Search PubMed⌕ Search

Biomedical subjects

M A Israel

Publications and source records attributed to M A Israel.

At least 127 records · Page 7Linked to original sources
PubMed1985

Tyrosine phosphorylation within the amino-terminal domain of pp60c-src molecules associated with polyoma virus middle-sized tumor antigen.

We have examined the in vitro phosphorylation of cellular src protein (pp60c-src) molecules associated with the polyoma virus middle-sized tumor antigen in polyoma virus-transformed cells. These pp60c-src molecules possessed an enhanced tyrosyl kinase activity, migrated aberrantly on NaDodSO4/polyacrylamide gels, and contained a novel site of tyrosine phosphorylation within the amino-terminal region of the molecule. The pp60c-src molecules not associated with the middle-sized tumor antigen were phosphorylated exclusively on a tyrosine residue within the carboxyl-terminal domain of pp60c-src. A similar modified form of the middle-sized tumor antigen-associated pp60c-src protein was detected in lysates from polyoma virus-transformed cells labeled in vivo with [32P]orthophosphate in the presence of sodium orthovanadate, an inhibitor of phosphotyrosyl phosphatases.

Animals↗
PubMed1985

Increased pp60c-src tyrosyl kinase activity in human neuroblastomas is associated with amino-terminal tyrosine phosphorylation of the src gene product.

We have observed a 20- to 40-fold increase in pp60c-src tyrosyl kinase activity in human neuroblastoma cell lines over that found in either human glioblastoma cells or human fibroblasts. The level of c-src gene transcripts and pp60c-src protein synthesis in the neuroblastoma cells was not significantly increased when compared to the levels found in glioblastoma cells. Approximately one-half of the pp60c-src molecules synthesized during a 4-hr [35S]methionine or [32P]orthophosphate labeling period in neuroblastoma cells were found to migrate more slowly on NaDodSO4/polyacrylamide gels than pp60c-src molecules labeled in glioblastoma cells. Peptide and phosphoamino acid analysis of the in vivo phosphorylated c-src molecules from these two cell types revealed that pp60c-src molecules from the neuroblastoma cells possess in the amino-terminal portion of the protein at least one unique tyrosine phosphorylation site not found in pp60c-src derived from glioblastoma cells.

Cell Line↗
PubMed1985

Middle tumor antigen of polyomavirus transformation-defective mutant NG59 is associated with pp60c-src.

We have found that lysis of mouse embryo cells infected with the polyomavirus host range transformation-defective (hr-t) mutant NG59 under gentle conditions that avoid ionic detergents results in detectable NG59-encoded middle tumor antigen (MTAg) associated with pp60c-src. This MTAg-pp60c-src complex could be immunoprecipitated from NG59-infected cell lysates by either sera from animals bearing polyomavirus-induced tumors or by monoclonal antibodies directed against MTAg. Immune complex kinase assays revealed that, whereas the pp60c-src associated with NG59 MTAg possessed tyrosyl kinase activity, the NG59 MTAg in this complex was not phosphorylated in these in vitro reactions. These results demonstrate that the point insertion mutation found in this transformation-deficient strain of polyomavirus encodes MTAg molecules capable of associating with pp60c-src and defines a limited region within MTAg which appears to be critical for stable MTAg-pp60c-src interactions.

Amino Acids↗
PubMed1985

A determinant of polyomavirus virulence enhances virus growth in cells of renal origin.

We have identified a strain of polyomavirus, Py(L), which is unusual in causing acute morbidity and early death after inoculation of newborn mice. We determined that these animals died of kidney failure associated with extensive, virus-mediated destruction of renal tissue. Interestingly, the Py(L) strain infects baby mouse kidney cell cultures more efficiently than do other strains.

Aging↗
PubMed1984

In vitro association and phosphorylation of polyoma virus middle T antigen by cellular tyrosyl kinase activity.

We have observed increased phosphorylation of tyrosine residues on the polyoma virus middle tumor antigen (MTAg) in in vitro kinase assays of the immune complexes immunoprecipitated from lysates of polyoma virus-infected mouse embryo cells to which increasing amounts of uninfected mouse embryo cell lysate had been added. The components from uninfected mouse cells responsible for increased MTAg phosphorylation were localized by subcellular fractionation to the plasma membrane and found to be sensitive to protease digestion, N-ethylmaleimide, and 5'-p-fluorosulfonylbenzoyladenosine inactivation. The majority of the membrane-associated activity responsible for the increased MTAg phosphorylation in these assays could be cleared from lysates of uninfected mouse cell lysates by centrifugation after reaction with Sepharose-bound monoclonal antibodies which recognize pp60c-src. These results suggest that MTAg can associate with cellular tyrosyl kinases in vitro and be phosphorylated by these enzymes in immune-complex kinase assays. The identity of at least one of these cellular tryosyl kinases which can associate with MTAg in vitro is likely to be pp60c-src.

Adenosine↗
PubMed1984

Enhancement of cellular src gene product associated tyrosyl kinase activity following polyoma virus infection and transformation.

We examine the interaction between polyoma-virus-encoded middle tumor antigen and the cellular src gene product, pp60c-src, using a series of monoclonal antibodies that recognize mammalian pp60c-src. Our results show that infection of mouse cells with transformation-competent strains of polyoma virus results in the stimulation of pp60c-src kinase activity severalfold over that observed in uninfected mouse cells and mouse cells infected with transformation-deficient polyoma virus. A similar degree of enhancement of pp60c-src kinase activity was found in polyoma-virus-transformed rodent cells. No differences were detected in the level of pp60c-src synthesis in polyoma-virus-infected and uninfected mouse cells or polyoma-virus-transformed and normal rodent cells. These studies demonstrate that polyoma-virus-encoded middle tumor antigen is associated with pp60c-src in lysates of polyoma-virus-infected and polyoma-virus-transformed cells and suggest a novel mechanism for the functional activation of a cellular proto-oncogene product, namely, that the interaction between middle tumor antigen and pp60c-src leads to a stimulation of pp60c-src tyrosyl kinase activity.

Animals↗
PubMed1983

Inhibition of polyoma virus middle T antigen-associated tyrosyl kinase activity by N-ethylmaleimide.

The middle T antigen (MT Ag) encoded by polyoma virus has an associated protein kinase activity which transfers a phosphoryl group from ATP or GTP to a tyrosine residue on MT Ag in immunoprecipitates formed between polyoma virus-infected or transformed cell extracts and serum from animals bearing polyoma-induced tumors. Incubation of such immunoprecipitates or polyoma-transformed cell extracts prior to immunoprecipitation with the sulfhydryl reagent, N-ethylmaleimide (NEM), resulted in a significant inhibition of MT Ag-associated kinase activity. Inactivation of this enzyme activity by NEM was found to be dependent upon the incubation pH, time of incubation, and NEM concentration. ATP, GTP, and ADP in the presence of Mg2+ were found to decrease the rate of NEM-mediated inactivation of MT Ag-associated kinase activity, while CTP and UTP did not detectably alter the rate of enzyme inhibition by NEM. These results suggest that the MT Ag-associated kinase possesses at least one NEM-sensitive sulfhydryl group essential for phosphotransferase activity which may be present at or near the enzyme catalytic site.

Adenine Nucleotides↗
PubMed1983

Structural analysis of integrated polyomavirus DNA in a polyomavirus-induced hamster tumor cell line.

The nucleotide sequence and structural organization of integrated polyomavirus DNA and flanking host chromosomal DNA in a polyomavirus-induced hamster tumor cell line were evaluated. Although the proximal portion of the early transcriptional region was intact, the remainder of the integrated viral sequences was characterized by a series of rearrangements, including deletions, insertions, and a tandem duplication. Short direct repeats of different nucleotide sequences located on each side of the host-viral DNA junctions we examined were observed. Tumor cell chromosomal DNA flanking the integration site contained regions of patchy homology with viral DNA sequences which could have participated in the recombinational events leading to stable integration. The presence of several unusual structural features of tumor cell chromosomal DNA at the site of viral DNA integration, which were reminiscent of papovavirus DNA integrated into transformed cells, suggests that the recombinational mechanisms operative in vivo are accurately reflected by those functioning in vitro.

Animals↗
PubMed1982

Stability of polyoma DNA sequences and virus-coded proteins during tumor formation.

To determine the stability of polyoma viral DNA in transformed rat cells during their growth in vivo, we compared the state and arrangement of polyoma virus DNA sequences in virus-transformed rat cell lines before and after their passage in vivo. In cell lines from 12 independent tumors induced by the inoculation of animals with three different transformed cell lines, we could detect no significant changes in the arrangement of viral DNA sequences associated with the in vivo passage of these cell lines. In 13 of 14 tumor cell lines examined, the pattern of polyoma virus tumor antigens, characterized by the presence of the polyoma virus large, middle, and small tumor antigens, was unchanged.

Animals↗
PubMed1980

Characterization of polyoma viral DNA sequences in polyoma-induced hamster tumor cell lines.

The polyoma (PY) viral DNA sequences present in a series of hamster tumor cell lines were evaluated using the blotting technique of Southern ((1975) J. Mol. Biol. 98, 503-517). Remarkably, no cell line contained an intact distal portion of the early gene region which encodes a portion of the large T antigen. All cell lines examined contained the PY DNA Bum I fragment which contains most of the genetic information encoding PY small and middle T antigens, as well as the origin of viral DNA replication. These results provide an explanation for our previous observation that PY virus-induced hamster tumors do not contain the large species of T antigen.

Animals↗
PubMed1980

Polyoma large tumor antigen is not required for tumorigenesis mediated by viral DNA.

The arrangement of viral DNA sequences in a hamster cell line derived from a tumor induced by a recombinant plasmid DNA preparation containing the entire polyoma virus genome was examined. In the recombinant plasmid employed, viral DNA sequences specifying the large species of polyoma tumor antigen but not the small and middle tumor antigens were interrupted by the insertion of plasmid DNA at the EcoRI restriction endonuclease site. Blot-hybridization analyses of tumor cell DNA indicated that the "joints" linking viral and plasmid DNAs in the original recombinant plasmid used in animal inoculation had been preserved. Integration into the hamster cell genome had apparently occurred within plasmid DNA sequences. These results indicate that polyoma large tumor antigen is not required for tumorigenesis mediated by viral DNA.

Animals↗
PubMed1980

Cloning of human papilloma virus genomic DNAs and analysis of homologous polynucleotide sequences.

The complete DNA genomes of four distinct human papilloma viruses (human papilloma virus subtype 1a [HPV-1a], HPV-1b, HPV-2a, and HPV-4) were molecularly cloned in Escherichia coli, using the certified plasmid vector pBR322. The restriction endonuclease patterns of the cloned HPV-1a and HPV-1b DNAs were similar to those already published for uncloned DNAs. Physical maps were constructed for HPV-2a DNA and HPV-4 DNA, since these viral DNAs had not been previously mapped. By using the cloned DNAs, the genomes of HPV-1a, HPV-2a, and HPV-4 were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm = --28 degrees C), no homology was detectable among the genomes of these papilloma viruses, in agreement with previous reports. However, under less stringent conditions (i.e., Tm = --50 degrees C), stable DNA hybrids could be detected between these viral DNAs, indicating homologous segments in the genomes with approximately 30% base mismatch. By using specific DNA fragments immobilized on nitrocellulose filters, these regions of homology were mapped. Hybridization experiments between radiolabeled bovine papilloma virus type 1 (BPV-1) DNA and the unlabeled HPV-1a, HPV-2a, or HPV-4 DNA restriction fragments under low-stringency conditions indicated that the regions of homology among the HPV DNAs are also conserved in the BPV-1 genome with approximately the same degree of base mismatch.

Base Sequence↗
PubMed1980

A cloned polyoma DNA fragment representing the 5' half of the early gene region is oncogenic.

The two polyoma DNA fragments generated by cleavage with BamHI and EcoRI were cloned in pBR322, and their oncogenic potential was tested in vivo and in vitro. Only recombinant plasmid DNA containing a polyoma DNA fragment which extends clockwise from 58 to 0 map units and include approximately the 5'-proximal half of the early gene region produced tumors in newborn hamsters and transformed rat embryo cells in tissue culture. Southern blotting analysis indicated that the entire 2.2-kilobase polyoma BamHI-EcoRI fragment was intact in both a tumor cell line and a cell line transformed in culture which we examined. The presence of polyoma middle and small T antigen in these lines was demonstrated by immunoprecipitation and tryptic peptide mapping. DNA from a recombinant plasmid containing a polyoma genome deleted between 90 and 4 map units failed to induce tumors or transform cells.

Animals↗