Peter T. Mora

Nuclear preparations of SV40-transformed cells contain tumors-pecific transplantation antigen activity

Abstract Nuclear preparations from human SV40-transformed cells containing high levels of the tumor antigen (TA) were found to be enriched for tumor-specific transplantation antigen (TSTA).

The phosphoprotein p53 is down-regulated post-transcriptionally during embryogenesis in vertebrates

The phosphoprotein p53 has been investigated mainly because of its relationship with tumorigenic transformation. In this communication, we report that, during the embryonal development of mouse and chicken, there is a decline in the steady-state levels of the p53 protein and an equal decline in p53 mRNA. During the development of the chicken, the relative rates of p53 transcription appear to be constant. p53 mRNA is relatively stable (half-life greater than 12 h) in both chicken and mouse embryos. We conclude that (i) the down-modulation of p53 mRNA (and of protein) during embryonal development has been well conserved during the evolution of the vertebrate, implying that the p53 protein may have a function in embryonal development; and (ii) the mechanism of control is apparently mainly on a post-transcriptional level.

Ganglioside biosynthesis in mouse cells: Glycosyltransferase activities in normal and virally-transformed lines

Abstract The activities of four glycosyltransferases involved in ganglioside biosynthesis were measured in two different established mouse cell lines and in the SV40 and polyoma transformed variants of these lines. The only consistent change observed was the very reduced activity of UDP-GalNAc: hematoside N-acetyl-galactosaminyltransferase in all of the transformed cell lines (15–20% of normal cells). There was no significant differences in any of the glycosyltransferases with increased cell density and cell-to-cell contact.

Alteration in ganglioside pattern and synthesis in sv40- and polyoma virus-transformed mouse cell lines.

Abstract Analysis of mouse cell lines revealed that m all the cell lines investigated which were transformed in tissue culture by the tumorigenic DNA viruses, the highest ganglioside homolog N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (GD1a) is decreased or is nearly absent, and concomitant with this N-acetylneuraminylgalactosylglucosylceramide (GM3) becomes the principal ganglioside. In the untransformed control or parent cell lines, or in cells transformed “spontaneously” in culture, GD1a occurs in well quantifiable amounts. Incorporation studies with the radioactive precursors N-acetyl- d -[ 3 H]mannosamine and N-acetyl- d -[ 3 H]glucosamine confirmed these results. We postulate that these changes in the membrane glycolipids are expressions of a common virus-regulated biochemical function in SV40- and polyoma virus-transformed mouse cells.

A common biochemical change in sv40 and polyoma virus transformed mouse cells coupled to control of cell growth in culture.

Abstract In mouse cell lines after transformation with SV40 or polyoma virus there was shown to be a reduction in the content of higher ganglioside homologs and in the activity of an enzyme (uridine diphosphate N -acetylgalactosamine: hematoside 2 N -acetylgalactosaminyltransferase) necessary for the synthesis of the higher gangliosides. These changes were found to be reversibly coupled to the changes in growth properties in tissue culture: in the flat derivatives of the virus transformed cell lines which reverted to normal phenotypic growth properties in culture there was a trend of reversion in ganglioside pattern and in enzymatic activity near to that of the parent 3T3 line, in spite of the continued presence of the viral DNA. Mouse cells productively infected with polyoma virus did not show decrease in the aminosugar transferase activity. Cocultivation of virally transformed cells with enzymatically active cells did not result in reduced enzyme activity in the enzymatically active cells, or in the restoration of enzyme activity in the virally transformed cells. Similarly, no change in enzyme activity occurred when admixing homogenates of the two type of cells. These biochemical findings are consistent with a similar repressor-like mechanism in both SV40 and polyoma virus induced cell transformation .

Continuous spectrophotometric assay for retroviral proteases of HIV-1 and AMV.

Ac-Lys-Ala-Ser-Gln-Asn-Phe(NO2)-Pro-Val-Val-NH2 (peptide I) and Thr-Phe-Gln-Ala-Phe(NO2)-Pro-Leu-Arg-Glu-Ala (peptide II) undergo hydrolysis between the p-nitrophenylalanyl and prolyl residues catalyzed by the proteases of HIV-1 and AMV, respectively. The specific hydrolyses of peptides I and II are accompanied by a decrease in their uv absorption at 269 nm (delta epsilon = 1000) and an increase at 316 nm (delta epsilon = 600). The use of microspectrophotometric cells allows continuous uv measurements on a volume (60 to 120 microliters) comparable to that required for the HPLC point assay currently used. At the highest substrate concentration possible under the assay conditions, good first-order kinetics were observed with both proteases, and the values of Vmax/Km were obtained.

Chemical synthesis and expression of the HIV-1 protease gene in E.coli

The 297bp HIV-1 protease gene was constructed from five discrete synthetic fragments and expressed in E. coli. A soluble protein product of 11.5 Kd was detected by immunoblotting using protease specific antisera. A quantitative assay system, utilizing a synthetic nonapeptide spanning the cleavage site between p17-p24 in the gag polyprotein, was used to measure the specific protease activity in crude extracts. The protease hydrolyzed tyrosyl-proline bonds with an approximate specific activity of 43 pmoles/min/micrograms of total protein. The chemical synthesis of the protease gene and its expression provides a feasible method for rapid mutant analysis, important for structure-function studies and rational design of potential inhibitors.

Reversible inhibition of enzymes by interaction with synthetic polysaccharide macroanions

Abstract Charged polyglucose derivatives, such as polyglucose sulfates, with different molecular weights, branching, and degree of substitution precipitated basic proteins, such as lysozyme, ribonuclease, protamine, thymus peptide, polymyxin B, an ACTH preparation, cytochrome c, and serum albumin from aqueous solution at low pH and salt concentration. Enzymic activity of lysozyme, ribonuclease (both on ribonucleic acid and on a cyclic phosphate), and hyaluronidase was inhibited in solution. The inhibition required macromolecular size and polyanionic structure and depended on the type and amount of charged groups on the synthetic polysaccharide derivative. Highly substituted sulfates were the most effective inhibitors, while sulfates with similar molecular size but lower charge density or derivatives with weaker dissociating anionic groups (carboxyls) had less inhibitory activity. The inhibition appears to be due to electrostatic interaction forces between the oppositely charged macromolecules and can be reversed by a polymeric cation stronger than the substrate.

Substitution mutations of the highly conserved arginine 87 of HIV-1 protease result in loss of proteolytic activity

The 297bp gene coding for the HIV-1 protease was chemically synthesized and expressed in E. coli. Single amino acid substitutions (Arg 87 - greater than Lys; Arg 87 - greater than Glu) were introduced in the C-terminally located conserved region GlyArgAsn of the protease gene in the wild-type clone. The products of the mutant and the wild-type clones were expressed at approximately similar levels at 30 minutes of induction but the mutant protease proteins accumulated as a function of time of induction unlike the wild-type protease which declined after 60 minutes. The mutants were completely devoid of proteolytic activity as determined in assays employing as substrates a synthetic nonapeptide and a gag related recombinant polyprotein.

Cytolytic microassays and studies of SV40 tumor-specific transplantation antigen

Abstract A rapid in vitro assay for SV40 virus-associated tumor-specific transplantation antigen (TSTA) has been developed using SV40-transformed cell lines from inbred mice. This assay, which is based on antibody-complement-mediated cytolysis, permits quantitative measurement of the new antigen on the surface of living cells and in subcellular fractions. The procedure can be done in 5–10 μl of solution in 3 hr using microtest tissue culture plates, with cytolysis indicated either by trypan blue dye uptake or by the release of 51 Cr from labeled target cells. Mouse antiserum kills only those mouse cells which have been transformed by SV40 virus. It also kills hamster cells which carry SV40 T antigen and TSTA; however, it does not kill hamster cells which carry the “S” antigen but neither the T antigen nor the TSTA. The assay is suitable for monitoring analytical scale biochemical work on solubilized TSTA by the method of cytolysis-inhibition. The cytolytic activity of the mouse antiserum elutes from Sephadex G-200 with the immunoglobulin G-containing fractions. Incubation of SV40-transformed cells with the specific antiserum, without complement, enhances the tumorigenicity of these cells when they are injected into syngeneic animals. This assay has permitted the direct measurement of TSTA on the surfaces of cells infected, abortively transformed, or stably transformed by SV40 virus.