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Biomedical subjects

S Mukherji

Publications and source records attributed to S Mukherji.

At least 55 records · Page 3Linked to original sources
PubMed1994

Regulation of Ca2+/calmodulin-dependent protein kinase II by inter- and intrasubunit-catalyzed autophosphorylations.

Autophosphorylation of CaM kinase II on Thr286 is known to occur by an intraholoenzyme mechanism, but it is not known whether this reaction is intra- or intersubunit-catalyzed in the native heteromeric enzyme containing 10-12 alpha/beta subunits. In this study inactive CaM kinase II beta subunit, generated by mutation of Lys43 to Ala, and active kinase alpha subunit were expressed separately (homomeric kinases) or co-expressed (heteromeric kinase) using the baculovirus/Sf9 cell expression system and purified on CaM-Sepharose. Ca2+/CaM-dependent autophosphorylation of heteromeric alpha/beta kinase, which activated the enzyme, produced rapid autophosphorylation on Thr286 in both the active alpha and inactive beta subunits; the latter could only occur by intersubunit catalysis. Ca2+/CaM-independent autophosphorylation of nonactivated heteromeric kinase was slow, resulted in partial loss of total kinase activity, occurred only in the alpha subunit, and existed on Thr306 but not Thr286. This result demonstrates intrasubunit catalysis of Thr306 autophosphorylation. These observations that regulatory autophosphorylations of Thr286 and Thr306 were inter- and intrasubunit-catalyzed, respectively, have important consequences for structure/function models of CaM kinase II and for involvement of CaM kinase II autophosphorylation and activation during synaptic plasticity in neural systems.

Amino Acid Sequence↗
PubMed1994

Changes in UsnRNA biosynthesis during rat liver regeneration.

Partial hepatectomy (P.H.) induces a partially synchronized growth response of liver under normal regulation of growth. In this phase changes in cellular morphology, radial distribution pattern of cells and other biological as well as major biochemical changes are well documented [24]. Here, we have shown that the cellular content of UsnRNAs altered during this proliferative phase as well. The level of spliceosomal UsnRNAs (U1, U2, U4-U6) gradually decreased by 30-50% upto 48 hrs of P.H. followed by gradual increase to reach the normal level within one month of P.H. The U3 snRNA level on the other hand, was nearly equal to that in normal liver at 48 hrs of P.H. but in 24 and 72 hrs of P.H. its level was high (4 fold) in contrast to that in other UsnRNAs. Thus, it is clear from our data that the level of all the six UsnRNAs decreased during 48 hrs of P.H. compared to that after first 24 hrs. This has been correlated in the kinetics of UsnRNAs' synthesis (in terms of labelling) in isolated hepatocytes, where the rate of labelling of all the six UsnRNAs increased 20-30% in 24 hrs regenerating hepatocytes (R.H.) followed by sharp decrease by 30-50% within next 24 hrs, compared to that in the normal hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

3-Aminobenzamide delays rejoining of DNA strand breaks in gamma-irradiated lymphocytes from patients with breast cancer and not cervical cancer.

An inhibitor of poly(ADP-ribose) polymerase is 3-aminobenzamide (3AB), treatment by which normally results in the decreased levels of rejoining of DNA single strand breaks. We have studied the effect of 3AB in gamma-irradiation induced damage and the subsequent repair of DNA along with the corresponding changes in poly (ADP-ribose) polymerase activity in lymphocytes of patients with the cancer of breast and uterine cervix. The presence or absence of 3AB prior to gamma-irradiation did not influence the extent of DNA damage. On the other hand, single strand breaks rejoining in breast cancer cases was found to be slower as compared to that of cervical cancer cases and normal individuals. The relative ADP-ribosylation in the presence of 3AB was much lower in the breast cancer cases as compared to normal but there was no significant change in the cervical cancer cases. Overall the maximum relative ADP-ribosylation was slower in the presence of 3AB. This suggests that the lower activity of poly (ADP-ribose) polymerase in breast cancer cases might delay the first phase of single strand breaks (SSBs) rejoining.

Adenosine Diphosphate↗
PubMed1994

Fast spin-echo MR in the detection of vertebral metastases: comparison of three sequences.

PURPOSE: To examine the relative capabilities for the detection of vertebral metastases of three available fast spin-echo sequences: T1-weighted fast spin-echo, short tau inversion recovery (STIR) fast spin-echo, and T2-weighted fast spin-echo sequences with chemical shift selective saturation pulse fat suppression. METHODS: Fourteen patients were evaluated prospectively over a 2-month period with T1-weighted fast spin-echo (four echo train, four acquisitions, 1 min 59 sec-2 min 37 sec). STIR fast spin-echo (16 echo train, four acquisitions, 2 min 30 sec-3 min 19 sec), and T2-weighted fast spin-echo (16 echo train, 4 acquisitions, 2 min 27 sec-3 min 16 sec). For all three pulse sequences, measurements were obtained of the signal intensities of normal marrow, abnormal marrow, fat, and noise posterior to the spine. Contrast-to-noise ratios were calculated for metastases in each case. Lesions were evaluated by three observers and rated for size, location, and conspicuity. RESULTS: Signal intensities of fat, normal marrow, and noise were highest for T1-weighted fast spin-echo sequences. STIR fast spin-echo and fat-suppressed T2-weighted fast spin-echo had approximately similar fat-suppression capabilities. Though contrast-to-noise ratios were highest overall for STIR fast spin-echo, the finding was not statistically significant and lesion conspicuity was deemed better with fat-suppressed T2-weighted fast spin-echo and T1-weighted fast spin-echo images. Discrete lesions were well identified on all three pulse sequences. CONCLUSION: Fast spin-echo sequences appear promising for the detection of vertebral metastases. Further work should be directed toward comparison with conventional spin-echo to determine whether fast spin-echo may replace conventional spin-echo sequences for evaluation of vertebral metastases.

Adult↗
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↗
PubMed1990

Visible light induced DNA-protein crosslinking in DNA-histone complex and sarcoma-180 chromatin in the presence of methylene blue.

Formation of DNA-protein cross-links by the action of visible light in the presence of methylene blue was studied in calf thymus DNA-calf thymus histone complex and sarcoma-180 chromatin. The extent of cross-link formation decreases with a decrease in the histone to DNA ratio in the DNA-histone complex. In chromatin, it is at a maximum (93%) at a dye to DNA nucleotide ratio (D/P ratio) of 0.04 and is appreciable even at a very low dye concentration (75% at a D/P ratio of 0.0033). Sepharose 4B-CL column chromatography indicates that methylene blue acts as a mediator in the cross-linking process, but not as a linker in the DNA-protein cross-link. Dodecylsulphate-polyacrylamide gel electrophoresis patterns reveal that both histone and non-histone proteins are involved in cross-linking, but to a varied extent. Competition experiments with ethidium bromide demonstrated the necessity of intercalative binding of methylene blue in the formation of DNA--protein cross-links. Viscometric studies in 2 M NaCl indicate that the compact structure of chromatin is stabilized by cross-linking.

Animals↗
PubMed1989

Changes in the template activity of chromatin isolated from sarcoma-180 ascites cells treated with mitomycin C and gamma irradiation in vivo.

The murine ascites sarcoma 180 cells were used to test the in vivo effectiveness of mitomycin C (MMC) and gamma-radiation applied in combination. The action of intraperitoneal administration of MMC and/or whole-body gamma irradiation on sarcoma 180 tumor bearing Swiss albino mice was investigated by studying the template activity of isolated tumor chromatin. The Km value for transcription of 10 Gy-irradiated chromatin was found to decrease with time implying an increase in the template efficiency with respect to that of the unirradiated control. Maximum decrease in Km was observed after 24 h of irradiation. MMC treatment (7 mg/kg body weight of mouse) for 18 h resulted in an inhibition of the transcription rate. Severe inhibition in the template activity was found when cells were subjected to MMC treatment 18 h prior to irradiation with 10 Gy. Susceptibility of tumor chromatin to DNase II followed the same pattern as observed in the case of transcription indicating structural alteration of the treated chromatin. The data showed that DNA damage and its consequences produced in the ascites cells by prior treatment of MMC were not repaired during the 18 h period after which the application of radiation enhanced cytotoxicity.

Animals↗
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↗
PubMed1985

Approaches to prostatic cancer chemotherapy using the Dunning R3327H prostatic adenocarcinoma.

Androgen-responsive cells: To determine if testosterone or dihydrotestosterone is the main trophic hormone of prostatic adenocarcinoma, we have treated Dunning R3327H prostatic adenocarcinoma-bearing rats with 6-methylene progesterone, which blocks conversion of testosterone to dihydrotestosterone. Copenhagen-Fisher rats were treated with steroid (20 mg/Kg daily) immediately following implantation of tumor and thereafter for 117 days. There was a 92% inhibition of growth of tumors and a lesser effect upon prostate and seminal vesicles. Tumor-free body weights remained unchanged. Both treated and untreated tumors had equivalent DNA content on a per weight basis. This result supports the thesis that prostatic adenocarcinoma requires dihydrotestosterone for growth. Androgen-insensitive cells: Advanced prostate cancer does not respond to endocrine therapy but is temporarily controlled by the cytotoxic steroid estramustine. The latter shows significant selective binding to prostatic protein. To develop chemotherapeutic agents that will control androgen-insensitive cells and possess improved selectivity for prostatic protein, we have studied a number of steroids for their ability to displace 3H-labeled estramustine from prostatic cytosolic proteins. Surprisingly, a carbamido substituent at the C17 position was found to confer significant binding affinity for prostatic estramustine-binding protein. Extension of this structural characteristic to the estramustine type of molecule is being studied.

5-alpha Reductase Inhibitors↗