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A Perkins

Publications and source records attributed to A Perkins.

67 records · Page 4Linked to original sources
PubMed1989

Structural and functional characterization of human immunodeficiency virus tat protein.

Site-directed mutagenesis was used to identify functional domains present within the human immunodeficiency virus (HIV) tat protein. Transient cotransfection experiments showed that derivatives of tat protein with amino acid substitutions either at the amino-terminal end or at cysteine residue 22, 37, 27, or 25 were no longer able to transactivate HIV long terminal repeat-directed gene expression. Incubation of Tat expressed in Escherichia coli with zinc demonstrated that both authentic Tat and cysteine mutation derivatives could form metal-protein complexes. The tat proteins that contained alterations within the cluster of positively charged amino acid residues retained their ability to transactivate gene expression, albeit at markedly reduced levels. Indirect immunofluorescence showed that the authentic tat protein and the amino-terminal and cysteine substitution mutants all localized in the nucleus, with accumulation being most evident in the nucleolus. In contrast, nuclear accumulation was greatly reduced with the basic-substitution mutations. Consistent with this result, a fusion protein that contained amino acids GRKKR, derived from the basic region, fused to the amino-terminal end of beta-galactosidase also accumulated within the nucleus. These results demonstrate that the 14-kilodalton tat protein contains at least three distinct functional domains affecting localization and transactivation.

Amino Acid Sequence↗
PubMed1989

Activation of NF-kappa B by the HTLV-I trans-activator protein Tax requires an additional factor present in lymphoid cells.

The Tax protein of human T-cell leukemia virus is a potent transcriptional activator of viral and cellular genes, including the genes for interleukin 2 and interleukin 2 receptor alpha chain (IL2R alpha). The NF-kappa B protein has been implicated in Tax-mediated activation of IL2R alpha gene expression. We show that activation of NF-kappa B by Tax is an indirect process that requires prior activation of a preexistent factor that is present in lymphoid cells. Deletion mutagenesis revealed that the carboxyl-terminal acidic region of Tax is required for activation of IL2R alpha-directed gene expression but dispensable for activation of the long terminal repeat (LTR). Our findings suggest that activation of viral and cellular gene expression by Tax is achieved through a cascade of events that involves multiple protein-protein associations and that the specificity of these associations is conferred through different domains of the Tax protein.

B-Lymphocytes↗
PubMed1989

Structural and functional characterization of the human immunodeficiency virus rev protein.

Expression of the human immunodeficiency virus (HIV) rev protein is required for expression of virus structural proteins. Site-directed mutagenesis was used to localize regions important for Rev function. We found that proteins with single amino acid substitution mutations concentrated within the amino termini and midportion of Rev were for the most part nonfunctional. Indirect immunofluorescence revealed that Rev was localized predominantly in the nucleolus. However, a deletion mutant that lacked the basic stretch of amino acids comprising residues Arg-Arg-Arg-Arg-Trp accumulated in the cytoplasm and was no longer functional. Consistent with this observation, a beta-galactosidase fusion protein containing this basic rich peptide at its amino termini was targeted to the nucleus. These observations indicate that the HIV rev protein has a distinct nuclear localization sequence and suggest that Rev-mediated regulation of gene expression involves nuclear events.

Amino Acid Sequence↗
PubMed1987

Biochemical and functional analysis of soluble human interleukin-2 receptor produced in rodent cells. Solid-phase reconstitution of a receptor-ligand binding reaction.

The binding of interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) on human T-cells is a key regulatory event which is absolutely required for T-cell-mediated immune responses. To understand further this binding event, we modified the human IL-2R gene to encode a secreted form of IL-2R. Secreted IL-2R was then expressed at very high levels (approximately 11 micrograms/10(6) cells/48 h) in rodent cells using gene-linked co-amplification. The soluble forms of IL-2R were shown to retain IL-2 affinity shown by cell-surface IL-2R (Kd approximately 18 nM) and were purified to homogeneity using IL-2 affinity chromatography. Purified, recombinant IL-2R and biotinylated IL-2 were used to establish a solid-phase receptor binding assay. Binding of IL-2-biotin was demonstrated to be dose-dependent at concentrations ranging from 10 to 1000 ng/ml, and the specificity of receptor-ligand binding was demonstrated by competition with non-biotinylated IL-2 and with anti-receptor antibodies known to block IL-2 binding in vivo. This immunosorbent receptor assay offers a simple and rapid method for studying the binding of IL-2 to human IL-2R.

Animals↗
PubMed1987

Trans-activation of human immunodeficiency virus gene expression is mediated by nuclear events.

Human immunodeficiency virus encodes a gene product termed tat that is able to activate viral gene expression when present in trans. The mechanism of action of the tat gene product appears to be bimodal, resulting in both an increase in the steady-state level of viral mRNA and the enhanced translation of that RNA. In this report we have examined the mechanism by which tat elevates viral mRNA levels. Data are presented demonstrating that tat acts by increasing the rate of viral transcription, rather than by modulating the stability of viral mRNA. Indirect immunofluorescence was used to show that tat is predominantly localized in the nucleus of expressing cells, a location consistent with a role in the regulation of viral transcription. These results suggest that tat could play a role in human immunodeficiency virus replication essentially similar to that proposed for the trans-acting nuclear gene products described for several other virus species.

Cell Compartmentation↗
PubMed1984

Regulation of anti-hapten antibody responses in vivo: memory, carrier-specific Lyt-2+ T cells can terminate antibody production without altering the peak of response.

By using the classical hapten-carrier antigenic system (dinitrophenyl-keyhole limpet hemocyanin) (DNP-KLH), and positive immunoselective techniques for purification of lymphocyte subsets, we studied the kinetics and role of KLH-primed T cells in the termination of a secondary in vivo plaque-forming cell (PFC) response against DNP in the mouse. Separation of T cells by Lyt-2 phenotype and recombination of T cell subsets demonstrated that a single injection of KLH in complete Freund's adjuvant (CFA) concomitantly generated both helper and suppressor T cells in the spleens of primed mice. When these KLH-primed, purified T cells were transferred into irradiated recipients along with DNP-primed, purified B cells and DNP-KLH, helper activity was demonstrable first, with the early production of a high anti-DNP PFC response. Suppression of the PFC response was observed only after the response had fully developed; the PFC response was then "terminated" within a few days. Helper activity was obtained with the carrier-primed Lyt-2- T cell population, whereas suppression required the addition of Lyt-2+ T cells. Both activities were generated over a wide dose range, were specific for the priming antigen KLH, and could be detected as early as 8 days or as late as 9 mo after a single priming injection of KLH. Lyt-2+ T cells from unprimed donors or from animals primed with a different carrier did not have the capacity to terminate the response when added with the primed Lyt-2- helper T cells. Thus, we have now demonstrated the existence of carrier-specific, long-lived, "memory" suppressor T cells, which are initially generated in parallel with specific helper T cells. Whereas these memory suppressor T cells do not inhibit the induction of a secondary antibody response, they can effectively terminate it once it has developed, and may therefore account for the normal termination of antibody responses.

Animals↗
PubMed1983

Localisation of human osteosarcoma by antitumour monoclonal antibody.

Immunoscintigraphy using radioisotope-labelled monoclonal antibody prepared against osteosarcoma 791T cells was used to detect a primary osteosarcoma. The eight-centimetre tumour was detected using rectilinear scintigraphy of 131I-labelled antibodies. Image enhancement was achieved by subtraction of blood-pool radioactivity labelled with technetium-99m. The ratio of tumour to non-tumour uptake of radioactivity (5:1) suggested that antibody targeting of therapeutic agents is feasible.

Adolescent↗