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T A Walker

Publications and source records attributed to T A Walker.

At least 37 records · Page 2Linked to original sources
PubMed1988

In vivo evolution of adenovirus 2-transformed cell virulence associated with altered E1A gene function.

Neoplastic cell populations may evolve to a state of higher virulence in immunocompetent hosts. Transforming gene involvement in this process of tumor progression was evaluated using adenovirus type 2 (Ad2)-transformed hamster cells that are highly susceptible to destruction by natural killer cells and activated macrophages, due to Ad E1A gene function, and are nontumorigenic in immunocompetent animals. Cells selected for increased tumorigenicity retained parental cell patterns of viral gene integration and methylation and expressed Ad2 E1A proteins but exhibited altered E1A function evidenced by decreased susceptibility to killer cell-mediated lysis and inability to support E1A(-) mutant virus replication. The data suggest that an interruption in cellular pathways of E1A expression may result in increased transformed cell virulence.

Adenoviridae↗
PubMed1988

Recognition of adenovirus E1A gene products on immortalized cell surfaces by cytotoxic T lymphocytes.

The experiments described in this report were designed to examine whether target cells transfected with the adenovirus E1A gene and exhibiting increased susceptibility to lysis by natural killer cells and activated macrophages (J. L. Cook, T. A. Walker, A. M. Lewis, Jr., H. E. Ruley, F. L. Graham, and S. H. Pilder, Proc. Natl. Acad. Sci. USA 83:6965-6969, 1986) also express E1A proteins on their surfaces. MT1A, 12S, and 13S are strain Fischer baby rat kidney (BRK) cell lines immortalized by transfection with plasmids containing only the E1A gene of nononcogenic adenovirus. All of these cell lines were effective in stimulating the generation of cytotoxic T lymphocytes (CTL) in vitro, provided that the cultures were supplemented with an exogenous source of lymphokine and that the responding lymphocytes were from syngeneic Fischer rats previously immunized with a cell line containing the intact E1A gene. HrA2, a Fischer BRK cell line immortalized by transfection with a plasmid containing only exon 1 of the E1A gene, did not generate, nor was it lysed by, E1A-specific CTL. The cytolytic activity of E1A-specific CTL was blocked by antiserum from Fischer rats immunized with purified E1A proteins synthesized in Escherichia coli, supporting the conclusion that an epitope on E1A proteins encoded by the intact E1A gene constitutes part of the CTL target structure on adenovirus-transformed cells. These data suggest that in addition to their functions within host cells, E1A gene products are important immunogenic determinants on the surfaces of adenovirus-transformed cells.

Adenoviridae↗
PubMed1987

Adenovirus E1A gene induction of susceptibility to lysis by natural killer cells and activated macrophages in infected rodent cells.

Rodent cells immortalized by the E1A gene of nononcogenic adenoviruses are susceptible to lysis by natural killer (NK) cells and activated macrophages. This cytolysis-susceptible phenotype may contribute to the rejection of adenovirus-transformed cells by immunocompetent animals. Such increased cytolytic susceptibility has also been observed with infected rodent cells. This infection model provided a means to study the role of E1A gene products in induction of cytolytic susceptibility without cell selection during transformation. Deletion mutations outside of the E1A gene had no effect on adenovirus type 2 (Ad2) or Ad5 induction of cytolytic susceptibility in infected hamster cells, while E1A-minus mutant viruses could not induce this phenotype. E1A mutant viruses that induced expression of either E1A 12S or 13S mRNA in infected cells were competent to induce cytolytic susceptibility. Furthermore, there was a correlation between the accumulation of E1A gene products in Ad5-infected cells and the level of susceptibility of such target cells to lysis by NK cells. The results of coinfection studies indicated that the E1A gene products of highly oncogenic Ad12 could not complement the lack of induction of cytolytic susceptibility by E1A-minus Ad5 virus in infected cells and also could not block induction of this infected-cell phenotype by Ad5. These data suggest that expression of the E1A gene of nononcogenic adenoviruses may cause the elimination of infected cells by the immunologically nonspecific host inflammatory cell response prior to cellular transformation. The lack of induction of this cytolysis-susceptible phenotype by Ad12 E1A may result in an increased persistence of Ad12-infected cells in vivo and may lead to an increased Ad12-transformed cell burden for the host.

Adenovirus Early Proteins↗
PubMed1986

Expression of the adenovirus E1A oncogene during cell transformation is sufficient to induce susceptibility to lysis by host inflammatory cells.

Mammalian cells transformed by nononcogenic human adenoviruses exhibit high susceptibility to destruction by host mononuclear inflammatory cells. We have analyzed the viral gene regulation of the susceptibility of transformed cells to lysis by natural killer cells and activated macrophages. Comparisons of target cell lines transformed by overlapping segments of the adenovirus E1-transforming gene region revealed that isolated expression of a single oncogene, E1A, was sufficient to cause increased cytolytic susceptibility in the absence of detectable transformed cell-surface expression of viral transplantation antigens and irrespective of histocompatibility antigen identity between killer cells and target cells. These results suggest that oncogene functions that are not linked to the expression of previously recognized cell-surface target structures may actively induce neoplastic cell elimination by components of the host immune surveillance system.

Adenovirus Early Proteins↗
PubMed1986

Tumorigenicity of hamster and mouse cells transformed by adenovirus types 2 and 5 is not influenced by the level of class I major histocompatibility antigens expressed on the cells.

Inbred hamster and mouse cells transformed by the nononcogenic adenovirus (Ad) serotypes, Ad2 and Ad5, are nontumorigenic in syngeneic adult animals, while cells from these species transformed by the highly oncogenic Ad12 are tumorigenic in such rodents. By immunoprecipitation and flow cytometry, cells from four of six Ad2- and Ad5-transformed hamster and mouse lines expressed high levels of cell-surface class I major histocompatibility complex (MHC) antigens, while cells from two of these six lines expressed low levels of cell-surface class I MHC antigens. The levels of class I MHC proteins expressed by cells from these latter two lines were comparable to the levels of cell-surface class I MHC proteins expressed by cells from Ad12-transformed hamster and mouse lines. Moreover, an Ad2-transformed line that had become highly oncogenic after in vivo adaptation showed the same high level of MHC expression as the nononcogenic parent. The amounts of class I mRNA, analyzed by RNA blotting, were, in general, consistent with the levels of class I antigens expressed on the surfaces of these cells. These results indicate that there is no correlation between the tumorigenicity in immunocompetent syngeneic adult rodents of Ad2- and Ad5-transformed hamster and mouse cells and the level of class I MHC antigens expressed on the surfaces of these cells. Thus, the expression of different levels of class I MHC proteins does not seem to explain the differences in the oncogenicity between nononcogenic and highly oncogenic human Ad serotypes.

Adenoviruses, Human↗
PubMed1983

Location of 5.8 S rRNA contact sites in 28 S rRNA and the effect of alpha-sarcin on the association of 5.8 S rRNA with 28 S rRNA.

We have constructed phage M13mp7 clones each containing the coding strand from one of three restriction fragments which collectively span the mouse 28 S rRNA gene with the exception of the 3'-terminal approximately 500 base pairs. When hybridized to 28 S rRNA, only the fragment containing the 5'-terminal 1400 nucleotides of the gene inhibited the annealing of 5.8 S rRNA to the 28 S rRNA. The same results were obtained when either the 5'- or 3'-terminal fragment of 5.8 S rRNA was used in lieu of intact 5.8 S rRNA, each of which had been shown to contain an independent 28 S rRNA contact site. However, alpha-sarcin, a cytotoxin that inhibits protein synthesis by hydrolyzing a phosphodiester bond near the 3' end of 28 S rRNA, produces a 3'-terminal 488-nucleotide fragment which exhibits a marginal capacity to anneal to 5.8 S rRNA. These results indicate that 5.8 S rRNA interacts predominantly with a structural domain near the 5' end of 28 S rRNA. This conclusion is consistent with base-pairing interactions between 5.8 S rRNA and 28 S rRNA based on the proposed secondary structures for Escherichia coli 23 S and yeast 26 S rRNAs. However, alpha-sarcin treatment of ribosomes affects the stability of the binding of 5.8 S rRNA to the 28 S rRNA, even though the toxin hydrolyzes a phosphodiester bond several thousand nucleotides from the proposed contact regions. Finally, mouse 5.8 S rRNA was shown to lack two internal nucleotides reported to be present in rat 5.8 S rRNA.

Animals↗
PubMed1982

Enzymatic and chemical structure mapping of mouse 28S ribosomal ribonucleic acid contacts in 5.8S ribosomal ribonucleic acid.

Secondary structure mapping experiments using S1 nuclease, RNase T1, and diethyl pyrocarbonate as conformational probes have identified those regions in mouse 5.8S rRNA containing major sites of interaction with 28S rRNA. One site encompasses the 3'-terminal 20 nucleotides and corresponds to the region identified previously as a component of an RNase-resistant 5.8S/28S rRNA junction complex. A second site, located at the 5' terminus, has not been defined precisely but is believed to involve approximately 20--30 nucleotides. The existence of these sites of interaction is supported by comparing sequences of eukaryotic 5.8S and 28S rRNA with those of the prokaryotic 23S rRNA. Evidence for the occurrence of at least three helical regions in the central portion of the mouse 5.8S rRNA molecule is also presented.

Animals↗
PubMed1982

Independent binding sites in mouse 5.8S ribosomal ribonucleic acid for 28S ribosomal ribonucleic acid.

Limited digestion of mouse 5.8S ribosomal RNA (rRNA) with RNase T2 generates 5'- and 3'-terminal "half-molecules". These fragments are capable of independently and specifically binding to 28S rRNA, so there exist at least two contacts in the 5.8S rRNA for the 28S rRNA. The dissociation constants for the 5.8S/28S, 5' 5.8S fragment/28S, and 3' 5.8S fragment/28S complexes are 9 x 10(-8) M, 6 x 10(-8) M, and 13 x 10(-8) M, respectively. Thus, each of the fragment binding sites contributes about equally to the overall binding energy of the 5.8S/28S rRNA complex, and the binding sites act independently, rather than cooperatively. The dissociation constants suggest that the 5.8S rRNA termini from short, irregular helices with 28S rRNA. Thermal denaturation data on complexes containing 28S rRNA and each of the half-molecules of 5.8S rRNA indicate that the 5'-terminal binding site(s) exist(s) in a single conformation while the 3'-terminal site exhibits two conformational alternatives. The functional significance of the different conformational states is presently indeterminate, but the possibility they may represent alternative forms of a conformational switch operative during ribosome function is discussed.

Animals↗
PubMed1982

Relatedness of acyl carrier proteins shown by amino acid compositions.

1. Relatedness among the following carrier proteins was assessed on the basis of amino acid compositions: eight acyl carrier proteins (ACP's) associated with fatty acid synthesis, ACP's associated with citrate lyase and citramalate lyase, a biotin carboxyl carrier protein and cytochrome 552. Two independent indices of amino acid composition were used. 2. The fatty acid synthesis-associated ACP's of many organisms and the lyase-associated ACP's show a high degree of relatedness among one another. 3. The ACP's show no relatedness to biotin carboxyl carrier protein or cytochrome 552.

Acyl Carrier Protein↗
PubMed1981

Kinetic studies of the fatty acid synthetase multienzyme complex from Euglena gracilis variety bacillaris.

A fatty acid synthetase multienzyme complex was purified from Euglena gracilis variety bacillaris. The fatty acid synthetase activity is specifically inhibited by antibodies against Escherichia coli acyl-carrier protein. The Euglena enzyme system requires both NADPH and NADH for maximal activity. An analysis was done of the steady-state kinetics of the reaction catalysed by the fatty acid synthetase multienzyme complex. Initial-velocity studies were done in which the concentrations of the following pairs of substrates were varied: malonyl-CoA and acetyl-CoA, NADPH and acetyl-CoA, malonyl-CoA and NADPH. In all three cases patterns of the Ping Pong type were obtained. Product-inhibition studies were done with NADP+ and CoA. NADP+ is a competitive inhibitor with respect to NADPH, and uncompetitive with respect to malonyl-CoA and acetyl-CoA. CoA is uncompetitive with respect to NADPH and competitive with respect to malonyl-CoA and acetyl-CoA. When the concentrations of acetyl-CoA and malonyl-CoA were varied over a wide range, mutual competitive substrate inhibition was observed. When the fatty acid synthetase was incubated with radiolabelled acetyl-CoA or malonyl-CoA, labelled acyl-enzyme was isolated. The results are consistent with the idea that fatty acid synthesis proceeds by a multisite substituted-enzyme mechanism involving Ping Pong reactions at the following enzyme sites: acetyl transacylase, malonyl transacylase, beta-oxo acyl-enzyme synthetase and fatty acyl transacylase.

Acetyl Coenzyme A↗
PubMed1979

Precursor-specific nucleotide sequences can govern RNA folding.

An immediate precursor of 5S ribosomal RNA (rRNA) from Bacillus subtilis has 21 and 42 nucleotide precursor-specific segments associated with its 5' and 3' termini, respectively. On the basis of its nucleotide sequence, predicted secondary structure and location in the rRNA transcriptional unit, the 3' precursor element apparently functions during the termination of transcription. A portion of the 5' precursor element is shown to facilitate the native folding of the mature domain of the precursor. Precursor 5S rRNA molecules which lack the 5' terminal 8-9 nucleotides of the 5' precursor elements were fabricated. These abbreviated constructs assume a non-native conformation, as revealed by their behavior during polyacrylamide gel electrophoresis. The aberrant conformation is evidently forced upon the abbreviated constructs by the residual 5' precursor sequence, since its removal by the maturation endonuclease RNAase M5 precipitates the reordering of the mature domain into its native conformation. Inspection of the nucleotide sequence of the 5S precursor suggested the nature of the conformational aberration, and gel electrophoresis analyses of limited nuclease digests of end-labeled precursors in the native and aberrant conformations are consistent with the derived model. We conclude taht the 5' terminal six nucleotides in the intact 5S precursor assist in the folding of the mature domain by forming a base-paired duplex with neighboring nucleotides, thereby preventing that adjacent sequence from engendering the abnormal conformation. The involvement of precursor-specific sequences and conformational dynamics in RNA function are discussed.

Bacillus subtilis↗