PubMed Health⌕ Search

Biomedical subjects

P T Mora

Publications and source records attributed to P T Mora.

At least 19 recordsLinked to original sources
PubMed1991

Autoprocessing of the HIV-1 protease using purified wild-type and mutated fusion proteins expressed at high levels in Escherichia coli.

Various constructs of the human immunodeficiency virus, type 1 (HIV-1) protease containing flanking Pol region sequences were expressed as fusion proteins with the maltose-binding protein of the malE gene of Escherichia coli. The full-length fusion proteins did not exhibit self-processing in E. coli, thereby allowing rapid purification by affinity chromatography on cross-linked amylose columns. Denaturation of the fusion protein in 5 M urea, followed by renaturation, resulted in efficient site-specific autoprocessing to release the 11-kDa protease. Rapid purification involving two column steps gave an HIV-1 protease preparations of greater than 95% purity (specific activity approximately 8500 pmol.min-1.micrograms protease-1) with an overall yield of about 1 mg/l culture. Incubation of an inactive mutant protease fusion protein with the purified wild-type protease resulted in specific trans cleavage and release of the mutant protease. Analysis of products of the HIV-1 fusion proteins containing mutations at either the N- or the C-terminal protease cleavage sites indicated that blocking one of the cleavage sites influences the cleavage at the non-mutated site. Such mutated full-length and truncated protease fusion proteins possess very low levels of proteolytic activity (approximately 5 pmol.min-1.micrograms protein-1).

ATP-Binding Cassette Transporters↗
PubMed1991

Purification of HIV-1 wild-type protease and characterization of proteolytically inactive HIV-1 protease mutants by pepstatin A affinity chromatography.

Recombinant wild-type protease of human immunodeficiency virus, type 1 (HIV-1) expressed in E. coli was purified by pepstatin A affinity chromatography. An 88-fold purification was achieved giving a protease preparation with a specific enzymatic activity of approximately 3700 pmol/min/micrograms. Two proteolytically inactive HIV-1 mutant proteases (Arg-87----Lys; Asn-88----Glu) were found to bind to pepstatin A agarose, and and they were purified as the wild-type protease. A third mutant protease Arg-87----Glu) was apparently unable to bind to pepstatin A under similar conditions. Binding to pepstatin A indicates the binding ability of the substrate binding site and the ability to form dimers. These features may be used to purify and to characterize other mutated HIV-1 proteases.

Amino Acid Sequence↗
PubMed1989

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.

Arginine↗
PubMed1989

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.

Avian Leukosis Virus↗
PubMed1989

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 it's expression provides a feasible method for rapid mutant analysis, important for structure-function studies and rational design of potential inhibitors.

Amino Acid Sequence↗
PubMed1988

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.

Actins↗
PubMed1986

The transformation-related protein p53 is not bound to the SV40 T antigen in BALB 3T12 cells expressing T antigen.

In most murine cells transformed by the SV40 virus, virtually all of the cellular phosphoprotein p53 is in a complex with the SV40 T antigen. Here, we report that, in SV40-infected T-antigen-positive Balb 3T12 mouse cells, most (approximately 80%) of the p53 is not in complex. Complex formation was determined by measuring the amounts of [35S]methionine-labeled p53 which coprecipitated with T antigen when using monoclonal antibody to T antigen. The amount of complex formation was expressed as a percentage of total p53 present, measured by the amount of p53 precipitated with the monoclonal antibody to the p53. The values were confirmed by Western blotting procedure, in which the steady-state levels of the proteins were measured. In these measurements after complete precipitation with antibody to T antigen, the residual p53 in the supernatant was precipitated by antibody to p53, and this amount was denoted as free p53. There was no significant difference seen between the [35S]methionine-labeled tryptic peptides of complexed and the free p53 (or between complexed and free T antigens) as determined by two-dimensional gel electrophoresis and chromatography. Virus rescue experiments and retransformation by the rescued virus showed that there was no mutation in the SV40 DNA coding for the T antigen which could account for the lack of complex formation. Both p53 and T antigen were underphosphorylated in cells which exhibited reduced complex formation. Tumorigenicity in syngeneic mice and anchorage-independent cell growth in culture of various cloned mouse cells with or without T antigen expression was compared. The changes in the biologic properties were explainable solely on the basis of known or expected effects of expression of the T antigen and were independent of complex formation or of absence of complex formation between p53 and T antigen.

Animals↗
PubMed1986

Immunologic selection of simian virus 40 (SV40) T-antigen-negative tumor cells which arise by excision of early SV40 DNA.

A clonal line of highly oncogenic spontaneously transformed mouse cells (104C) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned (106CSC). This 106CSC cell line expressed T antigen and transplantation antigen but was about 100 times less tumorigenic than the 104C parent. When 10(5) 106CSC cells were injected into immunocompetent syngeneic mice, tumors were produced. From such tumors, cell lines were established in culture, all of which were consistently negative for T antigen. We found previously by solution DNA hybridization methods that the tumor cells were depleted in the early region of SV40 DNA which codes for the T antigen. We postulated that this loss occurs through a DNA rearrangement of unknown mechanism in one or a few 106CSC cells and that the tumors are then produced from such a cell or cells, whereas all the T-antigen-positive 106CSC cells are rejected by immunologic means. In this investigation we showed by the DNA transfer method using appropriately selected SV40 DNA probes that indeed the tumor cell clone (130CSCT) we selected to investigate came from one 106CSC cell in which the T-antigen-coding SV40 DNA sequences (but not all the early SV40 DNA sequences) were lost by an excision and recombination mechanism. We also showed that the 130CSCT cells, which are highly tumorigenic, could again be transformed by SV40 and that the resulting T-antigen-positive cloned derivative cells became much less tumorigenic (approximately 10(5)-fold), apparently again because of immunologic recognition and rejection. Indeed, when 10(7) T-antigen-positive cloned cells were injected, all the T-antigen-positive cells were rejected and the tumor was produced again from one or more T-antigen-negative cells. Thus, a one-step in vivo transplantation experiment allowed a selection (for tumorigenicity and against the SV40 T antigen) of a mutant mammalian cell with a DNA deletion at a definable site.

Animals↗
PubMed1983

Immunoaffinity chromatography of a cellular tumor antigen from mouse neuroblastoma cells.

The cellular tumor antigen p53 was isolated from mouse neuroblastoma cells and was found in a form not complexed to another protein. The p53 in these cells was stable, turning over about every 10 h. Its methionine-labelled tryptic peptides were very similar to those of the p53 isolated from SV40-transformed mouse cells. The labelled protein was purified from neuroblastoma cells by immunoaffinity using specific monoclonal antibodies and was about 80% radiochemically pure. Furthermore, the purified p53 sedimented in sucrose gradients with a sedimentation coefficient of approximately 8S. This correlated with the sedimentation coefficient of p53 prior to purification, showing that the purified protein retained its native size.

Animals↗
PubMed1982

The amount of a specific cellular protein (p53) is a correlate of differentiation in embryonal carcinoma cells.

A specific cellular protein of molecular weight of 53-55,000 (p53) has been shown to be induced in all SV40 transformed cells. A similar protein has also been shown to be present in embryonal carcinoma cells and in midgestation murine embryo primary cells, which are not infected by SV40. In embryo cell primaries the amount of the protein was shown to decrease with the increase in the stage of embryo development. As differentiation or decrease in cell growth rate can account for this, and since the growth rate of embryo primary cells cannot be measured, we chose to investigate various embryonal carcinoma cells. We report that the p53 is present in a pluripotent embryonal carcinoma cell OTT6050, and in its differentiated parietal endoderm derivative, PYS-2 cells. The amount of p53 is higher in the undifferentiated EC stem cells than in the differentiated PYS-2 (parietal endoderm) cells. The amount of the protein decreases in F9 embryonal carcinoma cells induced to differentiate to a parietal endoderm cell type by treatment with retinoic acid, as it does following spontaneous differentiation of OTT6050 EC cells. To determine if a change in growth rate, rather than differentiation, might account for the diminished levels of this protein, the amount of p53 was measured in growing and in growth arrested cell populations. When the growth rate of F9 cells was reduced by treatment with 8-bromocyclic AMP there was no change in the amount of p53. The half life of the p53 was compared in the undifferentiated and the differentiated cell types to determine if a change in stability might account, in part, for the altered levels of this protein. The p53 is found to be most stable in the SV40 transformed established clonal cells. It is less stable in the fibroblast clonal cells which were not transformed by SV40. The results of these experiments indicate that a decrease in the amount of p53 primarily correlates with differentiation in the embryonal carcinoma cell lines studied and not with cell growth rate. Furthermore, the decrease appears to be related (in part) to the decreased stability of the p53.

Animals↗
PubMed1982

Quantitation of a 55K cellular protein: similar amount and instability in normal and malignant mouse cells.

Quantitative expression of a specific 55,000 (55K)-molecular-weight cellular protein was studied in two groups of mouse embryo fibroblast (clonal) cells originating from two parent clones, one of which possessed high tumorigenicity and the other of which possessed very low tumorigenicity. From the clone with low tumorigenicity, tumor lines and clones were obtained by selecting rare spontaneously transformed highly tumorigenic (mutant) cells. Cells were labeled during exponential growth for 3 h at 37 degrees C, with [35S]methionine, and the cellular 55K protein was immunoprecipitated with a monoclonal antibody and quantitated. There were low and approximately equal amounts of 55K protein in cells (clones) with both low and high tumorigenicity from both groups of cells, and there was no correlation at all between quantitative expression of 55K protein and of cellular tumorigenicity. There was approximately 10- to 20-fold more 55K protein in all simian virus 40-transformed T antigen-positive derivative clones, as shown previously. The T antigen-negative revertant tumor lines and clones obtained by an immunological in vivo selection method had low amounts of 55K protein, similar to the parent cell before simian virus 40 transformation. In all of the T antigen-negative cells, including the highly tumorigenic cells, degradation (turnover?) of the 55K protein was rapid, and a half-life of 15 to 60 min was estimated from pulse-chase experiments. In all of the T antigen-positive cells the 55K protein was stable (half-life greater than 10 h). In primary cells established from the tumors induced by highly tumorigenic cells there was a very low or no detectable amount of the 55K protein. This is in contrast to the primary cells obtained from early murine embryos in which we have reported high amounts of (stable) 55K proteins.

Animals↗
PubMed1982

Generation of cytotoxic lymphocytes by SV40-induced antigens.

In order to study the correlation of in vivo tumor transplantation immunity and in vitro immunologic assays, cell-mediated cytotoxicity against SV40-transformed cells was studied in AL/N strain mice by using 51Cr-release assay. Killing of SV40-transformed AL/N fibroblast cells was observed by spleen cells of AL/N mice immunized with syngeneic SV40-transformed cells. Immunization with the solubilized SV40 tumor-specific transplantation antigen (TSTA) that induced transplantation immunity in vivo did not elicit cytotoxic spleen cells in vitro. However, the spleen cells from mice immunized with solubilized TSTA and then sensitized in vitro with SV40-transformed cells became cytotoxic against SV40-transformed fibroblasts. Similarly, SV40 TSTA (T antigen) purified by immunoprecipitation was able to prime the lymphocytes in AL/N mice: the primed lymphocytes could differentiate into cytotoxic lymphocytes upon in vitro stimulation by SV40-transformed cells. These data indicate that SV40 TSTA (T antigen) plays a role in the induction of cytotoxic lymphocytes.

Animals↗
PubMed1981

Cells selected for high tumorigenicity or transformed by simian virus 40 synthesize heparan sulfate with reduced degree of sulfation.

Cell lines, selected from two independent clones of an established mouse embryo cell line by their ability to grow as solid tumors in immunocompetent syngeneic hosts, were found to have the same alteration in anion exchange properties as was previously reported for simian virus 40 (SV40)-transformed subclones. One tumor cell line (219CT) and one SV40-transformed subclone (215CSC) were selected for further detailed comparison with their common parent clone (210C). Cellulose acetate electrophoresis at pH 1.0 showed that 215CSC heparan sulfate had a slight overall decrease in sulfation compared with heparan sulfate from 210C; however, no gross difference in sulfation could be detected between heparan sulfate from 219CT and 210C. Analysis of the products of deaminative cleavage of heparan sulfate by nitrous acid under conditions where cleavage occurs quantitatively at N-sulfated glucosamine residues showed that, although heparan sulfate from the three cell lines gave similar yields of O-sulfated disaccharides, both 215CSC and 219CT had only about half as many O-sulfate residues in higher molecular weight oligosaccharides compared to heparan sulfate from 210C. Enzymatic degradation of heparan sulfate with a mixture of enzymes from Flavobacterium heparinum showed that this common alteration in heparan sulfate from both 215CSC and 219CT resulted from a 30% decrease in glucosamine residues bearing 6-O-sulfate groups. As this decrease in 6-O-sulfate glucosamine residues occurs in regions of the chain containing relatively few sulfate groups, it is clear that certain sequences of charged groups present in heparan sulfate frm 210C will be found only rarely in heparan sulfate from 215CSC and 219CT. It is suggested that this will result in alterations of the interaction of heparan sulfate with other molecules in the microenvironment at the cell surface which may be important in the control of such phenomena as cell growth and adhesion.

Animals↗
PubMed1981

Surface proteins of simian-virus-40-transformed cells.

Mammalian cells transformed in tissue culture by SV40 were shown to contain, in addition to the SV40-coded 94,000 d large T antigen and the 20,000 d small t antigen, a approximately 56,000 d cellular protein, which specifically precipitates with sera of animals bearing SV40-induced tumor(s) (tumor or T serum). We investigated the presence of these three proteins at the surface of logarithmically growing SV40-transformed cloned mouse cells, after metabolic labelling with [35S]-methionine for 3 h. The 56,000 d protein was found to be susceptible to digestion by trypsin under conditions which did not disrupt the cells, while no small t antigen was found to be digested. Both the 56,000 d cellular protein and the SV40 large T antigen were susceptible to lactoperoxidase-catalyzed iodination from the outside of intact cells. Trypsin treatment removed both the iodinated 56,000 d protein and the iodinated SV40 large T antigen. These experiments indicated that (a certain amount of) the 56,000 d protein and a relatively small amount of the large T antigen (which is present mainly in the nucleus) are present on the cell surface. The results confirm and extend independent experiments using subcellular fractionation techniques (Luborsky and Chandrasekaran, 1980; Soule and Butel, 1979). After heat treatment (at 50 degrees C for 30 min) of the whole-cell extract the 56,000 d cellular protein was precipitated by the tumor serum in the absence of precipitation of SV40 large T antigen. This result showed that the 56,000 d protein is more (thermo)stable (in the whole-cell extract) than the SV40 large T antigen, and also indicated that the tumor serum employed had antibodies against the 56,000 d cellular antigen. The heat-treated whole-cell extract of Sv40-transformed mouse cells was able to immunize and fully protect mice against a lethal tumorigenic dose of SV40-transformed cells. These results suggest the need for further experiments to characterize the chemical and immunologic properties of the 56,000 d protein.

Animals↗
PubMed1981

Quantitation and characterization of a species-specific and embryo stage-dependent 55-kilodalton phosphoprotein also present in cells transformed by simian virus 40.

A 55-kilodalton (kDal) protein was detected recently in primary cultures of day 12 mouse embryos by immunoprecipitation with serum from simian virus 40 (SV40) tumor-bearing hamsters (T serum), Preliminary evidence suggested that this protein was similar to a cellular 55-kDal protein induced after SV40 transformation of mouse cells. We now show that specific approximately 55-kDal [35S]methionine-labeled proteins precipitate from primary cultures of midgestation mouse, rat, and hamster embryos on addition of T serum or monoclonal antiserum prepared against the SV40-induced mouse 55-kDal proteins. The two-dimensional maps of the [35S]methionine-labeled tryptic peptides of the mouse, hamster, and rat embryo proteins are similar to the maps of the corresponding proteins from SV40-transformed cells. Primary cells from midgestation mouse, hamster, or rat embryos contain one-third to one-half as much 55-kDal protein as a SV40-transformed mouse fibroblast cell and nearly the same amount as F9 mouse embryonal carcinoma cells. The amount of 55-kDal protein is greatly reduced on replating the mouse, rat, or hamster embryo primary cells. The amount of this protein in mouse embryos is dependent on the stage of the embryo. The embryo proteins are phosphoproteins.

Animals↗