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Biomedical subjects

A K Datta

Publications and source records attributed to A K Datta.

At least 73 records · Page 4Linked to original sources
PubMed1988

Reaction kinetics and inhibition of adenosine kinase from Leishmania donovani.

The reaction kinetics and the inhibitor specificity of adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) from Leishmania donovani, have been analysed using homogeneous preparation of the enzyme. The reaction proceeds with equimolar stoichiometry of each reactant. Double reciprocal plots of initial velocity studies in the absence of products yielded intersecting lines for both adenosine and Mg2+-ATP. AMP is a competitive inhibitor of the enzyme with respect to adenosine and noncompetitive inhibitor with respect to ATP. In contrast, ADP was a noncompetitive inhibitor with respect to both adenosine and ATP, with inhibition by ADP becoming uncompetitive at very high concentration of ATP. Parallel equilibrium dialysis experiments against [3H]adenosine and [gamma-32P]ATP resulted in binding of adenosine to fre enzyme. Tubercidin (7-deazaadenosine) and 6-methyl-mercaptopurine riboside acted as substrates for the enzyme and were found to inhibit adenosine phosphorylation competitively in vitro. 'Substrate efficiency (Vmax/Km)' and 'turnover numbers (Kcat)' of the enzyme with respect to specific analogs were determined. Taken together the results suggest that (a) the kinetic mechanism of adenosine kinase is sequential Bi-Bi, (b) AMP and ADP may regulate enzyme activity in vivo and (c) tubercidin and 6-methylmercaptopurine riboside are monophosphorylated by the parasite enzyme.

Adenosine↗
PubMed1987

Isolation and characterization of adenosine kinase from Leishmania donovani.

Adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) has been purified 3250-fold from Leishmania donovani promastigotes using ion-exchange, gel filtration, and affinity chromatography techniques. Both native and sodium dodecyl sulfate-gel electrophoresis of the enzyme revealed a single polypeptide of around 38,000 molecular weight. Biophysical and biochemical analyses of the enzyme reveal unique characteristics different from those of adenosine kinases from other eukaryotic sources. The isoelectric pH of the enzyme is 8.8. In native acrylamide gels the enzyme moves with an RF of about 0.62. The enzyme displays a maximum activity at pH between 7.5 and 8.5 and is dependent upon an optimum ATP/Mg2+ ratio. ATP at high concentration inhibits the reaction. Adenosine and Mg2+ are not inhibitory. EDTA completely knocks off the activity. Enzyme activity is dependent upon the presence of active thiol group(s) at or near the active center. Under a defined set of conditions the enzyme exhibited an apparent Km for adenosine and ATP of 33 and 50 microM, respectively. Of the nucleoside triphosphates tested ATP and GTP were the most effective phosphate donors. Marginal inhibition of activity was detected with other nucleosides as competitors. However, adenosine analogs, such as 7-deaza-adenosine (tubercidin) and 6-methylmercaptopurine riboside at very low concentrations, were found to be excellent inhibitors and substrates as well. S-Adenosylhomocysteine does not inhibit the reaction even at very high concentration.

Adenosine Kinase↗
PubMed1987

Appearance of a methoxy mycolate-like component by the acid methanolysis of Mycobacterium leprae.

It has been reported that Mycobacterium leprae contains two types of mycolic acid, namely, alpha- and keto-mycolic acids, thus it is taxonomically similar to M. bovis BCG. However, there was some controversy about the presence of methoxy mycolic acid which was observed in small amounts only in the case of experimentally infected (W45) armadillo-derived M. leprae. To investigate this fact, mycolic acids were extracted from the cell-wall structure of M. leprae and characterized using chromatographic techniques. The results showed the appearance of a methoxy mycolate-like component for both purified bacilli and infected human skin tissue materials. However, this appearance occurred only when the acid methanolysis procedure was followed for the release of mycolic acids from these bacilli. No such component appeared on the chromatogram when the alkaline methanolysis procedure was followed. Nevertheless, the consistent presence of this methoxy mycolate-like component by acid methanolysis is an important finding which has to be kept in mind while identifying this pathogen when using chromatographic techniques.

Chromatography, Thin Layer↗
PubMed1985

Comparative study on the thin-layer chromatographic pattern of methyl mycolates of M. leprae and related species.

The thin-layer chromatographic (TLC) pattern of methyl mycolates of Mycobacterium leprae has been compared with the lipids extracted from M. leprae-infected tissue material and also with those of other cultivable mycobacterial species. It has been found that M. leprae-derived methyl mycolates give two spots, namely, alpha- and keto-derivatives, after charring the TLC plates with K2Cr2O7/H2SO4 spray. Lipids extracted from infected tissue material also show spots with the same Rf values when compared with those of armadillo-derived M. leprae. Normal skin tissue extracts, however, do not show any presence of mycolates. This TLC pattern of M. leprae-derived methyl mycolates is characteristic and is not shown by any other mycobacterial species isolated from leprosy lesions. The strain and species which have been earlier found to resemble M. leprae antigenically or biochemically could readily be distinguished with the help of the TLC pattern of their methyl mycolates.

Cell Wall↗
PubMed1984

A metronidazole-resistant strain of Trichomonas vaginalis and its sensitivity to Go 10213.

In the present study the comparative in-vitro and in-vivo efficacy of Go 10213 was compared with those of metronidazole, secnidazole, tinidazole, ornidazole and nimorazole against a metronidazole-resistant strain of T. vaginalis. Go 10213 was found to be superior in activity. It appears more promising than the drugs mentioned above on the basis of the evidence presented.

Animals↗
PubMed1983

Phosphorylation of acyclovir in vitro in activated Burkitt somatic cell hybrids.

Acyclovir [9-(2-hydroxyethoxymethyl)guanine] (ACV), a potent antiviral compound, was phosphorylated to the same extent by extracts from untreated and iododeoxyuridine-treated Epstein-Barr virus-containing latent D98/HR-1 somatic hybrid cells. ATP was the preferred phosphate donor over other nucleoside triphosphates. The cytosol extract from D98/HR-1 cells effected optimum phosphorylation of thymidine at pH 8.0, whereas ACV was phosphorylated equally well over a wide pH range. Electrophoretic analysis of thymidine kinase-, deoxycytidine kinase-, and ACV-phosphorylating activities from both untreated and iododeoxyuridine-treated cell extracts displayed identical properties. A small part (5 to 10%) of the loaded ACV-phosphorylating activity seemed to migrate with the deoxycytidine kinase activity from cytosol. dTTP and dCTP, at relatively high concentrations, partially inhibited ACV-phosphorylating activity. The results suggest that Epstein-Barr virus does not code for its own thymidine kinase and that phosphorylation of ACV in Epstein-Barr virus-producing cells is carried out by multiple or as yet unidentified ATP-dependent nonspecific cellular phosphotransferases.

Acyclovir↗
PubMed1982

Perspectives on interactions of acyclovir with Epstein-Barr and other herpes viruses.

Acyclovir [9-(2-hydroxyethoxymethyl)guanine] inhibits Epstein-Barr virus (EBV) replication in lymphoblastoid cells at concentrations nontoxic to cellular growth. The mode of action of the drug against EBV differs from the mechanism described in herpes simplex virus systems. Due to the absence of virus-specified thymidine kinase, the drug is poorly phosphorylated in EBV-infected cells. The extent of monophosphorylation is similar both in mock-infected and EBV-infected cells. Despite weak phosphorylation of the drug, the replication of linear EBV DNA is inhibited due to exquisite sensitivity of the viral DNA polymerase. Activation of acyclovir does not require phosphorylation by virus-specified thymidine kinase, inhibition of different herpes-group viruses depends on three variable factors: degree of phosphorylation, cellular metabolism of the drug, and degree of sensitivity of the viral polymerase. Interaction of acyclovir-triphosphate with EBV DNA polymerase is reversible. Cells infected with EBV and treated with acyclovir resume virus replication following removal of the drug even after long exposure. Acyclovir inhibits replication of linear genomes and stops production of virus, but has no effect on latent cellular infection. These results lead us to predict that acyclovir will suppress, but not cure, EBV infection.

Acyclovir↗
PubMed1982

Identification and characterization of a DNase induced by Epstein-Barr virus.

The diterpene ester promoter of mouse skin tumors, 12-O-tetradecanoyl-phorbol-13-acetate, induced a DNase activity in the Epstein-Barr virus-producer cell line P3HR-1. The elution patterns of the enzyme from DEAE-cellulose, phosphocellulose, and DNA-cellulose columns were different from virus-associated DNA polymerase activity. The partially purified activity could be neutralized to the extent of 90% by sera of patients with nasopharyngeal carcinoma. Purified immunoglobulin G from sera of nasopharyngeal carcinoma patients inhibited this enzyme and that obtained from superinfected Raji cells to the same extent. The partially purified enzyme preferred native DNA as a substrate over denatured DNA and 3'-terminally labeled activated calf thymus DNA. The activity was inhibited by high ionic strength. Phosphonoformic acid did not have any effect on this enzyme activity.

Burkitt Lymphoma↗
PubMed1981

The effects of digital nerve stimulation on the firing of motor units in human first dorsal interosseous muscle.

1. The effect of cutaneous stimulation on the firing of single motor units has been studied during voluntary contractions in human first dorsal interosseous muscle. 2. Electrical stimulation of the index finger at 3 x threshold for perception reduced the firing rate of most units recruited at voluntary contraction strengths less than 1.5 N and increased the firing rate of all units recruited at contraction strengths greater than 1.5 N. The firing rate of all slow twitch units (contraction time greater than 75 msec) was reduced. The behaviour of fast twitch units was mixed but at this stimulus strength all units recruited at contraction strengths greater than 1.5 N had their firing rate increased. 3. Tested at different stimulus strengths, the stronger the stimulus the more the firing rate of low threshold units was reduced. The firing rate of units recruited at high contraction strength was increased by weak stimuli but reduced by strong stimuli. 4. It is concluded that stimulation of the index finger shifts the weighting of synaptic input associated with a voluntary contraction to favour the activity of the more powerful fast twitch motor units in first dorsal interosseous muscle.

Action Potentials↗
PubMed1980

Induction of Epstein-Barr virus-associated DNA polymerase by 12-O-tetradecanoylphorbol-13-acetate. Purification and characterization.

The diterpene ester promoter of mouse skin tumors, 12-O-tetradecanoylphorbol-13-acetate (TPA), efficiently induces Epstein-Barr virus (EBV)-associated DNA polymerase (DNA nucleotidyltransferase) activity in the EBV-producing lymphoblastoid cell line, P3HR-1. With the use of intervent dilution chromatography followed by sequential DEAE-cellulose and phosphocellulose column chromatography, the virus-associated enzyme has been isolated and purified 300-fold. The partially purified EBV DNA polymerase activity could be distinguished from cellular polymerases by its activation with salt and its degree of sensitivity to N-ethylmaleimide and phosphonoacetic acid. The enzyme showed maximum activity for copying activated calf thymus DNA in the presence of 100 mM ammonium sulfate. In the absence of salt, the enzyme utilized with high efficiency deoxyoligomer-homopolymer templates, but failed to copy poly(rA) . oligo(dT)10 and oligo(dT)10, showing that the enzyme had properties distinct from DNA polymerase gamma, reverse transcriptase, and terminal deoxynucleotidyltransferase. The partially purified enzyme is strongly inhibited by acyclovir triphosphate and thus has properties similar to herpes simplex virus DNA polymerase.

Acyclovir↗
PubMed1980

Acyclovir inhibition of Epstein-Barr virus replication.

Acyclovir [9-(2-hydroxyethoxymethyl)guanine] triphosphate inhibits Epstein-Barr virus (EBV)-associated DNA polymerase (DNA nucleotidyltransferase; EC 2.7.7.7) to a greater extent than it inhibits host alpha and beta DNA polymerases. The affinity of the compound for viral polymerase is 100-fold higher than for alpha-polymerase. The extent of inhibition is dependent upon the base composition of the template-primer. The inhibition is prevented by increasing concentrations of deoxyguanosine triphosphate. The EBV-associated DNA polymerase reaction in the presence of the inhibitor, although depressed, proceeds at a linear rate over a long period of time. In contrast, the reaction of Escherichia coli DNA polymerase I in the presence of 2',3'-dideoxythymidine 5'-triphosphate, a DNA chain terminator, levels off after initial linearity. Preincubation of acyclovir triphosphate with DNA and enzyme does not increase its inhibitory activity. The virus-producing cell line P3HRF-1 consistently shows reduced viral genome numbers and viral capsid antigen on prolonged exposure to acyclovir. The number of EBV genomes returns to the control level when the cells are grown in drug-free medium. The results suggest that a competitive mechanism is the major mode of acyclovir inhibition of EBV replication.

Acyclovir↗