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Biomedical subjects

J Horvath

Publications and source records attributed to J Horvath.

At least 55 records · Page 3Linked to original sources
PubMed1991

Complementation of adenovirus early region 1a and 2a mutants by Epstein-Barr virus immortalized lymphoblastoid cell lines.

Human B-lymphocytes may be infected by both adenoviruses and the Epstein-Barr virus (EBV). Some of the immediate early and early proteins in the two viruses are similar in function even though their primary structures are different. As these viruses might infect the same B-cells in man, we asked if complementation could take place. The adenovirus mutant H5ts125 has a thermolabile DNA-binding protein and is defective in DNA replication at 39 degrees. Several EBV-transformed human lymphoblastoid cell lines and a tamarin cell line B95-8 were infected with H5ts125 and incubated at either the nonpermissive or the permissive temperatures. Adenoviral DNA replication and assembly of new virions were observed at both temperatures, suggesting complementation by the resident EBV gene products. The adenovirus E1a region is deleted in the mutant d1312. Complementation of this mutant was only obtained in the EBV producer B95-8 cells. Immortalization by EBV was apparently not sufficient for effective complementation. This supports an earlier observation that one of the EBV early proteins (MS-EA) behaves like adenovirus E1a and can transactivate the E4 promoter in a CAT assay. The complementation of mutant adenoviruses in EBV-transformed lymphocytes may help the rescue of new adenovirus serotypes in immunosuppressed patients.

Adenovirus Early Proteins↗
PubMed1991

Dominant suppression of adenovirus mediated transformation and insufficiency of p105Rb binding as a condition for oncogenic transformation.

An adenovirus-specific transformation resistant cell line (G2) expressing biologically active E1a proteins and originally isolated as a revertant from Ad2-transformed rat cells (F4), was shown to form stable Rb-E1a and 300K-E1a complexes in immunoprecipitation experiments. Consistent with the transformation resistant phenotype, cell hybrids between G2 and F4 were all nontumorigenic. Retrovirus insertion mutagenesis resulted in tumorigenic cell lines and identified a common locus responsible for the E1a-specific dominant tumor suppressor phenotype of G2 cells.

Adenoviruses, Human↗
PubMed1989

Non-p-glycoprotein-mediated multidrug resistance in detransformed rat cells selected for resistance to methylglyoxal bis(guanylhydrazone).

Three independent variants (G2, G4, G5), resistant to methylglyoxal bis(guanylhydrazone), an anticancer drug, have been isolated by single step selection from an adenovirus-transformed rat brain cell line (1). These variants display selective cross-resistance to several natural product drugs of dissimilar structure and action. Multidrug resistance has recently been shown to be caused by overexpression of the membrane-associated p-glycoprotein, most often caused by amplification of the mdr gene. Several types of experiments were conducted to determine whether the observed drug resistance in our cell lines could be due to changes at the mdr locus. The following results were obtained: (a) the mdr locus was not amplified; (b) transcription of the mdr gene and p-glycoprotein synthesis were not increased; (c) multidrug resistance cell lines, which carry an amplified mdr locus, were not cross-resistant to methylglyoxal bis(guanylhydrazone); (d) verapamil did not reverse the resistance of G cells or mdr cells to methylglyoxal bis(guanylhydrazone), nor that of G cells to vincristine; and (e) methylglyoxal bis(guanylhydrazone) resistance was recessive and depended on a block to drug uptake, as opposed to mdr cells which are dominant and express increased drug efflux. The results obtained suggest that the drug resistance in the G2, G4, and G5 cells was atypical and may be due to a mechanism distinct from that mediated by the mdr locus.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Activation of the major late promoter in adenovirus transformed cells by 5-azacytidine.

The adenovirus major late promoter (MLP) is normally not active in transformed cells. We investigated if it could be activated with 5-azacytidine. Three days of treatment with 10 microM 5-azacytidine induced transient activation of the MLP as shown by hybridization with an L1 r-strand-specific probe. The po/III-transcribed VA-RNAs were not activated. L1 activation was not accompanied by detectable changes in methylation of HpaII sites at the promoter or in the body of the transcript. Stably activated cell clones could be obtained at 20% frequency after long-term drug treatment.

Adenoviridae↗
PubMed1988

Radioimmunoassay for octapeptide analogs of somatostatin: measurement of serum levels after administration of long-acting microcapsule formulations.

The development of a long-acting delivery system for D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 (RC-160), an octapeptide analog of somatostatin, required the establishment of a method for determining the concentration of this analog in serum during treatment. A sensitive and specific radioimmunoassay (RIA) for RC-160 was developed and used for following the rate of liberation of this peptide from microcapsules of poly(DL-lactide-coglycolide). Antibodies were generated in a rabbit against RC-160 conjugated to bovine serum albumin with glutaraldehyde. At an antiserum dilution of 1:100,000, the antibodies bound approximately 25% of added radiolabeled RC-160. Somatostatin octapeptide analogs that had a disulfide bridge showed crossreactivity with the antiserum, but analogs without the disulfide bridge and other peptides tested did not crossreact. The minimum detectable dose of RC-160 was 10 pg. Intra- and interassay coefficients of variation ranged from 9.1% to 12.8% and from 14% to 30%, respectively. The RIA was suitable for direct determination of RC-160 in serum. Eleven prototype batches of microcapsules were tested in rats, and the rate of release of the analog from the microcapsules was followed. An improved batch of microcapsules made from RC-160 pamoate maintained high serum levels of RC-160 for more than 30 days after intramuscular injection. The RIA should be of value for monitoring levels of this analog in serum during long-term therapy.

Animals↗
PubMed1988

Nonpermissivity of human peripheral blood lymphocytes to adenovirus type 2 infection.

The capacity of freshly explanted human peripheral blood lymphocytes (PBL) to support the replication of human adenovirus type 2 (Ad2) was investigated. Unlike other types of human cells, PBL were found to be highly nonpermissive. Ad2 adsorbed 30 to 40% of both T and non-T cells. Virus uncoating was very slow and inefficient, resulting in a 40-fold reduction compared with HEp-2 cells. On a population basis, viral DNA synthesis was reduced 460-fold and infectious virus production was reduced 10(6)-fold. Only 0.35% of PBL produced infectious centers, yielding 0.8 PFU per infected cell. Phytohemagglutinin stimulation increased DNA synthesis 23-fold, infectious centers 11-fold, and virus yield 14-fold. We conclude that resting human PBL are highly nonpermissive to Ad2 infection and that phytohemagglutinin can only marginally lift this nonpermissiveness.

Adenoviruses, Human↗
PubMed1987

Isolation of variants resistant to methylglyoxal bis(guanylhydrazone) from adenovirus-transformed rat cells.

It is presumed that proteins encoded by viral oncogenes interact with proteins encoded by cellular genes to bring about the transformed phenotype. To demonstrate the existence of such cellular genes we attempted to isolate mutants with a nontransformed phenotype from an adenovirus-transformed rat cell line (F4) which contains multiple copies of the transforming E1 region. F4 cells were mutagenized with ethyl methanesulfonate and variants resistant to the anticancer drug methylglyoxal bis(guanylhydrazone) were selected. The proportion of such variants was about one in 10(6) and increased 5-fold after mutagenesis. Two variant clones (G1 and G2) were isolated and characterized: they were 5-fold more resistant to methylglyoxal bis(guanylhydrazone); they had a stable phenotype; they showed decreased drug uptake; they had a reduced ability to grow in soft agar, low serum, and nude mice; there was no detectable change in the restriction pattern of integrated viral genes or in the expression of the E1a and E1b proteins. These properties suggest that selection for methylglyoxal bis(guanylhydrazone) resistance may result in the isolation of variants with phenotypic characteristics of nontransformed cells. It was likely that these variants were altered in a cellular function required for the maintenance of the transformed phenotype.

Adenoviridae↗
PubMed1987

Effect of an anti-substance P serum on prolactin and gonadotropins in hyperprolactinemic rats.

The effects of the acute injection of a rabbit anti-substance P serum (ASPS) were studied in normal rats and rats with hyperprolactinemia induced by 5-hydroxytryptophan and estradiol given as a short or chronic treatment. The anti-substance P serum decreased the release of prolactin induced by 5-hydroxytryptophan when this serotonin precursor was injected 24 h, but not 1 h, after the administration of the antiserum. ASPS reduced the hyperprolactinemia induced by short and chronic treatment with estradiol in castrated rats. This effect was observed 24 h after the injection of the antiserum. On the other hand, the injection of ASPS induced a significant decrease in LH levels in serum of intact male rats injected with 5-hydroxytryptophan 24 h after ASPS, and in castrated rats treated with short-term and chronic administration of estradiol, 24 h after the injection of the antiserum. These results suggest that substance P may have a role in the control of prolactin secretion and could play a part in the hyperprolactinemic effects of estradiol. On the other hand, substance P, under certain circumstances, may stimulate LH release.

5-Hydroxytryptophan↗
PubMed1987

T6+ and HLA-DR+ cell numbers in epidermis of immunosuppressed renal transplant recipients.

The increased susceptibility of the skin of chronically immunosuppressed individuals to viral infections and sunlight-induced malignancies suggests specific drug-induced, dysfunction of local immune mechanisms within the sun-exposed skin of these individuals. To help understand the effect of immunosuppressive therapy alone in the absence of ultraviolet light on the immune system of skin, biopsies were collected from non-sun-exposed buttock skin of control, healthy volunteers and kidney transplant recipients immunosuppressed with either azathioprine/prednisone or cyclosporin A/prednisone and examined for incidences of T6+, and HLA-DR+ cells. No significant differences in the incidences of these 2 cell types were found (a) between control individuals and transplants recipients, (b) between transplant recipients receiving either of the immunosuppressive drug regimes, or (c) between transplant recipients who either had or had not developed skin cancer.

Adolescent↗
PubMed1986

Effect of atrial natriuretic peptide on gonadotropin release in superfused rat pituitary cells.

Cardiac atrial muscle cells produce a polypeptide hormone that plays a role in the control of water and electrolyte balance and blood pressure. The circulating form of this hormone is the atrial natriuretic peptide (ANP), which contains 28 amino acids. Various immunohistochemical studies have shown that ANP is present in many areas of the central nervous system, including the median eminence. In our studies, we investigated the effect of ANP in a superfused rat pituitary cell system. When ANP was administered at increasing concentrations (0.01 microM to 1 microM), it caused a significant dose-related stimulation of the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The lowest effective dose of ANP in our system was 0.03 microM. When ANP and LH-releasing hormone were administered together, the response was prolonged and had the characteristics of ANP-stimulated LH and FSH release. In contrast with some previous reports, ANP in high concentration (1 microM) consistently induced a small but significant stimulation of the release of corticotropin. ANP did not influence the basal release of prolactin, growth hormone, and thyrotropin.

Adrenocorticotropic Hormone↗
PubMed1986

Purification and characterization of peptides with corticotropin-releasing factor activity from porcine hypothalami.

Ten polypeptides that stimulated the release of corticotropin from superfused rat pituitary cells and that are structurally related to porcine corticotropin-releasing factor were isolated from porcine hypothalami. The purification was carried out by gel filtration followed by reversed-phase HPLC using trifluoroacetic acid or heptafluorobutyric acid as the ion-pairing agent in water/acetonitrile solvent systems. The purified peptides were homogeneous by chromatography and by sequence analysis. One major polypeptide was characterized. Its structure is -H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Gl u-Val -Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys -Leu-Met-Glu-Asn-Phe-NH2 [Patthy, M., Horvath, J., Mason-Garcia, M., Szoke, B., Schlesinger, D. H. & Schally, A. V. (1985) Proc. Natl. Acad. Sci. USA 82, 8762-8766]. This 41-amino acid sequence is thought to represent porcine corticotropin-releasing factor. Based on automated gas-phase sequencing of the intact and CNBr-cleaved peptides, amino acid analysis, and carboxypeptidase Y digestion, the other nine polypeptides were found to be structurally similar to this 41-amino acid sequence. Modifications of this structure include deamidation of glutamine at position 26 or 29, oxidation of methionine at positions 21 and/or 38, a blocked N terminus, and deletion of phenylalanine amide at the C terminus. Eight of these nine modified peptides retained significant corticotropin-releasing factor activity as shown by the stimulation of corticotropin release from superfused rat and pig pituitary cells. Some of these peptides may be present in pig hypothalami, while the others could have been produced during the isolation.

Adrenocorticotropic Hormone↗
PubMed1986

Group C adenovirus DNA sequences in human lymphoid cells.

Human peripheral blood lymphocytes from healthy adults, cord blood lymphocytes, and lymphoblastoid cell lines were screened by hybridization for the presence of group C adenovirus DNA sequences. In 13 of 17 peripheral blood lymphocyte samples from adults, 1 of 10 cord blood samples, and seven of seven lymphoblastoid cell lines tested, results were positive for Group C adenovirus DNA (adenovirus 1 [Ad1], Ad2, Ad5, or Ad6). About 1 to 2% of the lymphocytes carried 50 to 100 viral genome copies per positive cell, as estimated by in situ hybridization. Infectious virus representing all members of group C were recovered, but cultivation in the presence of adenovirus antibody did not cure the cells of free viral genomes. Viral DNA was found in B, T, and N cells but only in 1 of 10 cord blood samples. The results suggest that group C adenovirus infections in childhood result in the persistence of the viral genome in circulating lymphocytes.

Adenoviruses, Human↗
PubMed1985

Adenovirus DNA synthesis is coupled to virus assembly.

The relationship between viral DNA synthesis and virion assembly was studied in adenovirus type 2 infected HEp2 cells. When cells were infected at the restrictive temperature with ts3, an assembly-negative mutant which permits normal viral DNA and protein synthesis, labeled and shifted to the permissive temperature, only de novo synthesized nonradioactive viral DNA was encapsidated. This suggested that only concurrently synthesized DNA is encapsidated. Blocking protein or DNA synthesis with cycloheximide or hydroxyurea after the temperature shift inhibited virus assembly. Therefore efficient virus assembly requires both concurrent protein and DNA synthesis. When DNA synthesis was arrested by shifting ts125 infected cells to the restrictive temperature, protein synthesis continued but assembly was completely blocked. Sucrose gradient sedimentation analysis of nuclear extracts of wt and ts3 infected cells provided evidence in support of a physical coupling between replication complexes and virus assembly complexes. Further evidence of coupling was also shown by preferential pulse labeling of the molecular right end of the genome isolated from reversibly cross-linked assembly intermediate particles. While DNA replication is not dependent on concurrent virion assembly, at least some significant proportion of replication complexes appear to be coupled to and are prerequisite for virion assembly.

Adenoviruses, Human↗