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Biomedical subjects

P Seth

Publications and source records attributed to P Seth.

At least 19 recordsLinked to original sources
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

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↗
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↗
PubMed1994

A simple and efficient method of protein delivery into cells using adenovirus.

Human adenovirus type 2 has been previously shown to increase the delivery of a variety of proteins into cells when Ad is co-internalized with the protein ligands. To increase the efficiency of adenoviral-mediated delivery of proteins, I have linked adenovirus, separately with two proteins, epidermal growth factor and an antibody against human transferrin receptor through disulfide and thioether linkages. Competition experiments indicate that the conjugates are taken up into the cells through adenovirus receptor. During the internalization of adenovirus-protein conjugates into KB cells, the conjugates were equally effective as the native adenovirus in disrupting endocytic vesicles and releasing their protein content into the cytosol. This implies the possible use of adenovirus to deliver a large number of protein molecules into the cells.

Adenoviruses, Human↗
PubMed1994

Alterations in the bioantioxidants following thrombosis.

The role of free radicals in rat thrombosis has been demonstrated by studying its scavenging enzyme system. Changes in the bio-antioxidants (GSH-redox cycle, total thiol groups, vitamins E and C) that also offer protection against the free radicals, have, however, not been studied so far. This investigation was undertaken to understand the involvement of these antioxidants during thrombosis. The tissues investigated following thrombosis were blood, platelets, polymorphonuclear leukocytes (PMNLs), heart, and lung. Glutathione (GSH) content in the platelets was observed to be depleted. However, oxidized glutathione (GSSG) contents in the platelets, PMNLs, and blood remain unaltered. In addition, in the whole blood GSH levels were increased significantly, whereas there was no change in the GSSG level. Activity of glutathione reductase (GR) was decreased significantly in platelets and lungs with an increase in the total thiol groups in the lung homogenate. Activity of Glutathione peroxidase (Gpx) remained unaltered in all the tissues studied. In addition, 24% and 15% decrease in the alpha-tocopherol concentration was observed in thrombocytes and PMNLs, respectively, with no change in the ascorbate levels in these cells. Results of this investigation suggest alterations in the GSH-redox cycle in blood, platelets, and lung after thrombosis in the rat.

Animals↗
PubMed1994

Gene transfer to freshly isolated human respiratory epithelial cells in vitro using a replication-deficient adenovirus containing the human cystic fibrosis transmembrane conductance regulator cDNA.

Cystic fibrosis (CF) results from mutations of the CF transmembrane conductance regulator (CFTR) gene and subsequent defective regulation of cAMP-stimulated chloride (Cl-) permeability across the apical membrane of epithelial cells. In vitro transfer of normal CFTR cDNA corrects this defect, and studies in experimental animals have shown successful gene transfer to airway epithelium in vivo using a recombinant adenoviral vector containing the human CFTR cDNA (AdCFTR), supporting the feasibility of in vivo AdCFTR-mediated gene therapy for the respiratory manifestations of CF. One step in applying this therapy to CF patients is to evaluate the safety and efficacy of AdCFTR-mediated gene transfer in the actual target for human gene therapy, human airway epithelium. The present study demonstrates that AdCFTR restores cAMP-stimulated Cl- permeability in human CF bronchial epithelial cells. In addition, the study utilizes freshly isolated human airway epithelial cells from the nose and/or bronchi of normal individuals and/or individuals with CF to demonstrate that after in vitro AdCFTR-mediated gene transfer: (i) AdCFTR DNA does not replicate as a function of dose and time; (ii) CF epithelial cells express AdCFTR-mediated normal human CFTR mRNA; and (iii) CF epithelial cells, including terminally differentiated ciliated cells (the most common airway epithelial cell type), express the normal human CFTR protein. Together, these data support the use of AdCFTR in human gene therapy trials and suggest that biologic efficacy should be achievable in vivo.

Adenoviridae↗
PubMed1994

Mechanism of enhancement of DNA expression consequent to cointernalization of a replication-deficient adenovirus and unmodified plasmid DNA.

Given the knowledge that replication-deficient adenoviruses can mediate the delivery of unlinked plasmid DNA into eukaryotic cells (K. Yoshimura, M. A. Rosenfeld, P. Seth, and R. G. Crystal, J. Biol. Chem. 268:2300-2303, 1993), this study focuses on the role of receptor-mediated endocytosis in this process. AdCFTR (an E1- E3- adenovirus type 5-based replication-deficient adenovirus containing the 4.5-kb human cystic fibrosis transmembrane conductance regulator cDNA) was added to Cos-7 cells together with plasmid pRSVL (containing the Rous sarcoma virus long terminal repeat promoter followed by the luciferase cDNA), and luciferase activity was quantified as a measure of the expression of the plasmid DNA. When AdCFTR was bound to Cos-7 cells at 4 degrees C and the cells were subsequently incubated at 37 degrees C in the presence of pRSVL, the expression of luciferase activity was increased in proportion to the amount of AdCFTR added, reaching > 10(4)-fold at 3,000 PFU per cell. AdCFTR-mediated increase in pRSVL was inhibited by addition of purified adenovirus fiber but not hexon, suggesting cell surface adenovirus receptors were involved in the cointernalization process. Cell lines with a high number of adenovirus receptors (Cos-7 and HeLa) showed significant AdCFTR-dependent pRSVL expression, while cell lines with low numbers of adenovirus receptors (NIH 3T3 and U-937) showed little. AdCFTR-mediated increase in the expression of pRSVL was prevented when AdCFTR was heat treated and exposed to antibody against adenovirus or when the cointernalization process was evaluated in the presence of chloroquine, conditions all known to prevent adenovirus-mediated disruption of endocytic vesicles. In contrast, the uptake of AdCFTR into Cos-7 cells was not affected by any of these conditions. When AdCFTR was exposed to UV light, its ability to grow in 293 cells was obviated, but AdCFTR-dependent increase in pRSVL expression was minimally reduced. Finally, empty capsids of AdCFTR were able to enhance the delivery and expression of plasmid pRSVL into Cos-7 cells, suggesting that the adenovirus genome is not required for AdCFTR-mediated plasmid cointernalization. Together, these observations suggest that the ability of a replication-deficient recombinantly adenovirus to mediate the cointernalization and expression of plasmids is mediated by the receptor-mediated endocytosis pathway.

Adenoviruses, Human↗
PubMed1993

TGF beta and HIV infection.

TGF beta is a cytokine which is involved with the regulation of different aspects of host defense responses to injury. Overexpression of TGF beta can lead to the conversion of its protective functions to pathogenetic manifestations. TGF beta is a potent factor in promoting anabolic aspects in connective tissue metabolism, and uncontrolled production of TGF beta has been associated with the development of fibrosis. With respect to its effects on immune and inflammatory responses, TGF beta is an important endogenous immunosuppressive factor which physiologically may protect the organism from tissue damage caused by chronic activation of leukocytes. As a result of overproduction in HIV infection, this function of TGF beta can contribute to noncytopathic mechanisms of immunodeficiency. TGF beta is involved with several aspects of HIV disease and promotes virus replication and spreading through multiple distinct mechanisms. It directly stimulates virus replication in infected monocytes and peripheral blood mononuclear cells under certain in vitro conditions. It may stimulate the production of other cytokines that enhance virus replication and it may be the mediator of other HIV-stimulating agents such as cocaine. It enhances recruitment of mononuclear phagocytes as cells susceptible to virus infection. Through its profound and broad inhibitory effects on different antiviral defense mechanisms, it facilitates more rapid progression of virus infection and increases susceptibility to opportunistic infections and malignancies. Although these findings are largely based on in vitro systems, the demonstration of TGF beta overexpression in HIV-infected patients supports the notion that this cytokine is an important pathogenetic mediator in HIV infection and its associated diseases. Therapeutic strategies to interfere with these functions of TGF beta are the development of TGF beta-neutralizing antibodies and soluble TGF beta-binding proteins and receptors as well as approaches directed at reducing TGF beta gene expression.

Animals↗
PubMed1993

Monoclonal anti-idiotype antibody to HSV-1 neutralizing monoclonal antibody: production and characterization.

This study is an attempt to produce and characterize murine monoclonal antibodies directed against the paratope of HSV-1 neutralizing monoclonal antibody. Monoclonal antibody 138 C5G10 which was neutralising and directed to 120 K antigen gB of HSV-1 was used as the idiotype. We were able to produce three Ab-2 monoclonal antibodies as characterized using immunofluorescence, ELISA and RIA. The findings of the present study suggest that two antiidiotypes 3AiB3E10 and 3AiB5D10 share the same unique fine specificity while 3AiB3C9 has a different specificity on 138 C5G10 paratope. The utility of such 'surrogate' antigens in serological assays and modulation of immune response is discussed.

Animals↗
PubMed1993

Monoclonal anti-idiotypes to herpes simplex virus type 1 capable of antigen specific priming & stimulatory activity.

Anti-idiotypic antibodies (Ab-2) to HSV-1 (herpes simplex virus 1) neutralizing monoclonal antibody were raised by hybridoma. These Ab-2 were found to represent an epitope of glycoprotein B (gB-1) of this virus. To further characterise this antibody for its ability to mimic the antigenic epitope, in vitro lymphoproliferation assays were done. In this assay (i) antigen specific lymphocyte priming activity of the three monoclonal Ab-2 and (ii) the in vitro stimulating ability of these Ab-2 for gb-1 primed mouse lymphocytes were tested. We could identify two monoclonal Ab-2 which were able to prime the mouse lymphocytes in vivo. These antibodies were able to recognise the in vitro stimulation signal of the antigen gB-1 and consequently could proliferate. The stimulation index was comparable to that with the antigen. These two Ab-2 were also recognized by the antigen primed mouse lymphocytes in a specific manner.

Animals↗
PubMed1992

Supersensitivity of spinal dopaminergic receptors in rat after chronic haloperidol.

In order to examine the effect of chronic neuroleptics on spinal dopaminergic system, rats were treated with haloperidol (0.5 mg/kg IP) for 21 days and the monosynaptic mass reflex (MMR) as well as dopamine (DA) metabolism were investigated. MMR, recorded from ventral root L6 following supramaximal stimulation to ipsilateral dorsal root L6 in spinalized rats, were found to be unaffected following chronic haloperidol treatment when compared to control. Apomorphine (0.1 mg/kg IV) caused 10-20% depression of MMR in control animals which was augmented to 40-50% in chronically haloperidol-treated animals suggesting an upregulation of DA receptors in the spinal cord. DA content of lumbar region of the spinal cord was unaffected whereas its major metabolite, homovanillic acid, was significantly reduced in chronic haloperidol-treated animals. This decreased utilization of DA may compensate the upregulation of DA receptors to maintain the physiological homeostasis of the spinal dopaminergic system.

Animals↗
PubMed1992

Presynaptic dopaminergic inhibition of the spinal reflex in rats.

Dopaminergic influence on spinal monosynaptic transmission was examined in rats. Monosynaptic mass reflex (MMR) was recorded from the ventral root L6 following supramaximal stimulation (0.2 Hz; 0.1 ms) to the ipsilateral dorsal root L6 in spinalized rat under pentobarbitone sodium (40 mg/kg, i.p.) anaesthesia. MMR was inhibited by intravenous administration of the dopaminergic agonist, apomorphine (50-200 ug/kg) in a dose-dependent manner. The attenuatory effect of apomorphine (200 ug/kg i.v.) on the reflex could be reversed by the dopaminergic antagonist haloperidol (0.5 mg/kg, i.v.). Under tetanic stimulation (200 Hz; 15s), the pretetanic relative inhibition induced by apomorphine (200 ug/kg, i.v.) was increased only for a short period immediately after the cessation of tetanic stimulation. The results indicate existence of presynaptic dopamine receptors on the afferent terminals converging on the motoneurone which may functionally modulate the spinal motor output.

Animals↗
PubMed1992

Detection of human papilloma virus types 16 & 18 DNA in cervical lesions of Indian women using in situ hybridization.

Fifty one biopsies from women with malignant lesions of the uterine cervix and 9 biopsies fron non-malignant lesions were examined for the presence of HPV 16 and 18 DNA sequences by in situ hybridization method using 35S-labelled DNA probes. HPV 16 DNA sequences were detected in 82.4 per cent biopsies from women with malignant lesions, whereas HPV 18 DNA was detected in only 3 biopsies which were also positive for HPV 16 DNA. Two biopsies from non-malignant lesions were positive for HPV 16 DNA only. Data were also analysed according to the histologic type of cancer. It was observed that no significant correlation existed between HPV types and different histologic types of cervical cancer.

DNA Probes, HPV↗