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Biomedical subjects

S Chatterjee

Publications and source records attributed to S Chatterjee.

At least 37 records · Page 2Linked to original sources
PubMed1995

The role of subterminal sites of transposable element Ds of Zea mays in excision.

Transposition depends on DNA sequences located at or near the termini of the transposon. In the maize transposable element Ds, these sequences were studied by site-directed mutagenesis followed by a transient excision assay in Petunia protoplasts. The transposase-binding AAACGG motifs found in large numbers in the element are important, but none of them is in itself indispensable, for excision. However, mutation of an isolated motif at the 3' end considerably reduced excisability. The inverted termini were confirmed to be indispensable. Point mutations in regions outside the inverted termini of Ds and not located in the transposase-binding motifs had, in some cases, a pronounced effect on excision frequency. The implications of these findings are discussed.

Base Sequence↗
PubMed1995

The non-inducible nature of super-repressors of the gal operon in Escherichia coli.

We isolated and characterized mutant repressors (GalR) of the gal operon in Escherichia coli. These repressors (super-repressors), called GalRs, have a non-inducible phenotype. Repression of the gal operon by super-repressors cannot be lifted by inducer. The mutant galR genes, galRs, have been cloned and the mutational changes determined. Two of them, galRuv7s and galR78s, were located in the proposed sugar binding domains of the repressor. The repressor from wild-type (galR+), as well as from mutant galRuv7s, was purified and characterized biochemically. The results showed that, like wild-type GalR+, GalRuv7s binds to DNA normally and represses transcription from the P1 promoter and stimulates that from the P2 promoter of the gal operon. Nevertheless, compared to GalR+, GalRuv7s is much less sensitive to the presence of the inducer, D-galactose. The affinity of D-galactose to GalRuv7s is 10 to 30-fold lower, as measured by the effect of the inducer on GalR tryptophan fluorescence; GalR complexes with DNA and on GalR repression of transcription. Our results suggest that the super-repressor phenotype of GalRuv7s is because of a defect in D-galactose binding rather than a defect in the ligand-induced allosteric change or increased affinity for the operator.

Amino Acid Sequence↗
PubMed1995

Inhibition of etoposide (VP-16)-induced DNA recombination and mutant frequency by Bcl-2 protein overexpression.

Bcl-2 has been shown to inhibit apoptosis induced by several anticancer agents and to cause a dissociation between etoposide (VP-16)-induced protein-cross-linked DNA strand breaks and VP-16-induced cell death. We suggested previously that VP-16-induced cytotoxicity is mediated by a series of events leading from cleavable complex formation to aberrant DNA recombination, as measured by sister chromatid exchange (SCE) and Southern blot analysis of the hypoxanthine phosphoribosyl transferase (hprt) gene mutations. To further evaluate this hypothesis and to determine whether Bcl-2 could affect any steps leading to the aberrant DNA recombination process, we stably transfected an expression vector containing human Bcl-2 cDNA into V79 Chinese hamster cells. This transfection resulted in overexpression of the Bcl-2 gene product. We subsequently quantitated the relationship between VP-16-induced cytotoxicity, DNA strand breaks, SCE, and mutant frequency at the hprt locus in these Bcl-2-overexpressing cells. Two independent Bcl-2-overexpressing cell lines, BCL2/2 and BCL2/4, showed 3-5 times higher survival at 15 microM VP-16 compared with parental V79 cells or control NeoR cells that were obtained by transfecting V79 cells with the expression vector containing the G-418 resistance gene only. DNA single-strand breaks induced by VP-16 were similar in parental V79, control NeoR, BCL2/2, and BCL2/4 cells. In contrast, VP-16 induced significantly less SCE in Bcl-2-overexpressing cell lines compared with parental V79 and control NeoR cells. The SCE/chromosome induced by 15 microM VP-16 were 0.65, 0.42, 0.09, and 0.10, respectively, in V79, NeoR, BCL2/2, and BCL2/4. In addition, there was an excellent correlation between VP-16-induced SCE and cytotoxicity in all cell lines. Furthermore, VP-16-induced mutant frequencies at the hprt locus were 5-10 times less in BCL-2/2 and BCL-2/4 cells than those observed in the V79 or NeoR control cells. These results indicate that overexpression of Bcl-2 is associated with reduction in VP-16-induced genetic recombination, mutation, and cytotoxicity. Moreover, they suggest that Bcl-2 modulates cytotoxicity of VP-16 between cleavable complex formation and subsequent induction of DNA recombination events. Thus, our results provide important support for the hypothesis that VP-16-induced cytotoxicity is associated with aberrant recombination events, including gene deletions and rearrangements.

Animals↗
PubMed1995

Pulmonary function of different categories of chronic obstructive pulmonary diseases in railway workers of eastern India.

Pulmonary function tests were done on 146 established chronic obstructive pulmonary disease (COPD) patients and 151 normal non-smoker and smoker subjects. These patients were assembled from Garden Reach Railway Hospital (Calcutta), West Bengal, India, 79 of which suffered from chronic bronchitis, 8 from chronic bronchitis with mild emphysema (who were included in the chronic bronchitis group), 38 from emphysema and 21 from asthma. In the normal subjects 75 were non-smokers and 76 smokers. All the subjects were in the age range of 16-60 years. The mean values obtained in COPD patients were compared with those of normal non-smokers and smokers in each group and amongst the different categories of COPD patients. All the pulmonary function tests (PFT) were measured by the standard spirometric technique, and the Peak Expiratory Flow Rate (PEFR) was studied by Wright's Peak Flow Meter. The values were expressed in body temperature at ambient pressure saturated with Water Vapour Pressure (BTPS). The PFT values significantly deteriorated in all categories of COPD patients as compared to normal non-smokers and smokers, but a few parameters showed exceptions. Significant deterioration was observed in emphysematous patients when compared to other categories of COPD patients. No definite trend of reduction of pulmonary function test values were found according to the years of suffering from the disease. A product moment correlation matrix showed a highly significant positive correlation between FVC and FEV1, in all four groups of patients. These two parameters also showed a strong positive relationship with FEF25-75%, FEF200-1200ml, FEF75-85%, MVVF and PEFR. The regression equations for some of the closely related variables of high correlation coefficient were calculated in COPD patients and presented.

Adolescent↗
PubMed1995

Involvement of NAD-poly(ADP-ribose) metabolism in p53 regulation and its consequences.

We have used two different approaches to study the consequences of NAD/poly(ADP-ribose) deficiency on p53 expression and its activity in V79-derived cell lines. In the first approach, we have used two cell lines that are deficient in poly(ADP-ribose) (pADPR) synthesis because of deficiency in the enzyme poly(ADP-ribose) polymerase (PARP). In a second approach, we have used a cell line that is deficient in NAD/pADPR metabolism due to unavailability of NAD, the substrate for PARP. These NAD/PARP-deficient cell lines exhibit a significant reduction in both baseline p53 expression and its activity compared to their parental V79 cells. Furthermore, etoposide, a topoisomerase II inhibitor that was shown to cause an increase in p53 expression and subsequent apoptosis in V79 cells, failed to produce any significant increase in p53 expression or apoptotic DNA fragmentation in NAD/PARP-deficient cell lines. Thus, our studies suggest that NAD/pADPR synthesis may be involved in the regulation of p53 and its dependent pathways.

Animals↗
PubMed1995

Novel dipeptide aldehydes are proteasome inhibitors and block the MHC-I antigen-processing pathway.

Class I MHC (MHC-I) molecules present peptides derived from Ag that are processed in the cytosol. The proteasome is a multicatalytic protease complex that is present in the cytosol and has been implicated in cytosolic Ag processing. Novel dipeptide aldehydes were designed, synthesized, and demonstrated to specifically inhibit the chymotrypsin-like protease activity of isolated proteasomes, but produced relatively little inhibition of cathepsin B, a vacuolar cysteine protease. The inhibitors were membrane permeable and inhibited intracellular cleavage of a membrane-permeable fluorogenic substrate of the chymotrypsin-like proteasome activity. When a model Ag, OVA, was introduced into the cytoplasm of M12.B6 murine B cells by electroporation, the proteasome inhibitors blocked its processing for subsequent presentation by MHC-I molecules. The inhibitors had little effect on class II MHC processing of exogenous Ag. The potencies of different inhibitors for blockade of MHC-I Ag processing correlated directly with their potencies for inhibition of the chymotrypsin-like proteasome activity. In contrast, conventional inhibitors of vacuolar cysteine proteases (e.g., leupeptin and benzyloxycarbonyl-Phe-Ala-CHN2) had little effect on MHC-I processing or the chymotryspin-like activity of isolated proteasomes. These results directly demonstrate that inhibition of proteasome activity blocks MHC-I Ag processing, confirming a role for proteasomes in this pathway. Moreover, they suggest that the chymotrypsin-like activity of the proteasome may be of major importance to the cytosolic processing of at least some Ag.

Aldehydes↗
PubMed1995

Staphylococcal enterotoxin-B (SEB) alters [14C]-choline transport and phosphatidylcholine metabolism in cultured human kidney proximal tubular cells.

We studied the effects of SEB on [14C]-choline transport and metabolism of choline containing phospholipids in cultured human kidney proximal tubular (PT) cells. SEB increased the uptake of [14C]-choline in PT cells as a function of toxin concentration, incubation time, and pH. The maximum increase in uptake (3.5-5-fold compared to control) was observed at a toxin concentration of 10 micrograms/10(4) cells, at 4 h and at pH 7.4. Two toxins structurally related to SEB, Staphylococcal enterotoxin-A and toxic shock toxin (TST-1) failed to alter [14C]-choline uptake in PT cells, a finding which indicates that SEB-mediated alteration in choline uptake in PT cells has high specificity. We found that SEB markedly and significantly increased the incorporation of [14C]-choline into phosphatidylcholine, Iysophosphatidylcholine and sphingomyelin, but not into phosphatidylethanolamine. Maximum increase in the incorporation of [14C]-choline into phosphatidylcholine (3-fold compared to control) was observed at 4 h after incubation with toxin. In contrast, SEB did not alter the incorporation of [14C]-choline in phosphatidylethanolamine. The cellular level of phosphatidylcholine was also increased (2-fold compared to control) in PT cells incubated with SEB. This was accompanied by a 3-to-4-fold increase in CTP; phosphocholine, cytidyltransferase activity. In sum, SEB specifically stimulates phosphatidylcholine synthesis in PT cells by increasing choline uptake or by activating CTP: phosphocholine, cytidyltransferase, or both. We believe this is the first-ever report indicating that a toxin can increase phosphatidylcholine synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport, Active↗
PubMed1995

Connecting a promoter-bound protein to TBP bypasses the need for a transcriptional activation domain.

Biochemical analyses have suggested potential targets for transcriptional activation domains, which include several components of the RNA polymerase II machinery, as well as the chromatin template. Here we examine the mechanism of transcriptional activation in yeast cells by connecting a heterologous DNA-binding domain (LexA) to the TATA-binding protein (TBP). LexA-TBP efficiently activates transcription from a promoter containing a LexA operator upstream of a TATA element. Activation is promoter-specific and is sensitive to mutations on the DNA-binding surface of TBP; hence it is not due to a fortuitous activation domain on TBP. Thus a promoter-bound protein lacking an activation domain can stimulate transcription if it is directly connected to TBP. This suggests that recruitment of TBP to the promoter can be a rate-limiting step for transcription in vivo, and that interactions between activation domains and factors that function after TBP recruitment can be bypassed for activation.

Bacterial Proteins↗
PubMed1995

Report of a controlled clinical trial comparing automated percutaneous lumbar discectomy and microdiscectomy in the treatment of contained lumbar disc herniation.

STUDY DESIGN: The results of a randomized controlled trial comparing automated percutaneous lumbar discectomy (APLD) with lumbar microdiscectomy for the treatment of small contained lumbar discal herniations are reported. All patients gave full informed consent and were assessed by an independent observer. Seventy-one patients with radiologically confirmed small contained lumbar disc herniations were randomly assigned to undergo either APLD or lumbar microdiscectomy. All patients were formally assessed by the independent assessor using the Macnab outcome classification at 3 weeks, 2 months, and 6 months after the procedure with follow-up being continued for the duration of the study. OBJECTIVE: The objective was to complete the first randomized and blinded study with sufficient numbers to provide a valid statistical evaluation of these procedures. SUMMARY OF BACKGROUND DATA: No previous randomized controlled study comparing these methods has been previously reported. METHODS: Each procedure was performed by the same surgeon using standard techniques. Statistical analysis was by the chi-square method. RESULTS: In the APLD group only 9 of 31 (29%) had satisfactory outcomes as compared to 32 of 40 (80%) for the microdiscectomy group. Of those patients in the APLD group who had an unsatisfactory outcome and who then opted to undergo surgery (20 of 22 patients), the final success rate was only 65%. Thus, the cumulative success rate of the group initially randomized to APLD including those undergoing either APLD alone or APLD and microdiscectomy after unsuccessful APLD was 22 of 31 (71%). CONCLUSION: In this group of patients, APLD is seen to be ineffective in the treatment of contained lumbar disc herniation.

Adult↗
PubMed1995

Comparison of genetically engineered herpes simplex viruses for the treatment of brain tumors in a scid mouse model of human malignant glioma.

Genetically engineered viruses and viral genes inserted into retroviral vectors are increasingly being considered for experimental therapy of brain tumors. A primary target of these viruses and vectors is human gliomas, the most frequently occurring primary human brain tumor. To investigate the potential of genetically engineered herpes simplex viruses (HSVs) in the therapy of these tumors, we compared the attributes of two viruses, a recombinant from which the gamma 1(34.5) gene had been deleted (R3616) and a recombinant in which the gamma 1(34.5) gene had been interrupted by a stop codon (R4009). Previous studies have shown that these recombinants were completely devoid of the ability to multiply in the central nervous system of rodents. To pursue these studies, we developed a scid mouse glioma model. Tumor cell response (survival) for 10(3), 10(4), and 10(5) implanted MT539MG glioma cells was 38, 23, and 15 days, respectively. The results were as follows: (i) both R3616 and R4009 replicate and cause cytolysis in diverse glioma cell lines of murine and human origin in vitro, and (ii) Winn-type assays 10(5) MT539MG cells coinoculated with R3616 or R4009 as compared to saline significantly prolonged survival in a dose-dependent fashion. Mice that received only tumor cells or the wild-type parent strain of the recombinants, HSV-1(F), died within 15 days. Survival was greatest with R4009. These experiments define both a model for screening oncolytic viruses and a genetically engineered virus of significant potential use as an oncolytic agent.

Animals↗
PubMed1995

Neutral sphingomyelinase: localization in rat liver nuclei and involvement in regeneration/proliferation.

We have studied the localization of neutral sphingomyelinase (N-SMas) in rat liver nuclei. The levels of neutral sphingomyelinase in regenerating liver nuclei were also assessed. We found that rat liver nuclei contain a sphingomyelinase having a pH optima of 7.2 and a kDa of 92. In intact nuclei, neutral sphingomyelinase was associated predominantly with the nuclear envelope. In regenerating/proliferating rat liver (during DNA synthesis), neutral sphingomyelinase was translocated from the nuclear envelope to the nuclear matrix. The levels of sphingomyelin in whole nuclei decreased in reverse proportion to an increase in the levels of neutral sphingomyelinase. By contrast, there was a corresponding increase in the levels of ceramide and sphingosine during cell regeneration/proliferation. Thus, endogenous nuclear neutral sphingomyelinase may play a role in the regulation of sphingomyelin levels and in relevant signal transduction reactions involving cell regeneration/proliferation. The potential significance of ceramide generation may be aimed at programmed cell death to allow the regeneration of liver mediated via target proteins such as, ceramide activated protein kinases/phospholipases or other unknown mechanisms.

Animals↗
PubMed1995

Effect of inhibitors of poly(ADP-ribose) polymerase on the induction of GRP78 and subsequent development of resistance to etoposide.

We have recently demonstrated that cell lines deficient in poly(ADP-ribose) synthesis due to deficiency in the enzyme poly(ADP-ribose) polymerase (PADPRP) or depletion of its substrate NAD+ overexpress GRP78. Furthermore, this overexpression of GRP78 is associated with the acquisition of resistance to topoisomerase II-directed drugs such as etoposide (VP-16); (S. Chatterjee et al., Cancer Res., 54: 4405-4411, 1994). Thus, our studies suggest that interference with NAD+-PADPRP metabolism could provide an important approach to (a) define pathways of GRP78 induction, (b) study the effect of GRP78 on other cellular processes, (c) elucidate the mechanism of GRP78-dependent resistance to topoisomerase II targeted drugs, and (d) modulate responses to chemotherapy in normal and tumor tissues. However, in the in vivo situation, it is impractical to interfere with NAD+-PADPRP metabolism by mutational inactivation of PADPRP or by depletion of its substrate NAD+. Therefore, we have examined several inhibitors of NAD+-PADPRP metabolism including 3-aminobenzamide, PD128763, and 6-aminonicotinamide for their ability to reproduce the results obtained with cell lines deficient in NAD+-PADPRP metabolism relative to the induction of GRP78 and subsequent development of resistance to VP-16. Our studies show that 6-aminoicotinamide treatment is highly effective in the induction of GRP78 and subsequent development of resistance to VP-16, whereas treatment with 3-aminobenzamide or PD128763 does not induce GRP78 and thus does not result in VP-16 resistance.

6-Aminonicotinamide↗
PubMed1995

Determination of Km and kcat for signal peptidase I using a full length secretory precursor, pro-OmpA-nuclease A.

An effective method for the determination of the activity of signal peptidase I (SPase I) of Escherichia coli is established using the hybrid protein pro-OmpA-nuclease A as substrate. Pro-OmpA-nuclease A, a hybrid secretory precursor was purified to homogeneity under denaturing conditions. When this protein was refolded, it could be quantitatively processed by purified SPase I. The Km of signal peptidase I was 0.0165 mM. The kcat was 8.73 s-1. The Km is 50 to 100 times lower than that obtained with peptide substrates indicating that SPase I has a significantly greater affinity for the protein substrate. The turnover number, kcat, is two to four orders of magnitude greater as well. Thus, the specificity constant, kcat/Km is six orders of magnitude greater with pro-OmpA-nuclease A than with peptide substrates. This is the first determination of kinetics of SPase I with a protein substrate.

Amino Acid Sequence↗
PubMed1995

An improved assay method for the measurement and detection of sphingomyelinase activity.

We have developed an improved assay method to measure sphingomyelinase activity and to detect this enzyme separated on polyacrylamide gels. The assay of sphingomyelinase activity involved immobilizing [N-methyl-14C]sphingomyelin on polyvinyldiflouride (PVDF) membrane, incubation with sphingomyelinase, and the measurement of radioactivity associated with [14C]phosphocholine. The enzyme activity was dependent on the concentration of sphingomyelin, enzyme, pH, and temperature. Thirty minutes of incubation time was optimal for enzyme activity. This enzyme had a bimodal pH optima, in that optimum enzyme activity was measured at pH 5.4 and 7.4. The detection of sphingomyelinase was pursued by separating the enzyme on a polyacrylamide gel and carrying out the enzyme assay by exposure to [14C]sphingomyelin blotted on a PVDF membrane. The enzyme activity on the PVDF membrane was visualized by autoradiography. A white band (depicting hydrolytic removal of [14C]sphingomyelin from PVDF was observed. Our method of detecting sphingomyelinase by immobilizing sphingomyelin on PVDF membrane may serve as a prototype for assaying various other enzymes in which the hydrolytic product is released into the aqueous phase. Moreover, our method for detecting sphingomyelinase on polyacrylamide gels may be helpful in further studies on the molecular biochemistry of this and related phospholipases.

Acrylic Resins↗
PubMed1995

Volume changes of the molten globule transitions of horse heart ferricytochrome c: a thermodynamic cycle.

Volume changes among the unfolded (U), native (N), and molten globule (MG) conformations of horse heart ferricytochrome c have been measured. U to N (pH 2 to pH 7) was determined in the absence of added salt to be -136 +/- 5 mL/mol protein. U to MG (pH 2, no added salt to pH 2, 0.5 M KCl) yielded + 100 +/- 6 mL/mol. MG to N was broken into two steps, N to NClx at pH 7 by addition of buffered KCl to buffered protein lacking added salt (NClx = N interacting with an unknown number, X, of chloride ions), and MG to NClx by jumping MG at pH 2 in 0.5 M KCl to pH7 at the same salt concentration. The delta V of N to NClx was -30.9 +/- 1.4 mL/mol protein, whereas MG to NClx entailed a delta V of -235 +/- 6 mL/mol. Within experimental error, the results add up to zero for a complete thermodynamic cycle. We believe this to be the first volumetric cycle to have been measured for the conformational transitions of a protein. The results are discussed in terms of hydration contributions from deprotonation of the protein, other hydration effects, and the formation and/or enlargement of packing defects in the protein's tertiary structure during the steps of folding.

Animals↗