Biomedical subjects

H M Prosser

Publications and source records attributed to H M Prosser.

7 recordsLinked to original sources

PubMed2001

Epileptogenesis and enhanced prepulse inhibition in GABA(B1)-deficient mice.

The recent cloning of two GABA(B) receptor subunits, GABA(B1) and GABA(B2), has raised the possibility that differences in GABA(B) receptor subunit composition may give rise to pharmacologically or functionally distinct receptors. If present, such molecular diversity could permit the selective targeting of GABA(B) receptor subtypes specifically involved in pathologies such as drug addiction, spasticity, pain, and epilepsy. To address these issues we have developed a GABA(B1) subunit knockout mouse using gene targeting techniques. In the brains of GABA(B1) null mice, all pre- and postsynaptic GABA(B) receptor function was absent demonstrating that the GABA(B1) subunit is essential for all GABA(B) receptor-mediated mechanisms. Despite this, GABA(B1) null mice appeared normal at birth, although by postnatal week four their growth was retarded and they developed a generalized epilepsy that resulted in premature death. In addition, GABA(B1) heterozygote animals showed enhanced prepulse inhibition responses compared to littermate controls, suggesting that GABA(B1) deficient mice exhibit increased sensorimotor gating mechanisms. These data suggest that GABA(B) receptor antagonists may be of benefit in the treatment of psychiatric and neurological disorders in which attentional processing is impaired.

Action Potentials↗

PubMed1993

Analysis of T cell repertoire and function in mice transgenic for the human V beta 3 TCR.

We have constructed mice containing the human V beta 3 TCR gene from the influenza virus haemagglutinin specific human CD4+ T cell clone HA1.7. Similar cell yields were obtained from transgenic and non-transgenic lymphoid tissue, with normal levels of T cells and with no unusual bias of the CD4 or CD8 subpopulations. Immunostaining and FACS analysis of transgenic thymocytes, spleen, and mesenteric lymph nodes revealed that the majority of T cells expressed the human V beta 3 TCR on the cell surface. Small numbers of cells expressing murine TCR beta chain were also detected. Polymerase chain reaction analysis revealed that an extensive V alpha TCR repertoire was used in the human V beta 3 transgenic mice. Lymphocytes from the spleen and mesenteric lymph nodes of transgenic mice were assessed for functional activity in vitro. Isolated cells were stimulated with mitogen or superantigen, as well as directly through the TCR-CD3 complex, and their ability to proliferate and secrete lymphokines analysed. Cells from transgenic mice responded well after stimulation with phytohaemagglutinin, concanavalin A, anti-CD3 antibody, anti-CD3 antibody with phorbol ester, and Staphylococcus aureus enterotoxin B, and also showed alloreactivity in a mixed lymphocyte reaction. Minimal levels of response were detected after stimulation with murine TCR beta antibody. Together, these data suggest that a human TCR beta chain is able to associate with a murine TCR alpha chain, to form a fully functional surface TCR-CD3 complex.

Animals↗

PubMed1993

Regulation of human T cell receptor beta gene expression by Ets-1.

Expression of the human TcR beta gene is controlled by an enhancer located 6kb 3' to the C beta 2 gene segment. The activity of this enhancer has been shown to be inducible with phorbol esters. Within the enhancer the beta E2 element is responsible for the major part of the inducibility, multimerised beta E2 alone is also highly phorbol ester inducible. The beta E2 element contains a consensus ets-binding site as well as a core motif, and we have shown that the beta E2 ets site binds both Ets-1 and Ets-2 in vitro and that purified core binding factor (CBF) can bind the core site present in beta E2. Mutations which specifically disrupt Ets-1 and Ets-2 binding abolish inducibility as well as reducing activity, whereas mutants which cannot bind CBF have only reduced basal activity. In Jurkat, which has a high level of endogenous Ets-1, multimerized beta E2 was inactive unless treated with PMA. However when transfected into cells with no detectable Ets-1 the beta E2 multimer was highly active in the absence of PMA. Co-transfection of an Ets-1 expression construct with the full enhancer into Jurkat cells led to a repression of enhancer activity, suggesting a repressive role for Ets-1. Co-transfection of Ets-1 was also able to repress strongly the activity of the beta E2 multimer. Repression of activity from both the full enhancer construct and the beta E2 multimer was most dramatic in the presence of PMA, suggesting that Ets-1 could block TcR beta activation. The Ets-1 expression construct used transactivated the HTLV-1 LTR which has also been shown to bind Ets-1. The repression of beta E2 activity by Ets-1 appears therefore to be specific. In conclusion, the combination of ets and core sites in beta E2 constitutes a novel inducible element, which is specifically transrepressed by Ets-1.

Base Sequence↗

PubMed1992

A phorbol ester response element within the human T-cell receptor beta-chain enhancer.

The activity of the T-cell receptor beta-chain gene enhancer is increased by activators of the protein kinase C pathway during T-cell activation. Analysis of mutant enhancer constructs identified two elements, beta E2 and beta E3, conferring phorbol ester inducibility. Multimerized beta E2 acted in isolation as a phorbol ester-responsive element. Both beta E2 and beta E3, which contain a consensus Ets-binding site, were shown to bind directly to the product of the c-ets-1 protooncogene. Both regions also bound a second factor, core-binding factor. Mutation of the beta E2 Ets site abolished the inducibility of the beta E2 multimer. beta E2 and beta E3 Ets site mutations also profoundly affected activity and inducibility of the enhancer. In contrast, enhancer activity but not its inducibility was affected by mutation of the beta E2 core-binding factor site. Cotransfection studies showed that Ets-1 specifically repressed activity of the multimerized beta E2 element and the complete T-cell receptor beta-chain enhancer. These data show that the T-cell receptor beta-chain enhancer responds to protein kinase C-mediated activation signals via a functional domain, composed of two elements, which contains binding sites for Ets transcription factors and which is negatively regulated by Ets-1.

Base Sequence↗

PubMed1992

Generation of monoclonal antibodies against a human T cell receptor beta chain expressed in transgenic mice.

The generation of a panel of monoclonal antibodies specific for different variable (V) regions of human T cell receptors will be of great importance in the study of T cell-mediated diseases. However, relatively few such reagents exist, due in part to the poor immunogenicity of TcRs on the surface of human T cells. We have employed a strategy in which T cells from a transgenic mouse line expressing a human V beta 3 C beta 1 TcR were used to immunise syngeneic conventional mice to generate two monoclonal antibodies specific for human T cell receptors. Binding of antibody JOVI.3, which stained approximately 5% of human peripheral blood CD3 positive T cells, correlated with the expression of the human TcR V beta 3 gene segment. Antibody JOVI.1 recognised a determinant on the majority of TcRs, staining 50-75% of peripheral blood T cells and T cell lines expressing different V beta regions. Some TcRs, however, failed to react with this antibody. Both antibodies immunoprecipitated detergent-solubilised TcR molecules and were capable of inducing proliferation of peripheral blood T cells.

Animals↗

PubMed1991

Identification and functional analysis of the transcriptional enhancer of the human T cell receptor beta gene.

The productive rearrangement and transcription of T cell receptor (TcR) beta genes is confined to T lymphocytes and is subject to both tissue-specific and developmental regulation. In addition to their function in transcriptional control, cis-acting elements are likely to play a role in the regulation of the rearrangement process. In this report we describe the location of a strong and inducible transcriptional enhancer 3' to the human TcR C beta 2 gene segment. The core enhancer, defined by deletion analysis using a transient transfection assay, resided within 362 bp of DNA. This enhancer core was able to activate transcription from a heterologous promoter and functioned well in T and B lymphocytes, but only minimally in HeLa cells. In contrast, a longer fragment containing the enhancer core showed marked T cell specificity. The enhancer was highly inducible by phorbol esters, the molecular basis for the inducibility residing within a 118-bp region of the enhancer core. This inducibility may be important in modulation of TcR beta gene expression during T cell differentiation and/or activation.

Base Sequence↗

PubMed1991

TCF-1, a T cell-specific transcription factor of the HMG box family, interacts with sequence motifs in the TCR beta and TCR delta enhancers.

We have recently identified and cloned TCF-1, a T cell-specific transcription factor with specificity for the AACAAAG motif in the CD3 epsilon enhancer and for the TTCAAAG motif in the TCR alpha enhancer. TCF-1 belongs to the family of transcription-regulating proteins which share a region of homology termed the HMG-box. Here, we show by gel retardation analysis that TCF-1 specifically recognizes the T beta 5 element of the TCR beta enhancer and the T delta 7 element of the TCR delta enhancer. Comparison of the sequences of all elements recognized by TCF-1 defines a consensus motif A/T A/T C A A/G A G. These observations imply that TCF-1 is involved in the control of several T cell-specific genes and might thus play an important role in the establishment and maintenance of the mature T cell phenotype.

Base Sequence↗