Biomedical subjects

P Seth

Publications and source records attributed to P Seth.

At least 127 records · Page 7Linked to original sources

PubMed1996

Regulation of insulin-like growth factor II P3 promotor by p53: a potential mechanism for tumorigenesis.

Human insulin-like growth factor (IGF)-II mRNA has been shown to be expressed at high levels in a variety of tumors, including rhabdomyosarcomas. In addition, many tumors have alterations in p53 expression. To investigate whether p53 regulates IGF-II gene expression, we transfected wild-type p53 expression vectors and luciferase constructs driven by IGF-II P3 promotors into multiple cell lines. We found that p53 reduced, in a dose-dependent manner, both endogenous IGF-II P3 transcripts and transfected P3 luciferase expression. The inhibition of P3 luciferase expression by p53 was more pronounced in the two cell lines that expressed mutant p53 protein, RD, and HTB114. The element responsible for this inhibition was mapped to the minimal promoter region. We also transfected an HPV-16 E6 expression plasmid into CCL13 cells containing functional p53 and found that E6 up-regulated IGF-II P3 activity. Wild-type, but not mutant, p53 interfered with the binding of TATA-binding protein to the TATA motif of P3, although both could directly associate with human TATA-binding protein. Our results suggest that p53 may play a role in regulation of IGF-II gene expression.

Gene Expression Regulation, Neoplastic↗

PubMed1996

Involvement of the presynaptic dopamine D2 receptor in the depression of spinal reflex by apomorphine.

The relative roles of D1 and D2 dopamine (DA) receptors in mediating apomorphine (APO)-induced changes in the spinal reflex was investigated. Low doses of APO, a DA receptor agonist (0.2 mg kg-1, i.v.), depressed the monosynaptic mass reflex (MMR) in spinalized rats. Pretreatment with the D2-specific antagonist, spiperone, 10 min before APO prevented the APO-induced MMR depression. Pretreatment with the D1 antagonist SCH 23390 failed to prevent the APO-induced depression. Interestingly, SCH 23390 pretreatment preferentially antagonized the depression induced by a high dose of APO (3 mg kg-1, i.v.). Pretreatment with SKF 38393, a selective D1 agonist, completely prevented the APO-induced MMR depression. These results suggest that inhibition of spinal transmission by low dose of APO may be mediated through its action on presynaptic D2 receptors and that D1 and D2 receptors are functionally coupled at the spinal level in modulating the spinal motor output.

Animals↗

PubMed1996

Evaluation of polymerase chain reaction for rapid diagnosis of clinically suspected tuberculous meningitis.

SETTING: Since conventional bacteriological methods rarely detect Mycobacterium tuberculosis in cerebrospinal fluid (CSF) and are of limited use in the diagnosis of tuberculous meningitis (TBM), clinical features suggestive of TBM supported by indirect evidence such as CSF examination and computerized tomography (CT) of the head have been used for the early diagnosis of TBM. OBJECTIVE: We evaluated the efficacy of polymerase chain reaction (PCR) in the diagnosis of TBM. METHODS: Coded CSF samples from 40 patients with TBM and from 49 patients with other neurological disorders were processed. In the absence of a reliable sensitive and specific test for M. tuberculosis in CSF, we used a set of established clinical criteria as the gold standard. Accordingly, the patients were divided into definite, highly probable, probable and possible TBM. The samples were decoded only after completion of the laboratory tests. RESULTS: PCR was positive in 2/4, 19/20, 13/16 patients with highly probable, probable and possible TBM respectively. None of the samples were positive by conventional bacteriological methods. However, 3/49 CSF samples from non-TBM patients were also found positive by PCR. PCR detected M. tuberculosis genomic DNA in the CSF of 85% of clinically suspected TBM cases and 6.1% of non-tuberculous controls. CONCLUSION: PCR, along with the suggested clinical criteria, offers a rapid and fairly accurate diagnosis of TBM.

Adolescent↗

PubMed1996

Adenovirus-mediated gene transfer of the tumor suppressor, p53, induces apoptosis in postmitotic neurons.

Programmed cell death is an ongoing process in both the developing and the mature nervous system. The tumor suppressor gene, p53, can induce apoptosis in a number of different cell types. Recently, the enhanced expression of p53 has been observed during acute neurological disease. To determine whether p53 overexpression could influence neuronal survival, we used a recombinant adenovirus vector carrying wild type p53 to transduce postmitotic neurons. A control consisting of the same adenovirus vector background but carrying the lacZ reporter expression cassette was used to establish working parameters for the effective genetic manipulation of sympathetic neurons. We have found that recombinant adenovirus can be used at titers sufficiently high (10 to 50 multiplicity of infection) to transduce the majority of the neuronal population without perturbing survival, electrophysiological function, or cytoarchitecture. Moreover, we demonstrate that overexpression of wild type p53 is sufficient to induce programmed cell death in neurons. The observation that p53 is capable of inducing apoptosis in postmitotic neurons has major implications for the mechanisms of cell death in the traumatized mature nervous system.

Adenoviridae↗

PubMed1996

Diversity of rotavirus strains infecting pediatric patients in New Delhi, India.

Polyacrylamide Gel Electrophoresis (PAGE) of rotavirus can provide information on variation in rotavirus strain prevalent in the community. In the present study 157 samples were collected from children below 5 years of age presenting with acute diarrhoea from May to December, 1990 at Safdarjung Hospital, New Delhi. Seventy-one (45 percent) of these stool samples were positive for rotavirus by ELISA. Sixty-seven samples showed discernible RNA pattern of group A rotavirus by PAGE. It was found that there were seven electropherotypes co-circulating in this 8-month period. Majority of the strains had a IIC pattern, but IIA, IID, IIE, IIG, IE, and IB electropherotype patterns were also seen. The simultaneous co-circulation of multiple group A strains in the community may lead to extensive genomic variation in rotavirus strains.

Acute Disease↗

PubMed1996

Interaction of nitric oxide synthase inhibitors and their D-enantiomers with rat neutrophil luminol dependent chemiluminescence response.

1. Formyl-methionyl-leucyl-phenylalanine (FMLP) or arachidonic acid (AA) induced luminol dependent chemiluminescence (LCL) response of rat polymorphonuclear leukocytes (PMNLs) was found to be inhibited by nitric oxide synthease inhibitors and their D-enantiomers. 2. Rat PMNLs LCL response was inhibited by NG-nitro-L-arginine methyl ester (L-NAME), D-NAME, NG-monomethyl-L-arginine (L-NMMA) or D-NMMA, in a concentration- and time-dependent manner. 3. It was observed that both L- and D-enantiomers of the arginine analogues (1000 microM) did not inhibit AA induced lucigenin-dependent chemiluminescence (LUCDCL) response and cytochrome c reduction, used for estimating the NADPH-oxidase activity in the cells and in the cell free system, respectively. 4. None of the L- and D-enantiomers had any effect on either rat basal PMNLs or AA-induced oxygen consumption. 5. In addition, neither the L nor D-enantiomers of NAME altered either AA-induced release or the activity of myeloperoxidase from rat PMNLs azurophilic granules. 6. The results obtained indicate that the attenuation of the LCL response by L- and D-enantiomers of arginine analogues, is a non-specific effect as there was no inhibition of NADPH-oxidase and MPO activity, MPO release or oxygen consumption. Therefore, the data obtained indicate that these agents should be used with caution to analyse the role of nitric oxide in rat PMNLs LCL response.

Animals↗

PubMed1996

Classification of rotavirus into G and P types with specimens from children with acute diarrhea in New Delhi, India.

Sixty rotavirus-positive stool specimens from children with diarrhea were classified into G and P genotypes. G typing was done by PCR and then by hybridization with G type-specific (G1 to G4) oligonucleotide probes, whereas nested PCR was performed for P typing. Thirty-nine samples could be classified into both G and P types, of which P8G1 and P4G2 (33% each) genotypes were predominant. The P6 genotype was detected in four children with diarrhea.

Acute Disease↗

PubMed1995

Consequences of p53 gene expression by adenovirus vector on cell cycle arrest and apoptosis in human aortic vascular smooth muscle cells.

p53 shows its tumor suppresser activity by inducing cell cycle arrest and/or apoptosis of tumor cells and these activities are in part mediated by p21 cyclin-dependent kinase inhibitor (also called as WAF1, Cip1 and SDI1). Using human aortic vascular smooth muscle cells, here we demonstrate that adenovirus vector expressing p53-induced p21, cell cycle arrest at G1 and G2/M boundary, and accumulation of cells in G1 subgroup. However, adenovirus vector expressing p21 induced only G1 cell cycle arrest. The adenovirus vector expressing p53 was 200 times more cytotoxic to human aortic vascular smooth muscle cells than adenovirus vector expressing p21. These results suggest that adenovirus expressing p53 induces cytotoxicity in human vascular smooth muscle cells by apoptosis and this cytotoxicity can not be fully accounted by p21 induction.

Adenoviridae↗

PubMed1995

Detection of group A rotavirus by reverse transcriptase and polymerase chain reaction in feces from children with acute gastroenteritis.

RT-PCR was employed to detect rotavirus infection in 450 fecal samples from children with acute diarrhoea. It was compared with enzyme-linked immunosorbent assay (ELISA) and polyacrylamide gel electrophoresis (PAGE) for rotavirus detection. A total of 67 samples were found positive by at least one of the three techniques. Of these 67 samples, 51 were positive by all three methods, 6 were positive by ELISA and RT-PCR but negative by PAGE, 3 were positive by PAGE and RT-PCR and negative by ELISA, 3 and 4 samples were exclusively positive by RT-PCR and ELISA respectively. These results indicate that RT-PCR is a sensitive and specific assay for detection of group A rotaviruses in stool samples from cases of acute diarrhoea.

Acute Disease↗

PubMed1995

Direct detection and characterization of rotavirus into subgroups by dot blot hybridization and correlation with 'long' and 'short' electropherotypes.

BACKGROUND: Enzyme-linked immunosorbent assay (ELISA) and polyacrylamide gel electrophoresis (PAGE) of viral RNA are well-established methods for detection of rotavirus in stool samples. Dot-blot hybridization has also been found to be a sensitive and specific technique for detection and characterization of rotaviruses. OBJECTIVES: To compare the performance of dot blot hybridization with ELISA and PAGE for detection of rotavirus in stool samples. To assess the use of dot blot hybridization for characterization of rotaviruses into subgroups. STUDY DESIGN: Stool samples were collected from 214 children presenting to the hospital with acute diarrhoea. These were assayed for rotavirus by ELISA and PAGE. Dot-blot hybridization was done with full length cloned radiolabelled c-DNA probes of gene segment 6 of SA-11 (subgroup I) and Wa (subgroup II) rotaviruses. RESULTS: Out of 214 stool samples 134 were found to be positive for rotavirus by one of the three methods. Among these 134 positive specimens 114 were positive by dot blot hybridization, this included 18 specimens which were positive only by dot blot assay. One-hundred-and-twelve of these 114 specimens could be subgrouped. Fifteen of these were classified as subgroup I, 97 as subgroup II and two had a dual subgroup specificity. Three subgroup 1 strains had a 'long' RNA pattern, whereas one subgroup II strain had a 'short' RNA pattern which has not been reported earlier for human rotaviruses. CONCLUSION: Dot blot hybridization as described here is a sensitive and specific assay for detection and subgrouping of rotaviruses. However, as there is a considerable genomic diversity among rotaviruses, the panel should include probes from all the genotypes of gene segment 6.

Journal Article↗

PubMed1995

Recombinant adenovirus vector expressing wild-type p53 is a potent inhibitor of prostate cancer cell proliferation.

OBJECTIVES: A recombinant adenovirus vector (AdWTp53) expressing wild-type p53 was evaluated for its cell growth inhibitory effects on metastatic human prostate cancer cells. METHODS: Human prostate cancer cells LNCaP, DU145, PC3, 1LN, and DUPro-1 were infected with AdWTp53 vector and expression of exogenous p53 in these cells was analyzed by immunoprecipitation and western blot assays. The cell growth inhibitory effects of AdWTp53 were determined by counting cell number on a hemocytometer or by crystal violet staining of cells after infection with AdWTp53. The p53-regulated gene WAF1 and DNA fragmentation were also analyzed in prostate cancer cells infected with AdWTp53. RESULTS: High levels of the AdWTp53 vector-derived p53 protein were present in metastatic prostate cancer cells, and the p53-regulated gene WAF1 was induced in these cells. Infection of these tumor cell lines with AdWTp53 vector resulted in severe growth inhibition and cell death in comparison to untreated or control adenovirus vector-infected cells. Furthermore, fragmentation of genomic DNA, a property associated with apoptosis, was also observed in prostate cancer cells infected with AdWTp53. CONCLUSIONS: AdWTp53 vector exhibited a potent inhibitory effect on the growth of all of human metastatic prostate cancer cells, and both cytostatic and cytotoxic effects of AdWTp53 were observed. The induction of p53-regulated gene WAF1 in AdWTp53-infected prostate cancer cells suggests the involvement of cellular p53 pathway in the cell growth inhibition. These results provide a molecular basis for further evaluation of antitumorigenic effects of AdWTp53 vector in animal models of prostate cancer.

Adenoviruses, Human↗

PubMed1995

A nonradioisotopic reverse phase dipstick hybridization method for detection of polymerase chain reaction amplified product.

A rapid and simple nonradioisotopic method has been developed for detection of polymerase chain reaction (PCR) amplified product. Digoxigenin-11-dUTP (DIG-11-dUTP) was incorporated in the amplified product by including it in the PCR reaction mixture. The PCR product was detected colorimetrically either directly or by reverse phase hybridization method where an unlabelled oligo-nucleotide probe was immobilized on a nitrocellulose dipstick and the digoxigenin labelled PCR product was in the liquid phase. With this system the PCR product could be detected even after 10 cycles of amplification by both direct and hybridization methods. The method was applied on the amplified product of DNA from peripheral blood mononuclear cells from 10 HIV-1 ELISA positive and 8 ELISA negative individuals. PCR was positive in all ELISA positive, Western blot positive individuals from whom HIV-1 was also isolated. PCR was negative in all ELISA negative individuals.

Base Sequence↗

PubMed1995

Effects of a recombinant adenovirus expressing WAF1/Cip1 on cell growth, cell cycle, and apoptosis.

To evaluate the effects of a p53-inducible gene WAF1/Cip1 on cell proliferation and apoptosis, a recombinant adenovirus vector (E1 minus) expressing WAF1/Cip1 cDNA (AdWAF1) was constructed and compared with a previously studied recombinant adenovirus vector expressing wild-type p53 (AdWTp53). Infection of normal and tumor cells of lung and mammary epithelial origin with AdWAF1 resulted in high levels of WAF1/Cip1 gene expression, which was comparable to that induced by AdWTp53. AdWAF1 and AdWTp53 inhibited growth of all cells studied; tumor cells devoid of endogenous p53 (H-358) or cells expressing endogenous mutant p53 (MDA-MB-231) were more sensitive to the inhibitory effect than tumor (MCF-7) or normal mammary epithelial cells expressing endogenous wild-type p53. Cell cycle analysis of AdWTp53-infected cells indicated a decline in the cell number in S phase and a significant increase in cell number in G2-M phase. AdWAF1 infection also led to a decline in the percentage of cells in S phase and a significant accumulation of cells in G1. AdWAF1 failed to induce apoptosis in any of the cells tested. In contrast, AdWTp53 induced apoptosis in H-358 and in MDA-MB-231 cells. These data suggest that AdWTp53-mediated WAF1/Cip1 induction and cytotoxicity are likely to be associated with WAF1/Cip1-mediated cell cycle arrest. However, because overexpression of WAF1/Cip1 protein failed to induce apoptosis, AdWTp53 effects on apoptosis apparently require cellular factors in addition to WAF1/Cip1 induction.

Adenoviridae↗

PubMed1995

Differential expression of multiple MDM2 messenger RNAs and proteins in normal and tumorigenic breast epithelial cells.

The MDM2 gene is a nuclear phosphoprotein that is regulated by p53 and functions, in one capacity, to inhibit the transcriptional activity of the wild-type p53 protein. Multiple MDM2 transcripts were detected in human breast epithelial cells. In estrogen receptor-negative normal, immortal, and tumorigenic breast epithelial cells, we found a good correlation between MDM2 mRNA levels and expression of wild-type p53. When wild-type p53 was overexpressed in estrogen receptor-negative tumor cells containing a mutant or no endogenous p53, MDM2 mRNA levels increased significantly, indicating that wild-type p53 positively influences MDM2 mRNA levels in these tumor cells. Because all estrogen receptor-positive breast tumor cells had high MDM2 mRNA levels regardless of the status of their endogenous p53 protein, other factors likely influence MDM2 expression in these cells. Distinct MDM2 proteins (range, Mr 54,000-68,000 and 90,000-100,000, respectively) were differentially expressed in human breast epithelial cells. The lower molecular weight MDM2 proteins were most abundant in the normal mammary cells but present at varying levels in many of the tumor cells examined. MDM2 was a nuclear protein; however, nuclear staining intensity did not always correlate with the amount of MDM2-immunoreactive protein as determined by Western blot analysis. This discrepancy suggests that MDM2 interacts with novel cellular proteins in different kinds of breast epithelial cells.

Breast↗

PubMed1995

Cytotoxic effects of adenovirus-mediated wild-type p53 protein expression in normal and tumor mammary epithelial cells.

To evaluate the effects of the wild-type p53 expression in normal and tumor cells, we have constructed a recombinant adenovirus vector (E1 minus) expressing human wild-type p53 cDNA (AdWTp53). Infection of normal and tumor cells of lung and mammary epithelial origin with AdWTp53 resulted in high levels of wild-type p53 expression. Production of p53 protein following infection was dependent on the dose of AdWTp53 with maximum amounts of p53 produced following infection with 50 plaque-forming units/cell. AdWTp53 infection inhibited the growth of all human cell lines studied. However, tumor cells that were null for p53 prior to infection (H-358 and MDA-MB-157) and tumor cells that expressed mutant endogenous p53 protein (MDA-MB-231 and MDA-MB-453) were more sensitive to AdWTp53 cytotoxicity than cells that contained the wild-type p53 (MCF-7, MCF-10, 184B5, and normal mammary epithelial cells). All cells exhibited WAF1/Cip1 mRNA and protein induction following AdWTp53 infection. AdWTp53-induced cytotoxicity of human tumor cell lines expressing mutant p53 was mediated by apoptosis as revealed by nucleosomal DNA fragmentation analysis. No detectable nucleosomal DNA fragmentation was observed following AdWTp53 infection of human cells expressing wild-type p53. These data suggest that endogenous p53 status is a determinant of AdWTp53-mediated cell killing of human tumor cells.

Adenoviruses, Human↗

PubMed1994

Mechanism of adenovirus-mediated endosome lysis: role of the intact adenovirus capsid structure.

Adenoviruses have been previously shown to enhance the delivery of many ligands including proteins and plasmid DNAs to the cells. The key biochemical step during this process is the ability of adenovirus to disrupt (lyse) the endosome membrane releasing the co-internalized virus and the other ligands into the cytosol (Seth et al, 1986, In: Adenovirus attachment and entry into cells, pp 191-195, American Society for Microbiology, Washington, D.C.). To understand the role of the adenovirus proteins involved in the endosome lysis, it is further shown here that empty capsids of adenovirus also possess this membrane vesicle lytic activity; though the activity is about 5-times lower than the adenovirus. Incubation of adenovirus with low concentration of ionic detergent or brief exposure to 45 degrees C destroyed this lytic activity without affecting the adenovirus binding to cell surface receptor, suggesting the lytic activity of adenovirus to be of enzymatic nature. However, exposing adenovirus to conditions that can disrupt adenovirus capsid structure such as heating at 65 degrees C, treating with 0.5% SDS, treating with different proteases, dialyzing against no glycerol buffer, treating with 6 M urea or with 10% pyridine, and sonication destroyed the adenovirus-associated lytic activity. Results suggest the requirement of an intact capsid structure for adenovirus-mediated lysis of the endosome.

Adenoviruses, Human↗

PubMed1994

Effect of platelet activating factor antagonists in different models of thrombosis.

Effect of three specific PAF antagonists, SR-27417, BN-50739 and ginkgolide derivative BN-52021 have been evaluated in models of thrombosis in the mouse, rat and cat. Thrombosis in the mouse was induced by intravenous infusion of collagen and adrenaline. In rats it was induced by inserting a metallic wire into the inferior vena cava. In the cat, thrombus formation was assessed in the extracorporeal shunt. All the antagonists offered a dose-dependent protection against pulmonary thromboembolism in mice (1, 3 and 10 mg/kg) and the thrombosis monitored in the extracorporeal shunt in cats (0.3, 1 and 3 mg/kg). In rats, no significant protection was observed with these antagonists even at the highest dose used.

Animals↗

PubMed1994

Modulation of rat peripheral polymorphonuclear leukocyte response by nitric oxide and arginine.

The effect of nitric oxide (NO) on the luminol-dependent chemiluminescence (LCL) response of rat polymorphonuclear leukocytes (PMNLs) was analyzed by using sodium nitroprusside (SNP), a NO donor, and L-arginine (L-arg), a NO precursor. A significant reduction in the LCL intensity was observed in presence of SNP (100 mumol/L) or L-arg (5 or 10 mmol/L) in arachidonic acid (AA) phorbol ester (PMA) and formyl-methionyl-leucyl-phenylalanine stimulated PMNLs. However, opsonized zymosan-induced LCL was not attenuated significantly. Reduction in hydroxyl radical and superoxide generation was also observed in SNP- or L-arg-pretreated cells. D-Arg (10 mmol/L) pretreatment did not inhibit PMNLs' LCL response. Furthermore, methylene blue (5 mumol/L) and L-NG-mono methyl-L-arginine (100 or 300 mumol/L) significantly attenuated the LCL response, as induced by various agonists. Cyclic GMP did not alter the reactive oxygen species generation from rat PMNLs. In addition, AA-induced release of myeloperoxidase, a marker of azurophilic granules, was found to be enhanced in L-arg- (10 mmol/L) pretreated PMNLs. The results suggest that NO inhibits free radical generation from rat PMNLs.

Animals↗