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Biomedical subjects

J E Kay

Publications and source records attributed to J E Kay.

At least 19 recordsLinked to original sources
PubMed1999

Binding of a putative and a known chaperone protein revealed by immunogold labeling transmission electron microscopy: A suggested use of chaperones as probes for the distribution of their target proteins.

Parvulins are a distinct family within the peptidyl-prolyl cis-trans isomerase group of proteins that catalyse the cis-trans isomerization of proline-containing peptides. The intracellular distribution of a novel human parvulin homologue (hEPVH) has been investigated in a human kidney cell line (HEK 293) by immunogold labeling transmission electron microscopy (TEM). This showed hEPVH to be distributed throughout HEK 293 cells but in highest concentration within mitochondria. Unexpectedly, preabsorption of anti-hEPVH antiserum with recombinant hEPVH exaggerated the observed immunolabel density in a pattern that mirrored that of the endogenous hEPVH. The hEPVH protein itself was then used to label sections, and the specificity of its binding was confirmed with the use of polyclonal and monoclonal antibodies in conjunction with homologous and irrelevant protein controls. The pattern of hEPVH binding also mirrored that of endogenous hEPVH. A known chaperone protein, Pin1, was also found to bind to cells in a pattern mirroring that of the endogenous protein. This lends considerable weight to our hypothesis that hEPVH is binding to its target protein(s) within the cell, reflecting its postulated chaperone function. Finally, we suggest that chaperone proteins in general might be used as TEM probes for their target (or substrate) proteins. (J Histochem Cytochem 47:1633-1640, 1999)

Absorption↗
PubMed1997

Cyclolinopeptide A (CLA) mediates its immunosuppressive activity through cyclophilin-dependent calcineurin inactivation.

The immunosuppressive cyclic nonapeptide cyclolinopeptide A inhibits calcium-dependent, but not calcium-independent, activation of T lymphocytes comparably to the actions of cyclosporin A and FK506. The concentration required for complete inhibition, however, is 10 times higher than that of cyclosporin A. In addition, we demonstrate that calcineurin, a phosphatase which plays an important role in T lymphocyte signalling, is inhibited in vitro by cyclolinopeptide A by a mechanism dependent on the peptidyl-prolyl cis-trans isomerase (PPIase) cyclophilin A but not FKBP12. Direct binding of cyclolinopeptide A to cyclophilin A was confirmed using tryptophan fluorescence studies and PPIase assays. These results represent a third example of the production of a natural product that neutralises calcineurin by a mechanism dependent on the primary binding to a PPIase.

Amino Acid Sequence↗
PubMed1996

Hypersensitivity to rapamycin of BJAB B lymphoblastoid cells.

Proliferation of the BJAB B-lymphoblastoid cell line was rapidly and almost completely suppressed by picomolar concentrations of the immunosuppressive macrolide rapamycin (50% inhibitory concentration 10-20 pM for cells reactivated from stationary phase). This cell line was considerably more sensitive to rapamycin than any other B-lymphoblastoid cell line tested, the Jurkat T-cell line or the HL60 promyelocytic cell line. BJAB cell proliferation was not affected by the related immunosuppressive macrolides FK506 or L-685,818, which bind to the immunophilin FKBP12 competitively with rapamycin and also inhibit its peptidylprolyl cis-trans isomerase activity. Excess FK506 or L-685,818 added simultaneously competitively antagonized rapamycin's anti-proliferative action. Levels of FKBP12 and uptake of rapamycin from the culture medium were also normal in BJAB cells. The hypersensitivity to rapamycin of BJAB cells thus reflects an unusual dependence on the intracellular signalling system targeted by the rapamycin-FKBP12 complex, and may provide a model system for elucidating the role played by this pathway in lymphocyte activation. The proliferation of BJAB cells reactivated from stationary phase can also be used as the basis for a highly sensitive bioassay for the presence of rapamycin in culture media or other biological fluids.

B-Lymphocytes↗
PubMed1993

Increased phosphorylation of eukaryotic initiation factor 4 alpha during early activation of T lymphocytes correlates with increased initiation factor 4F complex formation.

Mature porcine peripheral blood mononuclear cells (PPBMCs) exist in a resting state both in vivo and when maintained in culture, with low translation rates consistent with their non-proliferative state. When cultured in the presence of the appropriate mitogen, there is a 2-4-fold increase in the rate of protein synthesis per ribosome within 4 h of stimulation [Kay, J. E., Ahern, T. and Atkins, M. (1971) Biochim. Biophys. Acta 247, 322-334]. Studies on extracts prepared from unstimulated cells have suggested lesions in initiation factor activity, primarily affecting the binding of mRNA to ribosomes [Ahern, T., Sampson, J. and Kay, J. E. (1974) Nature 248, 519-521]. In these studies, we have demonstrated that activation of quiescent PPBMCs with the phorbol ester phorbol 12-myristate 13-acetate or concanavalin A leads to a rapid 2-4-fold increase in the rate of protein synthesis within 1 h or 4 h, respectively, which is insensitive to the transcriptional inhibitor, 5,6-dichlorobenzimidazole riboside. Relative to control cells, both phorbol ester and concanavalin A induce a 2-4-fold increase in labelling of the eukaryotic initiation factor eIF-4 alpha with phosphate in vivo, which primarily reflects a small net increase in phosphorylation rather than phosphate turnover on eIF-4 alpha. Similarly, with the human leukaemic T cell line JURKAT, stimulation of the T cell receptor with the monoclonal antibody, OKT-3, or treatment with phorbol ester induces a 2-3-fold increase in eIF-4 alpha phosphorylation within 30 min. Analysis of phosphorylation by two-dimensional gel electrophoresis and measurement of kinase activity towards synthetic peptides, indicate that this increased labelling also reflects increased eIF-4 alpha kinase activity rather than phosphate turnover on eIF-4 alpha. Of central importance is the finding that, concomitant with increased rates of protein synthesis following stimulation of PPBMCs with either phorbol ester or concanavalin A, there is a significant increase in the level of eIF-4 alpha recovered in high-molecular-mass complexes. These data suggest that, in quiescent PPBMCs, eIF-4F may be limiting and that the association of eIF-4 alpha and eIF-4 gamma into high-molecular-mass complexes is regulated by phosphorylation and may play a pivotal role in translational control.

Amino Acid Sequence↗
PubMed1993

Chlamydia trachomatis Mip-like protein has peptidyl-prolyl cis/trans isomerase activity that is inhibited by FK506 and rapamycin and is implicated in initiation of chlamydial infection.

The Mip-like protein of Chlamydia trachomatis has sequence similarity with both the Mip protein of Legionella pneumophila, a virulence factor necessary for optimal intracellular infection, and FK506-binding proteins (FKBPs) of both prokaryotic and eukaryotic origin. FKBPs contain a site for peptidyl-prolyl cis/trans isomerase activity, which is blocked upon binding of the drugs, FK506 or rapamycin. In this paper we report that the recombinant chlamydial Mip-like protein exhibits a peptidyl-prolyl cis/trans isomerase activity which is inhibited by either rapamycin or FK506. To assess the role of the Mip-like protein in chlamydial infection, rapamycin or FK506 (25 microM), were used in either treatment of chlamydial organisms prior to inoculation, or were present at different intervals through the infection. Pretreatment of organisms alone reduced infectivity for McCoy cells by 30%, with inhibition rising to 80% on more prolonged exposure from 0 to 8h and 8 to 16h post-inoculation and declining thereafter. When drug was present during the developmental cycle at intervals from 0 to 24h post-inoculation abnormal chlamydiae were induced in residual inclusions. The results suggest that inhibition of the isomerase of the Mip-like protein interferes with one or more early events in the infective process that determine productive intracellular infection.

Amino Acid Sequence↗
PubMed1991

Post-transcriptional regulation of cytoskeletal actin and T lymphocyte receptor beta chain mRNA by phorbol ester.

The protein kinase C activator phorbol 12-myristate 13-acetate (TPA) augments the level of cytoplasmic mRNA for cytoskeletal actin and the beta chain of the T cell antigen receptor (TCR beta) in peripheral blood lymphocytes. Calcium ionophore A23187 has an antagonistic effect on these increases. The immunosuppressive drug cyclosporin A blocked both positive and negative effects of A23187 without affecting those of TPA. TPA-mediated increases in cytoplasmic RNA for both genes can occur when transcription is blocked with actinomycin D and no increase is observed in the corresponding total cellular RNA. Activation of protein kinase C can thus mediate the accumulation of these mRNAs by a post-transcriptional mechanism.

Actins↗
PubMed1991

Mechanisms of T lymphocyte activation.

The proliferation of T lymphocytes in response to an antigenic stimulus requires the successive ligation of a range of T lymphocyte receptors by MHC-presented antigen, molecules expressed on the surfaces of accessory cells and lymphokines. A range of intracellular signalling systems are involved, with the possibility of alternative activation pathways utilising different intracellular signalling systems, though protein kinase C activation appears to play a key role. Appreciation of the complexity of the response may allow more selective clinical modulation of T lymphocyte-dependent immunological responses.

Antigens, CD↗
PubMed1991

Inhibition of T and B lymphocyte proliferation by rapamycin.

The immunosuppressive macrolide rapamycin shows marked structural similarity to FK-506, and like FK-506 inhibits the activation of cultured T and B lymphocytes at concentrations as low as 10(-10) M. However, rapamycin blocks T-lymphocyte proliferation at a much later stage than FK-506. It also inhibits human, porcine and murine T- and B-lymphocyte activation by all pathways tested, including pathways which are insensitive to FK-506, such as interleukin-2 (IL-2)-mediated proliferation of IL-2-dependent T-cell lines, activation of human peripheral blood T lymphocytes by phorbol ester and anti-CD28 and activation of murine B lymphocytes by bacterial lipopolysaccharide. Thus these two macrolides that bind competitively to the same major intracellular receptor protein inhibit T- and B-lymphocyte activation by quite distinct mechanisms.

Animals↗