapeutic site.

Leishmania donovani is a protozoal pathogen that belongs to the kinetoplastida order. Unlike in other eucaryotic systems, the first three enzymes of the de novo pyrimidine biosynthetic pathway are not components of a multifunctional protein system. The three enzyme activities in the crude extract were separated on a Sephacryl S-200 column. Aspartate carbamoyltransferase (EC 2.1.3.2) has been purified to apparent homogeneity. The enzyme has an approximate molecular weight of 135,000 and seems to be a tetramer of equivalent subunits of molecular weight 35,000. The enzyme shows strictly hyperbolic kinetics with both the substrates under a variety of conditions and is not inhibited by nucleotide phosphates. Km for carbamyl phosphate is 3.1 x 10(-4) M and for aspartate is 7.6 x 10(-3) M. Apparently, the enzyme has no regulatory role in pyrimidine biosynthesis. N-(Phosphonoacetyl)-L-aspartic acid is a powerful competitive inhibitor (Ki = 5 x 10(-7) M) for this enzyme with carbamyl phosphate as substrate. This inhibitor completely inhibits the growth of the vector form of organism at 60 microM and significantly affects the growth of the pathogenic form in a macrophage assay system. The potency of the inhibitor is comparable with allopurinol which is undergoing human clinical trial as an antileishmanial drug.

Animals↗
PubMed1987

Prognosis in breast carcinoma utilizing plasma carcinoembryonic antigen and histologic characteristics of the primary tumor.

Plasma carcinoembryonic antigen (CEA) concentrations in 128 patients with breast cancer were measured preoperatively. The data were related to the histologic features of the primary breast carcinoma and to the clinical follow-up data. Analysis of the plasma CEA values did not show a significant correlation with the histologic type and the histologic and nuclear grade of the primary tumor (n = 73) as well as to the presence or absence of keratin, necrosis, desmoplasia, tubule formation and mucin production. Furthermore, the results indicated that high CEA values (more than 10 ng/ml) may be associated with distant metastasis and not with the metastatic spread to lymph nodes. High CEA levels were also associated with reduced survival of the patients. This study confirms our previous report suggesting that high CEA levels are correlated with tumors of endodermal origin, whereas the CEA levels were within the normal range in the tumors of ectodermal origin. In agreement with other studies, however, it was found that the predictive value of plasma CEA concentrations in general is weak, so that the use of CEA measurement for prognosis is of limited value.

Breast Neoplasms↗
PubMed1986

UDP-glucose 4-epimerase from Saccharomyces fragilis. Presence of an essential arginine residue at the substrate-binding site of the enzyme.

UDP-glucose 4-epimerase from Saccharomyces fragilis was inactivated by the arginine-specific reagents phenylglyoxal, 1,2-cyclohexanedione, and 2,3-butanedione following pseudo first order reaction kinetics. The reaction order with respect to phenylglyoxal was 1.8 and that with respect to the other two diones was close to unity. Protection afforded by substrate and competitive inhibitors against inactivation by phenylglyoxal and the reduced interaction of 1-anilinonaphthalene 8-sulfonic acid, a fluorescent probe for the substrate-binding region after phenylglyoxal modification, suggested the presence of an essential arginine residue at the substrate-binding region. Experiments with [7-14C]phenylglyoxal in the presence of UMP, a ligand known to interact at the substrate-binding region, showed that only the arginine residue at the active site could be modified by phenylglyoxal. The characteristic coenzyme fluorescence of the yeast enzyme was found to be enhanced three times in phenylglyoxal-inactivated enzyme suggesting the incorporation of the phenyl ring near the pyridine moiety of NAD.

Arginine↗
PubMed1986

Mechanism of action of amphotericin B on Leishmania donovani promastigotes.

The growth of Leishmania donovani promastigotes in a liquid medium was completely inhibited by amphotericin B at a concentration of 0.3 microgram ml-1 (0.3 microM). Continuous release of small molecules that absorb at 260 nm and 280 nm was observed after contact with the drug. Uptake of [U-14C]glucose was inhibited in cells treated with the drug. An immediate release of isotopic glucose and its metabolites from preloaded cells could be demonstrated after incubation with amphotericin B (0.4 microM). Inhibition of respiration by the drug was a comparatively slower process. All the above effects could be effectively prevented in the presence of either cholesterol or ergosterol. The primary site of action of amphotericin B on L. donovani promastigote cells appears to be membrane sterols that result in a loss of the permeability barrier to small metabolites. An interesting biochemical similarity, thus, emerges between flagellated protozoa and fungi.

Amphotericin B↗
PubMed1984

A study of yoga as a nursing intervention in the care of patients with pleural effusion.

'Pranayama' or yogic breathing as a method of re-expansion of lungs in patients with pleural effusion was studied. Ten patients with pleural effusion practised alternate nostril breathing for 20 days after aspiration of fluid. An equal number matched for age and smoking habits underwent routine physiotherapy of the hospital for the same period. Lung function was measured: before aspiration; immediately after aspiration; and, 5, 10, 15 and 20 days after aspiration. The FVC, FEV1, MVV, PEFR, CE and RS, were used to measure lung function. The difference between the two groups in the gain in lung expansion as assessed by the above measures was tested for significance with appropriate nonparametric statistical tests at 0.1 level of significance. The results revealed that the patients practising Pranayama demonstrated a quicker re-expansion of the lungs in most of the measures of lung function. The findings are discussed in relation to implications for nursing care.

Adolescent↗