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Biomedical subjects

A D Beyers

Publications and source records attributed to A D Beyers.

18 recordsLinked to original sources
PubMed1994

Characterisation of phosphoprotein complexes associated with rat thymocyte Thy-1.

We have previously demonstrated the non-covalent association of the protein tyrosine kinases p56lck and p60fyn together with a number of substrates for phosphorylation with rat thymocyte Thy-1. Here we present evidence that one of these associated phosphoproteins, p85, is associated by disulphide bridging with another polypeptide, demonstrating that it is an integral membrane protein with an extracellular domain. We also show that phosphatidylinositol 3 kinase activity may be coprecipitated with Thy-1 in Brij 96 thymocyte lysates.

Animals↗
PubMed1993

Physical association of the cytoplasmic domain of CD2 with the tyrosine kinases p56lck and p59fyn.

In T lymphocytes, CD2 forms part of a loosely associated membrane complex which includes the T cell receptor (TcR) for antigen, the CD3 subunits, CD4 or CD8, CD5 and the protein tyrosine kinases p56lck and p59fyn. The interaction of CD2 with tyrosine kinases in this complex provides a possible mechanism for transmembrane signal transduction by CD2. We have investigated whether the interaction of CD2 with the kinases is dependent on other known members of the complex, or whether an independent association can be observed. Using in vitro kinase assays with immune complexes precipitated from cell lysates, we demonstrate that CD2 can associate with p56lck and p59fyn in a rat thymoma line that does not express CD4 or CD8, and in a TcR-negative Jurkat cell line. In TcR-positive Jurkat cells that express rat CD2, interaction of CD2 with p56lck and p59fyn was clearly seen, but it was absent in cells where the cytoplasmic tail of CD2 is truncated, indicating that the interactions are mediated by the cytoplasmic region of CD2. Furthermore, using cells expressing CD2 molecules with partial truncations in the cytoplasmic domain, we show that the association of CD2 with p56lck is progressively lost as the cytoplasmic domain is shortened, and that the capacity of the mutants to associate with p56lck correlates with their capacity to transduce transmembrane signals.

Animals↗
PubMed1993

The association of the protein tyrosine kinases p56lck and p60fyn with the glycosyl phosphatidylinositol-anchored proteins Thy-1 and CD48 in rat thymocytes is dependent on the state of cellular activation.

Cell surface glycoproteins anchored to the plasma membrane via glycosylphosphatidylinositol (GPI) structures, and hence having no cytoplasmic domains, can nevertheless transmit activation signals in lymphocytes. By immunoprecipitation from detergent lysates and in vitro immune complex kinase reactions the GPI-anchored molecules Thy-1 and CD48 are shown to be associated with multimolecular complexes of phosphoproteins including the protein tyrosine kinases p56lck and p60fyn in both rat and mouse thymocytes. Moreover, the kinase activity associated with Thy-1 on rat thymocytes is shown to be dependent on the activation state of the cells, with stimulation by the lectin, concanavalin A, producing a marked decrease in Thy-1-associated kinase activity. In such activated cells, there is an increased association of kinase activity with CD48, but this may be explained in terms of increased surface expression of CD48 and of increased total kinase activity. Additional phosphoproteins of 85, 36 and 32 kDa were consistently seen as components of the complexes.

Animals↗
PubMed1992

Molecular associations between the T-lymphocyte antigen receptor complex and the surface antigens CD2, CD4, or CD8 and CD5.

The T-cell antigen receptor (TCR) complex is the key structure involved in signal transduction in T cells. To analyze associations between the TCR complex and other molecules, immunoprecipitations were carried out, followed by phosphorylation of molecules in vitro by tyrosine kinases associated with the precipitated molecules. This provided a sensitive assay for molecular complexes, and associations were demonstrated between the TCR complex and the surface antigens CD2, CD4, or CD8 and CD5 in normal rat T cells. The complexes were readily seen in immunoprecipitates from Brij 96 but not Nonidet P-40 detergent extracts. The multimolecular complexes are associated with the internal tyrosine kinases p56lck and p59fyn. The presence of p56lck associated with CD4 or CD8 was also examined in early thymocytes, natural killer cells, and macrophages. The kinase was present in all cases except that of normal macrophages.

Animals↗
PubMed1991

Signal transduction by the CD2 antigen in T cells and natural killer cells: requirement for expression of a functional T cell receptor or binding of antibody Fc to the Fc receptor, Fc gamma RIIIA (CD16).

Crosslinking of CD2 antigen on T lymphocytes and natural killer (NK) cells leads to a rise in cytoplasmic-free Ca2+ concentration ([Ca2+]i). However, CD2 seems unlikely to interact directly with the second messenger pathways since signaling via CD2 is poor in T cells that lack the T cell receptor (TCR) and is absent in L cells or insect cells that express CD2. In contrast, NK cells that are also TCR- can be triggered via CD2, but it is unclear as to whether the CD16 Fc receptor (FcR) may facilitate this effect. The CD16 transmembrane molecule is expressed in a complex with the zeta homodimer or the zeta/gamma heterodimer and these dimers are also associated with the TCR complex. Thus, it seemed that zeta chains may provide the link between signaling on NK cells and T cells. This could be tested on TCR- cells since when CD16 is transfected into T cells it is expressed in a complex with TCR zeta homodimer or the zeta/gamma heterodimer. At first, potentiation of CD2 signaling was seen on TCR- Jurkat cells expressing CD16, but this was found to be dependent on trace levels (1%) of IgG in F(ab')2 antibody preparations. With pure F(ab')2, the effect was lost. Signaling on a rat NK cell line was also re-examined with F(ab')2 antibodies that had no IgG contamination, and again no signal transduction via CD2 was seen. We thus conclude that there is no clear evidence for potent signaling via CD2 on cells that lack a TCR complex and that TCR zeta chain expressed at the cell surface is not sufficient to potentiate signaling via CD2 as measured by an increase in [Ca2+]i.

Animals↗
PubMed1991

Autonomous roles for the cytoplasmic domains of the CD2 and CD4 T cell surface antigens.

CD2 and CD4 are single chain transmembrane T cell surface molecules that are involved in signal transduction. Chimaeric constructs from rat CD2 and CD4 antigens were expressed in the Jurkat human T cell line to examine the role of extracellular, transmembrane and cytoplasmic domains in mediating functions controlled by CD2 and CD4. The results show that the large rise in concentration of cytoplasmic free Ca2+ mediated via CD2 crosslinking is controlled by the cytoplasmic domain and does not require the CD2 transmembrane and extracellular domains. Similarly the CD4 cytoplasmic domain alone was shown to encode the specificity for binding to the p56lck tyrosine kinase and to control down-modulation of CD4 after treatment with phorbol ester. Evidence was obtained that down-modulation of CD4 occurs when p56lck dissociates from the cytoplasmic domain due to phosphorylation of Ser 405.

Amino Acid Sequence↗
PubMed1989

Activation of T lymphocytes via monoclonal antibodies against rat cell surface antigens with particular reference to CD2 antigen.

In the rat, monoclonal antibodies (mAbs) against the T-lymphocyte receptor (TCR), CD3 antigen and CD2 antigen are mitogenic for T lymphocytes. Antibodies against CD43 (leukosialin) and CD5 are not, but can enhance the effects of other stimuli. Activation of a T-lymphocyte cell line in terms of triggering an increase in free cytoplasmic Ca2+ [( Ca2+]i) was mediated by mAbs against Class 1 MHC and CD4 in addition to the mitogenic mAbs. In the presence of non-activating levels of anti-TCR mAb a strong synergistic signal was seen with anti-CD4, but incubation with anti-CD4 prior to activation with anti-CD3 inhibited the CD3 signal. Stimulation of DNA synthesis in resting T cells via anti-CD2 mAbs required a combination of two non-competitive antibodies and addition of a third anti-CD2 mAb inhibited the activation. The anti-CD2 mAbs were active on all types of mature T cells from peripheral lymphoid organs and the thymus, but cells in the thymus that lacked a TCR could not be activated via CD2. Rat CD2 was transfected into Jurkat cells and cross-linking with anti-rat CD2 mAbs gave an increase in [Ca2+]i similar to that seen with an anti-human CD3 mAb. All three types of anti-rat CD2 mAbs gave a signal including the mAb that inhibited mitogenesis. Signal transduction via CD2 was not seen in L cells and was only weakly observed in TCR-ve Jurkat cells that had been transfected with rat CD2. The cytoplasmic domain of transfected CD2 was shown to be important for the signal transduction since no increase in [Ca2+]i could be triggered in cells expressing mutant CD2 that lacked the cytoplasmic domain. Mutant forms with increasing lengths of cytoplasmic domain showed functions that progressively approached that of the native function.

Amino Acid Sequence↗
PubMed1988

Apparent involvement of phospholipase A2, but not protein kinase C, in the pro-oxidative interactions of clofazimine with human phagocytes.

The anti-leprosy agent, clofazimine, at concentrations of 0.1-5 micrograms/ml caused a dose-related, stimulus-non-specific (N-formyl-methionyl-leucyl-phenylalanine, calcium ionophore, opsonised zymosan, arachidonic acid and phorbol myristate acetate) potentiation of superoxide generation by human neutrophils in vitro without affecting basal oxidative responses. The pro-oxidative interactions of clofazimine with neutrophils were eliminated by the phospholipase A2 inhibitor 4-p-bromophenacyl bromide but not by the protein kinase C (PKC) inhibitor H-7. In support of these observations clofazimine promoted the release of radiolabeled arachidonic acid from neutrophil membrane phospholipids but did not influence the activity of PKC in cytosolic extracts of neutrophils or of purified PKC from rat brain. Pro-oxidative interactions of clofazimine with human phagocytes may contribute to the intraphagocytic antimycobacterial activity of this agent.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Characterization of protein kinase C and its isoforms in human T lymphocytes.

Protein kinase C (PKC) regulates numerous T cell functions and is present in abundance in normal human T cells and certain T cell lines. Although crude Triton X-100 soluble material obtained from T cell pellets contains minimal PKC activity, DEAE chromatography revealed that 12 to 37% of cellular PKC was membrane associated, probably due to removal of an inhibitor through column chromatography. As in other tissues, PKC from lymphoid tissue was phospholipid and Ca2+ dependent and diolein reduced the Ca2+ requirements for enzyme activity. Hydroxylapatite chromatography revealed that T cells possess two major peaks of PKC activity. Although, the enzyme in these peaks had similar m.w. and identical iso-electric mobility, the proteins differed with respect to their autophosphorylation sites and immunoreactivity toward an isoform specific antibody. Furthermore, differences in their activities in the presence of phospholipid, diolein, and limiting amounts of Ca2+ imply that these isoforms may be differentially activated. We discuss optimal conditions for activation of PKC and its isoforms for study of T lymphocyte cellular function.

Cell Line↗
PubMed1988

A role in transmembrane signaling for the cytoplasmic domain of the CD2 T lymphocyte surface antigen.

The CD2 antigen can mediate mitogenesis of T lymphocytes after binding combinations of monoclonal antibodies. To examine the importance of the cytoplasmic domain in signaling, rat CD2 cDNA has been transfected into the human Jurkat cell line and triggering of an increase in cytoplasmic free Ca2+ concentration [( Ca2+]i) has been assayed. In cells expressing full-length CD2, a clear signal was triggered with anti-CD2 monoclonal antibodies. In contrast, a barely detectable increase in [Ca2+]i occurred with mutant rat CD2 molecules that included only 6 or 40 amino acids of the full-length cytoplasmic domain of 116 residues. It thus appears that the CD2 cytoplasmic domain plays a role in the signaling event.

Amino Acid Sequence↗
PubMed1988

Promotion of DNA strand breaks in cocultured mononuclear leukocytes by protein kinase C-dependent prooxidative interactions of benoxaprofen, human polymorphonuclear leukocytes, and ultraviolet radiation.

At concentrations of 5 micrograms/ml and greater the nonsteroidal antiinflammatory drug benoxaprofen caused dose-related activation of lucigenin-enhanced chemiluminescence in human polymorphonuclear leukocytes (PMNL). Benoxaprofen-mediated activation of lucigenin-enhanced chemiluminescence by PMNL was increased by UV radiation and was particularly sensitive to inhibition by the selective protein kinase C inhibitor H-7. To identify the molecular mechanism of the prooxidative activity of benoxaprofen, the effects of the nonsteroidal antiinflammatory drug on the activity of purified protein kinase C in a cell-free system were investigated. Benoxaprofen caused a dose-related activation of protein kinase C by interaction with the binding site for the physiological activator phosphatidylserine, but could not replace diacylglycerol. When autologous mononuclear leukocytes (MNL) were cocultured with PMNL and benoxaprofen in combination, but not individually, the frequency of DNA strand breaks in MNL was markedly increased. UV radiation significantly potentiated damage to DNA mediated by benoxaprofen and PMNL. Inclusion of superoxide dismutase, H-7, and, to a much lesser extent, catalase during exposure of MNL to benoxaprofen-activated PMNL prevented oxidant damage to DNA. These results clearly demonstrate that potentially carcinogenic prooxidative interactions, which are unlikely to be detected by conventional assays of mutagenicity, may occur between phagocytes, UV radiation, and certain pharmacological agents.

Cells, Cultured↗
PubMed1987

Esophageal cancer: vitamin and lipotrope deficiencies in an at-risk South African population.

The nutritional status of individuals from areas of South Africa that are known for having a high incidence of esophageal cancer was investigated. Our results show that individuals living in high-risk areas differ significantly from those in low-risk areas with respect to vitamins A, E, and B12 in addition to folate. These results suggest that deficiencies in these nutrients may play a significant role in the etiology of esophageal cancer.

Avitaminosis↗
PubMed1986

Immunoreactive digitalis-like substance in pre-eclampsia.

An endogenous digitalis-like substance (DLS) may be involved in the pathogenesis of essential hypertension and pre-eclampsia. The digoxin levels in maternal and cord blood of 504 randomly selected patients were determined. Since none of the patients received digoxin, these levels indicated a cross-reacting substance (immunoreactive DLS). DLS levels were significantly higher in the cord blood of pre-eclamptic patients than in the cord blood of controls. DLS levels in cord blood increased with the severity of pre-eclampsia, and levels were higher in primigravidas than in multigravidas. The structure and biological activity of DLS must be determined before definite conclusions about its role in the pathogenesis of pre-eclampsia can be made.

Digitalis Glycosides↗
PubMed1984

Endogenous immunoreactive digitalis-like substance in neonatal serum and placental extracts.

Therapeutic levels of digoxin in the serum of untreated neonates delivered to mothers who had not received the drug prenatally were detected by radio-immunoassay. Digoxin levels in neonates should be interpreted with care because of the unknown contribution by the endogenous digitalis-like substance (DLS) to the level of the drug. Three commercially available radio-immunoassay kits were compared with regard to their sensitivity and reproducibility in detecting the endogenous DLS. The kit from Clinical Assays (Cambridge, Mass., USA) was selected for further investigations. In a series of 35 paired samples of maternal and cord blood the average DLS values in terms of digoxin were 0,52 +/- 0,07 and 0,81 +/- 0,27 ng/ml respectively. This difference is statistically highly significant. In the case of infants with DLS values of 1-1,5 ng/ml in terms of digoxin, approximately 1 week was required to reach nontherapeutic digoxin levels, i.e. below 0,5 ng/ml. Gel chromatography showed that the DLS in neonatal serum was more closely associated with protein than is authentic digoxin. In placental extracts it followed the elution profile of the protein completely, but it shifted to fractions with a lower molecular weight than haemoglobin after trypsinization. The level of DLS in neonatal serum was also increased by more than half its original value by trypsinization. Proteolysis therefore seems to have a releasing effect on DLS. The molecular size of this substance is probably in the same range as that of polypeptides, since it was not dialysable from trypsinized and untreated samples through a membrane with a 22 000 dalton molecular weight cut-off point.

Adult↗
PubMed1984

The possible role of endogenous digitalis-like substance in the causation of pre-eclampsia.

Digoxin levels have been reported in neonatal blood when neither the mother nor the baby had received digoxin. An endogenous digoxin-like substance (DLS) that may be causally related to hypertension has been described. Using a commercially available radioimmunoassay kit, we investigated the presence of an immunoreactive DLS in 21 pre-eclamptic mothers, 36 mothers with normal blood pressure (the control group) and their infants. We found mean DLS levels to be higher in cord blood from infants born to the pre-eclamptic mothers than in cord blood from those born to mothers in the control group. Levels were also higher in cord blood than in maternal blood in both the pre-eclamptic and the control groups. DLS seems to be associated with pre-eclampsia. Although further work is needed for verification, a hypothesis on the possible role of DLS in the causation of pre-eclampsia is presented.

Adolescent↗
PubMedDate not supplied

Association of esophageal cytological abnormalities with vitamin and lipotrope deficiencies in populations at risk for esophageal cancer.

Esophageal brush cytological screening was undertaken and blood concentrations of micronutrients (vitamin A, E, B12, folic acid and methionine) determined from adults at risk for esophageal carcinoma (EC) in Transkei and Ciskei, Southern Africa. Age-standardised EC rates per 100,000 per annum for both sexes in high, intermediate and low risk districts in Transkei were 74, 51 and 34, respectively. Corresponding rates in high and low EC risk districts in Ciskei were 129 and 9, respectively. Esophageal cytological changes including esophagitis, signs of folic acid deficiency, cellular atypia, dysplasia and cancer, were more prevalent in patients from high than from low EC risk areas. Dietary questionnaires revealed that corn was the main dietary staple in all populations, but that lower intakes of green vegetables, fruits and animal protein occurred in the high risk areas. Significantly lower concentrations of vitamins A, E, B12 and folic acid were present in the blood of patients presenting with cellular dysplasia or malignancy than in cytologically normal patients and in patients from low risk areas. Concentrations of red cell and plasma folate were significantly lower in patients presenting with cytological signs of folic acid deficiency or cellular atypia. The association of vitamin A, vitamin E and folic acid deficiencies with specific esophageal cytological abnormalities in populations at risk for EC is reported for the first time.

Adult↗