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P K Chatterjee

Publications and source records attributed to P K Chatterjee.

At least 19 recordsLinked to original sources
PubMed1996

Retrofitting high molecular weight DNA cloned in P1: introduction of reporter genes, markers selectable in mammalian cells and generation of nested deletions.

The bacteriophage P.1. cloning system is proving to be quite useful for the cloning and analysis of genomic DNA inserts of up to 95 kb in size. In an effort to use that DNA directly in biological experiments we have embarked on a scheme to retrofit the P.1. DNA using a mini-Tn10 transposon system. This transposon system is used in two ways: (i) to introduce a variety of sequence signals that are recognizable in mammalian cells, such as mammalian cell-responsive resistance markers and reporter genes, and (ii) to generate a nested set of deletions in a P.1. clone by using a ioxP site located within the transposon. In this report we show that such transpositions into P.1. DNA are efficient, distributed throughout the entire length of the genomic fragment and do not disrupt the DNA in any location other than the site of insertion of the transposon. The Tn10-based P.1. transduction system described here provides a general scheme for retrofitting any large genomic DNA cloned in a P.1. vector, thus facilitating the use of clones from the current P.1. recombinant libraries in cellular transformation studies.

Animals↗
PubMed1995

A general genetic approach in Escherichia coli for determining the mechanism(s) of action of tumoricidal agents: application to DMP 840, a tumoricidal agent.

We describe here a simple and easily manipulatable Escherichia coli-based genetic system that permits us to identify bacterial gene products that modulate the sensitivity of bacteria to tumoricidal agents, such as DMP 840, a bisnaphthalimide drug. To the extent that the action of these agents is conserved, these studies may expand our understanding agents is conserved, these studies may expand our understanding of how the agents work in mammalian cells. The approach briefly is to use a library of E. coli genes that are overexpressed in a high copy number vector to select bacterial clones that are resistant to the cytotoxic effects of drugs. AtolC bacterial mutant is used to maximize permeability of cells to hydrophobic organic molecules. By using DMP 840 to model the system, we have identified two genes, designated mdaA and mdaB, that impart resistance to DMP 840 when they are expressed at elevated levels. mdaB maps to E. coli map coordinate 66, is located between the parE and parC genes, and encodes a protein of 22 kDa. mdaA maps to E. coli map coordinate 18, is located adjacent to the glutaredoxin (grx) gene, and encodes a protein of 24 kDa. Specific and regulatable overproduction of both of these proteins correlates with DMP 840 resistance. Overproduction of the MdaB protein also imparts resistance to two mammalian topoisomerase inhibitors, Adriamycin and etoposide. In contrast, overproduction of the MdaA protein produces resistance only to Adriamycin. Based on its drug-resistance properties and its location between genes that encode the two subunits of the bacterial topoisomerase IV, we suggest that mdaB acts by modulating topoisomerase IV activity. The location of the mdaA gene adjacent to grx suggests it acts by a drug detoxification mechanism.

Amino Acid Sequence↗
PubMed1995

Characterization of Syrian hamster gastric mucosal H+,K+-ATPase.

Employing a simple one-step sucrose gradient fractionation method, gastric mucosal membrane of Syrian hamster was prepared and demonstrated to be specifically enriched in H+,K+-ATPase activity. The preparation is practically devoid of other ATP hydrolyzing activity and contains high K+-stimulated ATPase activity of at least 4-5 fold compared to basal ATPase activity. The H+,K+-ATPase showed hydroxylamine-sensitive phosphorylation and K+-dependent dephosphorylation of the phosphoenzyme, characteristic inhibition by vanadate, omeprazole and SCH 28080, and nigericin-reversible K+-dependent H+-transport--properties characteristic of gastric proton pump. One notable difference with H+,K+-ATPase of other species has been the observation of valinomycin-independent H+ transport in such membrane vesicles. It is proposed that such H+,K+-ATPase-rich hamster gastric mucosal membrane preparation might provide a unique model to study physiological aspects of H+,K+-ATPase function in relation to HCl secretion.

Adenosine Triphosphate↗
PubMed1995

Atrial natriuretic factor and angiotensin II stimulate nitric oxide release from human proximal tubular cells.

1. It has been recently reported that angiotensin II can enhance atrial natriuretic factor-stimulated cyclic GMP release from brain capillary endothelial cells and stimulate directly the release of cyclic GMP by Neuro 2a cells. A possible mechanism mediating such cyclic GMP release could be via the production of nitric oxide and the resultant stimulation of soluble guanylate cyclase. 2. The ability of angiotensin II, atrial natriuretic factor and c(4-23) atrial natriuretic factor to stimulate nitric oxide production was investigated in primary cultures of human proximal tubular cells. 3. Freshly prepared human proximal tubular cells were seeded onto 6-well plates and allowed to reach confluence. Cells were then incubated with incremental concentrations of either angiotensin II, atrial natriuretic factor or c(4-23) atrial natriuretic factor alone for 1, 4, 12 or 24h or in the presence of the nitric oxide synthase inhibitor NG-monomethyl-L-arginine. Angiotensin II was also incubated with human proximal tubular cells in the presence of the AT1 and AT2 receptor antagonists DuP 753 and PD 123319. 4. Incubation of human proximal tubular cells with angiotensin II, atrial natriuretic factor or c(4-23) atrial natriuretic factor produced a dose- and time-dependent increase in nitric oxide production, which was inhibited in the presence of NG-monomethyl-L-arginine. A similar increase in nitric oxide production was observed after incubation with atrial natriuretic factor or c(4-23) atrial natriuretic factor. 5. The angiotensin-induced increase in nitric oxide production was not inhibited in the presence of either the angiotensin AT1 or AT2 receptor antagonists DuP 753 or PD 123319. 6. This study demonstrates that primary cultures of human proximal tubular cells can be stimulated to produce nitric oxide by both atrial natriuretic factor and angiotensin II. Furthermore, the atrial natriuretic factor-induced response appears to be mediated via the atrial natriuretic factor-C receptor, while the angiotensin II-induced response appears to be mediated by a novel, as yet unidentified, angiotensin II receptor.

Angiotensin II↗
PubMed1995

Atrial natriuretic factor modulates nitric oxide production: an ANF-C receptor-mediated effect.

OBJECTIVES: To investigate the possible immunomodulatory and regulatory functions of atrial natriuretic factor (ANF) and the natriuretic peptide C (NPR-C) receptor in the control of cytokine-stimulated nitric oxide production in primary cultures of human proximal tubular cells. METHODS: Freshly prepared human proximal tubular cells were seeded on plastic plates and allowed to reach confluence. The confluent cells were then incubated with ANF or cyclic(4-23)ANF (c(4-23)ANF) alone, or preincubated with ANF or c(4-23)ANF before incubation with the nitric oxide-stimulating combination of cytokines interleukin-1 beta (10 u/ml), tumour necrosis factor-alpha (10 ng/ml) and interferon-gamma (100 u/ml). RESULTS: In the present series of experiments we have found that incubation of primary cultures of human proximal tubular cells with ANF or c(4-23)ANF stimulates nitric oxide production dose-dependently. Paradoxically, ANF acting via the NPR-C receptor also inhibits cytokine activation of the enzyme-inducible nitric oxide synthase via a cyclic GMP-independent mechanism. Both of these effects were reproduced by the NPR-C receptor-specific ligand c(4-23)ANF. CONCLUSIONS: These findings represent novel actions of ANF mediated via the NPR-C receptor. The results also provide a simple model system in which to study the subcellular mechanisms of NPR-C receptor activation.

Atrial Natriuretic Factor↗
PubMed1995

Using cell-fractionation and photochemical crosslinking methods to determine the cellular binding site(s) of the antitumor drug DMP 840.

In order to understand its mechanism of action we have begun an effort to better define the cellular target of action of the experimental antitumor agent DMP 840 (NSC D640430; (R,R)-2,2'-(1,2-ethanediylbis(imino-(1-methyl-2,1-ethanediyl)))-bi s(5- nitro-1H-benz(de)isoquinoline-1,3-(2H)-dione) dimethanesulfonate). Using a combination of gentle cell fractionation procedures and a previously unidentified photochemical crosslinking reaction, we have shown that after the drug is added to cultured Clone A cells, more than 80% of the drug that is found associated with cells partitions to the chromatin-containing structural framework of the cell and that the primary target after crosslinking with 360 nm light is DNA. While DMP 840 photoreacts quite efficiently with purified RNA in vitro, no photoattachment of the drug to RNA was observed in cells. In vitro photochemical studies also reveal that while GC-rich DNA is a preferred target for drug interaction, AT-rich DNA is more active in the photochemical crosslinking reaction. These results suggest that DMP 840 probably kills cells by interfering with DNA-metabolic processes, and that the drug and its derivatives are likely to be useful photoactive molecular probes for investigating higher order chromatin structures in cells.

Animals↗
PubMed1993

Purification of an active TATA-binding protein-containing factor using a monoclonal antibody that recognizes the human TATA-binding protein.

The human TATA-binding protein was expressed in Escherichia coli as a fusion with an N-terminal hexahistidine sequence, partially purified, and used to raise monoclonal antibodies. More than 50 hybridoma clones producing antibodies that reacted in immunoblot assays with HeLa cell TATA-binding protein and its bacterially synthesized derivative were identified. All antibodies examined recognized epitopes within the N-terminal 159 amino acids of the human TATA-binding protein. Further characterization of one monoclonal antibody, MTBP-6, established that it immunoprecipitates both native HeLa cell TATA-binding protein and TATA-binding protein extracted from cells in the presence of 0.5% SDS. Antibody MTBP-6 immunoprecipitates of native, human cell TATA-binding protein contained the TATA-binding protein and additional polypeptides. Immunoprecipitation of both the TATA-binding protein and several additional polypeptides was specifically blocked by bacterially synthesized, hexahistidine-tagged TATA-binding protein, suggesting that MTBP-6 can efficiently recognize the TATA-binding protein in TFIID and other complexes. Consistent with this conclusion, immunoaffinity chromatography on antibody MTBP-6 permitted purification, in active form, of a TATA-binding protein-containing factor required for transcription by RNA polymerase III. These properties suggest that MTBP-6 will be a useful reagent for the purification and characterization of the multiple TBP-containing complexes present in human cells.

Animals↗
PubMed1992

Specific transcription from the adenovirus E2E promoter by RNA polymerase III requires a subpopulation of TFIID.

The early E2 (E2E) promoter of adenovirus type 2 possesses a TATA-like element and binding sites for the factors E2F and ATF. This promoter is transcribed by RNA polymerase II in high salt nuclear extracts, but by RNA polymerase III in standard nuclear extracts, as judged by sensitivity to low and high, respectively, concentrations of alpha-amanitin. Transcription by the two RNA polymerases initiated at the same site and depended, in both cases, on the TATA-like sequence and upstream elements. However, RNA polymerase III transcripts, unlike those synthesized by RNA polymerase II, terminated at two runs of Ts downstream of the initiation site. Although they are not essential, sequences downstream of the initiation site increased the efficiency of E2E transcription by RNA polymerase III. Such RNA polymerase III dependent transcription required a subpopulation of the general transcription factor, TFIID: TFIID that binds weakly to phosphocellulose (0.3 M eluate) complemented a TFIID-depleted extract to restore RNAp III transcription, whereas TFIID tightly associated with phosphocellulose (1 M eluate) was unable to do so.

Adenovirus E2 Proteins↗
PubMed1990

Clinical and experimental mycotic keratitis caused by Aspergillus terreus and the effect of subconjunctival oxiconazole treatment in the animal model.

Aspergillus terreus was isolated from a case of Keratomycosis. The patient, a 50 year old, female presented with a large corneal ulcer with hypopyon. The direct microscopic examination of the scrapings revealed hyaline, thin, septate and branched hyphae. In vitro some antimycotics (amphotericin B, 5-fluorocytosine, oxiconazole, amorolfine and ketoconazole) were tested against A. terreus by agar dilution method. Ketoconazole with MIC of 3 micrograms/ml after 7 days of incubation was most effective followed by oxiconazole (10 micrograms/ml). Experimental corneal ulcer was produced by injecting intralamellary 0.1 ml of the spore suspension containing 10 x 10(6) cfu/ml into the eyes of previously immunocompressed albino rabbits. Histopathologic examination showed infiltration and large destruction of the corneal stroma. Subconjunctival oxiconazole therapy exhibited complete cure. Based on our findings, a clinical evaluation of oxiconazole in human keratomycosis seems to be justified.

Amphotericin B↗
PubMed1989

Clinical and experimental mycotic corneal ulcer caused by Aspergillus fumigatus and the effect of oral ketoconazole in the treatment.

Aspergillus fumigatus was isolated from a case of keratomycosis. The patient, a 12-year-old boy presented with large corneal ulcer with hypopyon. The direct microscopic examination of scrapings revealed hyaline, septate mycelium. In vitro some antimycotics (amphotericin B,5-fluorocytosine, oxiconazole, amorolfine and ketoconazole) were tested against A. fumigatus by agar dilution method. Ketoconazole with minimum inhibitory concentration of 30 micrograms/ml after 11 days of incubation was most effective against A. fumigatus. Experimental corneal ulcer was produced by injecting intralamellary spore suspension (2.5 x 10(6) c.f.u.) into the right eyes of previously immunosuppressed albino and black wild types of rabbits. The extent of ocular infection was graded up to 32 days. Histopathologic examination showed infiltration and large destruction of corneal stroma. Oral ketoconazole therapy exhibited partial response followed by relapse. The black type of rabbit appeared more suitable as an animal model for mycotic keratitis.

Administration, Oral↗
PubMed1989

Simultaneous determination of chlorzoxazone and acetaminophen in combined dosage forms by an absorbance ratio technique and difference spectrophotometry.

Two spectrophotometric methods have been developed for the simultaneous determination of chlorzoxazone and acetaminophen in their combined dosage forms. No preliminary separation step is required in either method. The first, an absorbance ratio technique using the isoabsorptive point as one of the wavelengths, together with "Q curve" analysis gives accurate and reproducible results for both drugs. The second, a difference spectrophotometric method is based on measurement of absorbance of an alkaline solution relative to that of an acidic solution of identical concentration of the sample at two different wavelengths. The method is suitable for routine analysis of such combinations of the drugs.

Acetaminophen↗
PubMed1988

DNA-binding properties of an adenovirus 289R E1A protein.

An adenovirus 2 289 amino acid (289R) E1A protein purified from Escherichia coli has been shown to interact with DNA by two independent methods. UV-crosslinking of complexes containing unmodified, uniformly 32P-labelled DNA and purified E1A protein induced efficient labelling of the protein with covalently attached oligonucleotides, indicating that the E1A protein itself contacts DNA. Discrete nucleoprotein species were also observed when E1A protein--DNA complexes were analysed by gel electrophoresis. Although the 289R E1A protein exhibited no significant binding to single-stranded DNA or to RNA, no evidence for its sequence-specific binding to double-stranded DNA was obtained with either assay. Identification of the sites of covalent attachment of 32P-labelled oligonucleotides by partial proteolysis of the crosslinked E1A protein indicated that the interaction of this protein with DNA is mediated via domain(s) in the C-terminal half of the protein. Such previously unrecognized DNA-binding activity is likely to contribute to the regulatory activities of this important adenoviral protein.

Adenovirus Early Proteins↗
PubMed1987

Adenovirus type 2 endopeptidase: an unusual phosphoprotein enzyme matured by autocatalysis.

A 19-kDa protein, present in low copy number in purified adenovirus type 2, has been characterized. Several criteria were used to establish that this protein is neither a degradation product of the known structural proteins of the virion nor a minor, unusually modified, form of protein VII. This 19-kDa protein, unlike other virion proteins, possesses alkali-resistant phosphoamino acids. Analysis by partial proteolysis indicated that it is related to a 23-kDa phosphoprotein present in H2ts-1 virions assembled in infected cells maintained at 39 degrees C. Affinity labeling with [3H]diisopropyl fluorophosphate showed that the 19-kDa protein contains the active site for a serine protease. We, therefore, conclude that the 19-kDa protein is the active form of the adenovirus-encoded endopeptidase, defined by the H2ts-1 mutation, and is synthesized as a 23-kDa precursor that appears to mature by autocatalysis.

Adenoviruses, Human↗
PubMed1986

Comparison of the interactions of the adenovirus type 2 major core protein and its precursor with DNA.

The interactions of the major core protein of adenovirus type 2 (Ad2) protein VII, and its precursor, protein pre-VII, with viral DNA, were studied using UV light induced crosslinking of 32P-labelled oligonucleotides to the proteins. Proteolytic fragments of these two proteins that contain DNA-binding domains were identified by virtue of their covalently attached, alkali-resistant 32P-radioactivity. The overall efficiency of crosslinking of protein pre-VII to DNA, in H2ts1 virions assembled at 39 degrees C, was comparable to that of the crosslinking of protein VII to DNA in Ad2 virions. However, a protease V8 fragment comprising the N-terminal half of protein pre-VII crosslinked to DNA at least ten times more efficiently than the corresponding N-terminal fragment of protein VII, which is truncated by the removal of 23 amino acids from the N-terminus of protein pre-VII during virion maturation.

Adenoviruses, Human↗
PubMed1986

Identification of proteins and protein domains that contact DNA within adenovirus nucleoprotein cores by ultraviolet light crosslinking of oligonucleotides 32P-labelled in vivo.

A new approach to the identification of DNA binding proteins has been developed and used to study the DNA-protein interactions within the nucleoprotein core of subgroup C adenoviruses. Virions labelled in vivo with [32P]orthophosphate were exposed to ultraviolet light and the DNA digested by chemical or enzymatic methods. Labelled phosphoamino acids of the virion phosphoproteins were selectively hydrolysed by alkali, permitting proteins crosslinked to DNA to be identified by virtue of their covalently attached, 32P-labelled nucleotides. In parallel experiments, [3H]arginine-labelled virions were crosslinked by exposure to ultraviolet light and analysed by more conventional methods. The results indicate that proteins VII and V lie in close contact with viral DNA within the core. The compact arrangement of the nucleoprotein core appears to be capable of trapping protein VII molecules that are not covalently attached to DNA after exposure to ultraviolet light, suggesting that viral DNA might be wrapped around clusters of protein VII molecules. The domains of protein VII that lie in contact with DNA were identified by partial proteolytic mapping of the sites of covalent-attachment of the 32P-labelled oligonucleotides. The implications of these data for the nature of the interactions that mediate the packaging of viral DNA within the nucleoprotein core of adenovirions are discussed.

Adenoviridae↗
PubMed1986

Adenoviral protein VII packages intracellular viral DNA throughout the early phase of infection.

The proteins associated with parental, adenoviral DNA in productively-infected HeLa cells have been examined both directly and indirectly. HeLa cells infected with 32P-labelled Ad2 were irradiated with u.v. light at various points in the infectious cycle. Following degradation of the DNA, nuclear proteins carrying cross-linked nucleotides, or oligonucleotides, were distinguished from virion phosphoproteins by the resistance of their 32P radioactivity to 1 M NaOH. The major core protein of the virion, protein VII, was found to be associated with viral DNA throughout infection, even when cells were infected at a multiplicity of 0.14. Micrococcal nuclease digestion of intranuclear viral DNA 4 h after infection liberated two nucleoprotein particles containing viral DNA, neither of which co-migrated with HeLa cell mononucleosomes. These results indicate that core protein VII remains associated with parental adenoviral DNA during productive infections. The observation that protein VII can be cross-linked to DNA in cells infected at very low multiplicity, together with the results of a comparison of proteins cross-linkable to viral DNA in cells infected by wild-type virus and a non-infectious mutant containing the precursor to protein VII, suggest that nucleoproteins comprising viral DNA and protein VII must be the templates for expression of pre-early and early viral genes.

Adenoviruses, Human↗
PubMed1986

Partition of E1A proteins between soluble and structural fractions of adenovirus-infected and -transformed cells.

The partition of E1A proteins between soluble and structural framework fractions of human cells infected or transformed by subgroup C adenoviruses was investigated by using gentle cell fractionation conditions. A polyclonal antibody raised against a trpE-E1A fusion protein (K.R. Spindler, D.S.E. Rosser, and A. J. Berk, J. Virol. 132-141, 1984) synthesized in Escherichia coli was used to measure the steady-state levels of E1A proteins recovered in the various fractions by immunoblotting. The relative concentration of E1A proteins recovered in the soluble fraction of adenovirus type 2-infected cells was at least fivefold greater than the relative concentration in the corresponding fraction of transformed 293 cells. The observed distribution of E1A proteins was not altered by the sulfhydryl-blocking reagent N-ethylmaleimide. E1A proteins were recovered in nuclear matrix, chromatin, and cytoskeleton fractions after further fractionation of the structural framework fraction. However, the E1A protein species that could be identified by one-dimensional gel electrophoresis were not uniformly distributed among the subcellular fractions examined. The results obtained when fractionation was performed in the presence of the oxidation catalysts Cu2+ or (ortho-phenanthroline)2 Cu2+ indicate that E1A proteins can be efficiently cross-linked, via disulfide bonds, to the structural framework of both adenovirus-infected and adenovirus-transformed cells.

Adenoviridae Infections↗