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P M Pitha

Publications and source records attributed to P M Pitha.

At least 127 records · Page 7Linked to original sources
PubMed1983

Two levels of regulation of beta-interferon gene expression in human cells.

We cloned alpha- and beta-interferon cDNA and used them as specific probes to determine the relative levels of interferon mRNA in human fibroblasts cells induced with poly(rI).poly(rC) or Newcastle disease virus to synthesize interferon. Both inducers activated only the beta-interferon gene; however, the half life of beta-interferon mRNA in cells induced with virus was substantially longer than in poly(rI).poly(rC)-induced cells. The transcription rate of beta-interferon RNA sequences was examined in nuclei isolated from poly(rI).poly(rC)-induced cells; it was found that the induction leads to transcriptional activation of the beta-interferon gene and that the shutoff period when no interferon synthesis or cytoplasmic betamRNA are detected. Thus, the synthesis of beta interferon in poly(rI).poly-(rC)-induced human fibroblasts is controlled both by activation of transcription of the beta-interferon gene and by alteration of the beta-interferon mRNA stability.

Cell Line↗
PubMed1982

Interferon treatment of murine Meth-A sarcoma cells: effects on the malignant phenotype and expression of tumor-specific and H-2 antigens.

A virus-free methycholanthrene-induced sarcoma (Meth-A) in BALB/c mice was grown in culture and treated with purified mouse interferon (alpha and beta mixture) prior to testing for oncogenicity in the host animal. Use of interferon in vitro caused growth inhibition, but not cytotoxic effects; such effects were fully reversible upon interferon removal from the system. There was a significant decrease in tumor incidence in mice challenged with interferon-treated cells, but this could be overcome by sufficiently increasing the number of cells in the inoculating dose. By transplanting these BALB/c sarcoma cells into Sprague-Dawley nude mice, the effects of interferon could be negated. Since these mice have an unaltered activity of NK cells, the results suggest that NK cells do not play a major role in rejection of the Meth-A tumor, but that the reduction in tumor incidence is dependent on the presence of functional T cells. Interferon caused a detectable reduction in the expression of the tumor-specific transplantation antigen (TSTA) associated with Meth-A cells, but increased the expression of H-2 antigens. It has recently been shown that H-2 antigens play a role in host recognition of the TSTA of methylcholanthrene-induced sarcomas. It is suggested that the increased expression of H-2 antigens on interferon-treated Meth-A cells could lead to an increased frequency of recognition by T-cells.

Animals↗
PubMed1982

Induction of human beta-interferon synthesis with poly(rI . rC) in mouse cells transfected with cloned cDNA plasmids.

Human genomic DNA and plasmids carrying portions of the cDNA gene for human beta-interferon have been introduced into mouse Ltk- cells by cotransfection with a herpes simplex virus thymidine kinase (TK) gene. One plasmid contains 840 base pairs of human DNA complementary to pre-beta-interferon mRNA inserted into pBR322, whereas the other plasmids have hybrid genes containing only the 560-base pair coding region inserted under the transcriptional control of the TK promoter. Constitutive interferon production could not be detected in any of the mouse TK+ cell lines tested. Nevertheless, synthesis of interferon could be induced by poly(rI . rC) treatment in at least 16 of these cell lines, including clones transfected with genomic DNA, the beta-interferon cDNA, and the TK-beta-interferon cDNA hybrid gene. The interferon produced was specific for human cells and could be neutralized by antiserum against human beta-interferon. In contrast to human fibroblast cells, in which the synthesis of induced beta-interferon is transient, the poly(rI . rC)-induced TK+ lines continued to produce beta-interferon for prolonged periods of time and did not respond to superinduction conditions. Therefore, in transfected mouse cells, the coding DNA sequence from the human beta-interferon gene, without any of the adjacent 3' or 5' flanking human DNA sequences, was sufficient both to direct synthesis of biologically active product and to respond to the specific induction system that operates in human cells. However, the mechanism that switches off the synthesis of induced interferon in human cells appears not to operate in mouse cells transfected with beta-interferon cDNA.

Cell Line↗
PubMed1982

Methylation and a polymorphic restriction site adjacent to human beta-interferon gene.

High-molecular-weight DNA from cultured human fibroblast cells and tissues was analyzed with the restriction endonucleases Msp I and Hpa II for the presence of methylated sites in the sequences flanking the beta-interferon gene. The majority of the DNAs analyzed were methylated in the restriction site (CCGG) for these two enzymes and thus were sensitive to cleavage by Msp I, but resistant to Hpa II. A polymorphic Msp I restriction site was identified approximately 1000 bp upstream from the beta-interferon gene. The results show an association of the beta-interferon gene with Msp I fragments of either 2.7 kb or 4.2 kb, which are inherited as Mendelian alleles. An unusual Msp I site upstream from the beta-interferon gene was present in 22% of the DNA from peripheral leukocytes of healthy individuals, and in 36% of the DNA from leukocytes of individuals with different forms of leukemia. Induction of beta-interferon with poly(rl X rC) did not alter the methylation pattern in the sequences flanking the beta-interferon gene, and the levels of beta-interferon induced in cells by poly(rl X rC) could not be directly related to the presence or absence of a polymorphic Msp I restriction site in the 5'-flanking region.

Base Sequence↗
PubMed1982

Radioimmunoassay of human beta interferon.

A solid phase radioimmunoassay (RIA) for human fibroblast-derived beta (beta) interferon is described using dinitrophenylated (DNP) IgG from rabbit anti-interferon serum and quantitation of the antigen-bound antibodies by subsequently measuring the attachment of 125I-labeled anti-DNP. The sensitivity limit of the assay is 50 u of interferon corresponding to 0.5 ng of interferon protein. Preparations of alpha leukocyte-derived interferon show only marginal cross-reactivity (less than 0.01%) while the mouse interferons (mixture of alpha and beta)are negative in this assay. The simplicity, reproducibility and the possibility of using 125I-labeled anti-DNP as a universal reagent for different anti-interferon sera may widen the development of RIA tests for interferons both in research and clinical areas.

Animals↗
PubMed1982

Effect of interferon on the replication of mink cell focus-inducing virus in murine cells: synthesis, processing, assembly, and release of viral proteins.

Treatment of mink cell focus-inducing (MCF) virus (isolate AK-13) producing SC-1 cells with mouse fibroblast interferon (150 to 600 U/ml) led to a 100-fold decrease in the release of infectious virus, whereas there was a 2.5- to 10-fold decrease in various parameters of virus particle release. Analysis of labeled virion proteins indicated that a temporal change in virion protein composition occurred after interferon treatment. After a 24-h exposure of chronically infected cells to interferon, the virions produced contained a 85,000-dalton glycoprotein (apparently of nonviral origin) which was in excess of the virus envelope glycoprotein gp70. Particles produced from cells treated with interferon for 32 to 48 h were nearly devoid of gp70 and contained substantially lower quantities of p30. Intracellular processing of viral precursor polyproteins to the mature virion structural proteins was not altered in the presence of interferon. However, an accumulation of the viral p30 and p12E proteins was observed in interferon-treated cells, consistent with an increase in cell-associated virions. Immunoprecipitation analysis of the tissue culture fluids from [35S]methionine-labeled control and interferon-treated cells revealed marked decrease in p30 and p15E/p12E released after interferon treatment. In contrast, gp70 did not accumulate in interferon-treated cells, but was released into the culture medium in a form that was neither pelletable nor associated with p15E/p12E.

Animals↗
PubMed1982

Human beta interferon gene localization and expression in somatic cell hybrids.

The human fibroblast interferon gene beta 1 was mapped to human chromosome 9. Sequence homology with a beta 1 cDNA clone was detected in both genomic DNA and induced mRNA of human/mouse or human/hamster somatic cell hybrids containing human chromosome 9, but not in lines lacking this chromosome or those retaining a complex translocation involving chromosomes 9 and 11. Interferon mRNA that did not share sequence homology with the beta 1 cDNA clone was detected in lines containing human chromosomes 2 and 5 but lacking chromosome 9, suggesting the presence of other unlinked interferon sequences in the human genome.

Animals↗
PubMed1981

Analysis of interferon mRNA in human fibroblast cells induced to produce interferon.

The levels of interferon mRNA as a function of interferon induction by poly(rI) . poly(rC) in human fibroblast cells were determined by RNA hybridization using a cloned beta interferon cDNA and by translation in Xenopus oocytes. Whereas previous studies analyzed mixtures of interferons, the availability of the cloned beta interferon cDNA and the antiserum to purified beta interferon enabled us to focus on the expression of only one class (beta) of interferon genes. The induction of interferon synthesis depends primarily on the accumulation of interferon beta mRNA in the cells, and the interferon beta mRNA rapidly disappears several hours after its appearance in the cytoplasm. No detectable interferon beta mRNA sequences are present in uninduced cells. The degradation of interferon beta mRNA in the induced cells requires ongoing protein synthesis; accumulation of interferon beta mRNA was observed in the continuous presence of cycloheximide. The interferon beta mRNA detected at the early stages of induction is 1100 nucleotides long and its size progressively decreases with time. By both the hybridization and the translational assay in Xenopus oocytes, only one size of interferon beta mRNA and one species of beta interferon could be identified.

Cells, Cultured↗
PubMed1981

Interferon heterogeneity resulting from differences in glycosylation.

35S methionine-labeled interferon synthesized in mouse C-243 induced by NDV in the presence and absence of glycosylation was analyzed after immunoprecipitation by gel electrophoresis. Interferons produced in the induced cells show large size heterogeneity in the region of 24,000-35,000-daltons. Interferon synthesized in the tunicamycin treated induced cells show two major bands of 15,000 and 18,000 daltons, which synthesis was also coded for by the interferon mRNA in cell-free system. The results indicate that the size heterogeneity of mouse interferons is due to the variability in the carbohydrate moiety of interferon polypeptides (alpha, beta).

Animals↗
PubMed1980

The messenger RNA sequences in human fibroblast cells induced with poly rI.rC to produce interferon.

Induction of human fibroblast cells with poly rI.rC induces interferon mRNA which can be translated into interferon precursor in wheat germ cell free system or in Xenopus oocytes into biologically active interferon. The extent of gene expression in the poly rI.rC induced cells was compared to that of the uninduced cells by hybridization of the mRNA to complementary DNA. Homologous template driven hybridization of cDNA revealed the presence of two clearly defined transitions in the total poly A RNA from the induced cells; abundant class and a scarce class comprising approximately 37,000 diverse species of RNA. Heterologus hybridization of the cDNA with total uninduced mRNA showed that the majority of the mRNA sequences are the same in both the induced and uninduced cells. The results of the hybridization using cDNA prepared to the fraction enriched for interferon mRNA, however, showed that about 4% of the sequences present in the interferon enriched fraction are not present in the uninduced cells. These differences may result from the poly rI.rC induced alterations in gene expression.

Fibroblasts↗
PubMed1980

Synthesis of new proteins associated with the induction of interferon in human fibroblast cells.

The relative amounts of translatable cellular mRNAs and newly synthesized cellular proteins were examined in poly(I) x poly(C)-induced human fibroblast cells early during induction. At this time interferon and interferon mRNA synthesis are maximal and cells have not acquired their antiviral thesis are maximal and cells have not acquired their antiviral state. Translation of the mRNA from poly(I) x poly(C)-induced cells in a wheat germ cell-free system led to the synthesis of a [35S]methionine-labeled 22,000-dalton protein that is precipitated by antiserum to highly purified human fibroblast interferon. The synthesis of this protein was detected only with the mRNA preparations that, when translated in Xenopus oocytes, coded for the synthesis of biologically active human interferon. Two-dimensional gel analysis of the [35S]methionine-labeled polypeptides translated from the total mRNA of the induced and uninduced cells revealed the presence of 23 new proteins that were translated from mRNAs of the induced cells but not from the mRNAs of the controls. These polypeptides ranged from 15,000 to 70,000 daltons. Thirteen of these proteins were detected in induced cells labeled with [35S]methionine. It is concluded that, in human fibroblasts, poly(I) x poly(C) induces, in addition to interferon, the synthesis of a variety of "interferon-associated" proteins.

Cell-Free System↗
PubMed1980

Effect of interferon on mouse leukaemia virus (MuLV). V. Abnormal proteins in virions of Rauscher MuLV produced in the presence of interferon.

Interferon treatment of JLSV-6 cells chronically infected with Rauscher MuLV leads to the formation of non-infectious particles ('interferon' virions) containing the structural proteins coded by the env and gag genes as well as additional virus polypeptides. The major glycoprotein detected in the control virions is gp71, but 'interferon' virions contain in addition an 85K mol. wt. (gp85) glucosamine-containing, fucose-deficient glycoprotein. This is recognized by antiserum to MuLV and may be related to env pr85. Surface iodination of intact virions indicates that gp71 and gp85 are the two major components of the external envelope. However, whereas in control virions gp71 associates with p15E (gp90), this complex was not detected in 'interferon' virions. Analysis of radio-labelled (3H-amino acids or iodinated) proteins from disrupted 'interferon' virions revealed the presence of 65K, 55K, 40K, 20K and 12K mol. wt. polypeptides which could be precipitated with antiserum against MuLV. There was a distinct difference in the patterns of incorporation of pulse-labelled 3H-amino acid polypeptides into virions in the presence and absence of interferon. Those polypeptides labelled in the presence of interferon and recovered in the extracellular virions in a chase with interferon appeared to have substantially fewer copies of p30 and more of gag pr55 polypeptide than the controls. These results indicate that in the presence of interferon there are changes in the proteolytic cleavage associated with virion assembly.

Animals↗