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Biomedical subjects

R A Franklin

Publications and source records attributed to R A Franklin.

At least 19 recordsLinked to original sources
PubMed1996

Seroprevalence of herpes simplex virus type 2 antibodies in New Zealand sexual health clinic patients.

AIM: To determine the prevalence of HSV-2 antibodies in STD clinic patients in Auckland and Christchurch between August 1991 and August 1992. METHODS: An unlinked anonymous HIV seroprevalence study was conducted in STD clinic patients in Auckland and Christchurch between August 1991 and August 1992. This cross-sectional seroprevalence study using stored sera and data from the HIV seroprevalence study, was conducted to determine the prevalence of antibody to herpes simplex virus type 2 (HSV-2). A random sample of 300 sera were analysed, using a type specific indirect enzyme-linked immunoassay (ELISA) to HSV glycoprotein G2 (gG-2) with Western blot confirmation of equivocal results. RESULTS: The seroprevalence was 25.7%. The seroprevalence increased up to age 50 years, but no significant differences were found for gender, or for European, Maori, or Pacific Island ethnic groups. The seroprevalence was significantly higher in sera obtained from patients attending the South Auckland STD clinic, than in that from Auckland central, west Auckland, or Christchurch clinics. CONCLUSIONS: We can conclude that infection with HSV-2 is common in STD clinic patients in New Zealand, and lies within the seroprevalence range of other similar studies from other countries.

Adolescent↗
PubMed1996

Nerve growth factor signal transduction in human B lymphocytes is mediated by gp140trk.

Nerve growth factor (NGF) plays an important role in the regulation of the immune system. Recent studies from this laboratory demonstrated the presence of functional NGF receptors on human B lymphocytes; in addition, NGF has been shown to enhance B lymphocyte proliferation. NGF caused both concentration- and time-dependent increases in tyrosine phosphorylation of five proteins of 140, 110, 85, 60 and 42 kDa, which were identified as phospholipase C-gamma 1, phosphatidylinositol-3 kinase and mitogen-activated protein kinase. To elucidate the contribution of the Trk family of tyrosine kinases to the phosphorylation events induced by NGF, we identified gp140trk in human B cells and in human B cell lines. Analysis of specific gp140trk immunoprecipitates indicated that addition of NGF to B cells induced a rapid increase in the tyrosine phosphorylation of gp140trk and inhibition of this phosphorylation prevented the tyrosine phosphorylation of other proteins. These data identify the central role of gp40trk in NGF signaling of human B lymphocytes.

B-Lymphocytes↗
PubMed1996

Activation of p42erk2 MAPK and p90rsk by IL-2 occurs independently of protein kinase C.

Stimulation of the human interleukin-2 (IL-2)-dependent cell line, Kit225, with IL-2 resulted in the rapid activation of microtubule-associated protein-2 kinase (MAPK), as demonstrated by the tyrosine phosphorylation of p42erk2, the reduced mobility of MAPK in SDS-PAGE gels, and the increased ability of lysates from these cells to phosphorylate myelin basic protein. Measurements of kinase activity in immunoprecipitates of p9O ribosomal S6 kinase (p90rsk) demonstrated that stimulation of this cell line with IL-2 also resulted in the activation of p90rsk. Further, increased MAPK activity occurred following IL-2 stimulation, but not following phorbol 12-myristate 13-acetate (PMA) stimulation in cells depleted of PKC by prolonged treatment with a high concentration of PMA. These results demonstrate that IL-2 stimulation results in the activation of MAPK and p90rsk and that this activation occurs in a PKC-independent manner.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1995

Nerve growth factor induces activation of MAP-kinase and p90rsk in human B lymphocytes.

A previous report from this laboratory demonstrated that human B lymphocytes expressed nerve growth factor (NGF) receptors on their surface. On the basis of NGF enhancement of B cell proliferation these receptors are presumed to be functional. We have now characterized one of the signaling pathways that NGF may utilize in the functional activation of B lymphocytes. Stimulation of three different human B-lymphoblastoid cell lines with NGF induced the tyrosine phosphorylation and activation of the p42erk-2 isoform of MAP-kinase (MAPK). In addition, NGF induced shifts in the mobility of p90 ribosomal S6 kinase (p90rsk) on immunoblots and increased p90rsk kinase activity in immunoprecipitates. NGF-induced shifts in p90rsk mobility displayed similar dose and time kinetics as NGF-induced MAPK activation. Activation of both MAPK and p90rsk occurred with doses of NGF as low as 400 pg/ml. Preincubation of NGF with anti-NGF Ab inhibited NGF-induced activation of MAPK and p90rsk. These results demonstrate that the interaction of NGF with its receptor on human B cells results in the stimulation of major components of the signaling pathway also initiated by NGF-receptor ligation in cells of neuronal origin.

B-Lymphocytes↗
PubMed1995

Microfilament assembly is required for anti-IgM dependent MAPK and p90rsk activation in human B lymphocytes.

Mitogen-activated protein kinases (MAPK) are important mediators of signal transduction from the cell surface to the nucleus. These MAPK pathways serve different receptor-mediated signaling pathways leading to dual phosphorylation on serine/threonine and tyrosine residues. The mechanisms linking cytoplasmic MAPK activation to later events is still unclear. In this study we demonstrate that the microfilament system has an active role in MAPK activation. Cross-linking of surface IgM or direct activation of PKC with PMA resulted in time and concentration-dependent increases in F-actin content, MAPK (p42erk-2) activation, and phosphorylation of p90rsk. Pretreatment of the B cells with cytochalasin D or botulinum C2 toxin, microfilament-disrupting agents, prevented the increases in F-actin content as well as MAPK and p90rsk activation. These data indicate a role for the microfilament system in the complex and divergent functions of MAPK.

Actin Cytoskeleton↗
PubMed1995

Platelet activating factor activates MAPK and increases in intracellular calcium via independent pathways in B lymphocytes.

Platelet activating factor (PAF)-stimulation of human B-lymphoblastoid cells results in the activation of microtubule associated protein 2-kinase (MAPK) and increases in intracellular calcium. Although increases in intracellular calcium induce MAPK activation in these cells, PAF can stimulate MAPK activation in the absence of detectable changes in intracellular calcium concentrations ([Ca2+]i). Treatment of the LA350 B-lymphoblastoid cell line with either pertussis toxin (PT) or cholera toxin (CT) blocked PAF-induced changes in [Ca2+]i. However, only PT blocked PAF-induced activation of MAPK as determined by shifts in the mobility of MAPK on immunoblots. In support of this finding, only PT but not CT blocked PAF-induced phosphorylation and activation of p90rsk, an event thought to be distal to MAPK activation. These results suggest that the PAF receptor is mediating MAPK activation through pathways separate from those mediating increases in intracellular calcium.

B-Lymphocytes↗
PubMed1994

Activation of MAP2-kinase in B lymphocytes by calcium ionophores.

The role of increases in intracellular calcium levels on tyrosine phosphorylation in human B lymphocytes was studied. Stimulation of normal, resting B lymphocytes or B lymphoblastoid cells with the calcium ionophores ionomycin or A23187 induced the tyrosine phosphorylation and the enzymatic activation of microtubule-associated protein-2 kinase (MAP2-K). Treatment of these cells with PMA induced tyrosine phosphorylation of a protein with the identical mobility, as well as the enzymatic activation of MAP2-K. Stimulation of these cells with ionomycin also resulted in increased ribosomal S6 kinase activity. Activation of MAP2-K in B lymphocytes by calcium ionophore was rapid (detectable within 1 min), transient (returning to background levels by 45 min), and dependent on extracellular calcium. These results demonstrate that transmembrane calcium flux induced by calcium ionophore results in the tyrosine phosphorylation and enzymatic activation of MAP2-K in human B cells.

B-Lymphocytes↗
PubMed1994

Monocytes express a non-neurokinin substance P receptor that is functionally coupled to MAP kinase.

The data presented in this paper demonstrate a new substance P (SP) binding site that is expressed on human monocytes. The apparent dissociation constant (Kd) for binding of 125I-labeled Bolton Hunter-SP (125I-BH-SP) to the receptor on monocyte membranes is 2.24 +/- 0.9 x 10(-7) M and the maximum binding capacity (Bmax) is 4.7 +/- 0.5 pmol/mg membrane protein. It could be excluded that this receptor is one of the known neurokinin (NK) type of receptors on the basis of binding characteristics for NK1, NK2, and NK3 agonists. Moreover, we demonstrate that the binding site is neither the bombesin receptor nor the serpin enzyme complex receptor nor the FMLP receptor. The order of potency for inhibition of 125I-BH-SP binding to the receptor on monocyte membranes is NK1 antagonist [D-Pro2,D-Trp7,9]SP > SP > NK3 agonist [MePhe7]SP > bombesin. Cross-linking studies with disuccinimidylsuberate, followed by SDS-PAGE analysis, revealed that 125I-BH-SP is specifically bound to a membrane protein with an apparent molecular mass of 47 kDa. At a functional level, SP induces the activation of MAP kinase in human monocytes. The ED50 for activation of MAP kinase positively correlated (r = 0.999, p < 0.0005) with the apparent affinity of the ligands applied in the 125I-BH-SP displacement studies. From these results, we conclude that this SP binding site on monocytes is a non-NK receptor protein that is functionally linked to the activation of MAP kinase.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1994

Cross-linking of surface IgM stimulates the Ras/Raf-1/MEK/MAPK cascade in human B lymphocytes.

The mechanism by which mitogen-activated protein kinase (MAPK) is activated in human B cells following cross-linking of the antigen receptor was investigated. Following anti-IgM antibody and phorbol 12-myristate 13-acetate (PMA) stimulation, we demonstrate the activation of Ras, Raf-1, and MAPK/ERK kinase (MEK), all of which are thought to participate in an important signaling cascade that leads to MAPK activation. We detected the kinase activities of Raf-1 and MEK toward purified recombinant substrates for each in this pathway (MEK for Raf-1 and MAPK for MEK). Following stimulation with either anti-IgM or PMA, Ras activation was observed, and the ability of Raf-1 to phosphorylate recombinant kinase-inactive MEK was increased by approximately 10-fold. Similarly, MEK activity toward kinase-active or -inactive recombinant MAPK also increased upon anti-IgM or PMA treatment. Furthermore, the activation of both MAPK and p90rsk was demonstrated under identical conditions in the B cells. We conclude that activation of B lymphocytes through the antigen receptor stimulates distinct members of the Ras/Raf-1/MEK cascade and this mechanism is likely to be responsible for MAPK and p90rsk activation in these cells.

B-Lymphocytes↗
PubMed1994

Autocrine stimulation of B lymphocytes by a platelet-activating factor receptor agonist, 1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine.

Based on previous data which demonstrated the ability of platelet-activating factor (PAF) antagonists to inhibit constitutive immunoglobulin synthesis in B-lymphoblastoid cell lines, we determined the capacity of these cells to synthesize PAF or 1-palmitoyl-2-acetyl-sn-glycero-phosphocholine (PAGPC), an acyl-PAF identified in various cell types. In two B-lymphoblastoid cell lines (LA350 and HSCE-), significant amounts of production of PAGPC were detected, whereas the amount of PAF was below the level of detection in our system. The biologic effects of PAGPC were examined in these cells, both of which have well-characterized PAF receptors. PAGPC induced a concentration-dependent increase in intracellular Ca2+ concentrations and activation of MAP-2 kinase (as detected by immunoblotting and measurements of kinase activity) in these cells. The kinetics and magnitude of these responses were similar to those induced by PAF, and they were inhibited by Web 2086, a PAFR antagonist. Phosphatidylcholine, which differs from PAGPC in that it contains a long fatty acid residue at position 2, did not induce any of these responses. A mutual cross-desensitization of the B lymphoblasts between PAGPC and PAF was observed for Ca2+ mobilization. To induce maximal cell stimulation, approximately 600-fold higher concentrations of PAGPC than of PAF were needed. Because the two B-lymphoblastoid cell lines synthesized significant amounts of PAGPC, this phospholipid may participate in an autocrine stimulation pathway in B cells. Furthermore, the data indicate that PAGPC is an agonist for the PAFR in B lymphoblasts and that the ether linkage in the PAF molecule is not an absolute requirement for activity, although it increases the potency of the ligand.

Amino Acid Sequence↗
PubMed1994

Human CD45RA+ and CD45R0+ T cells exhibit similar CD3/T cell receptor-mediated transmembrane signaling capacities but differ in response to co-stimulatory signals.

CD45RA+ cells have been described to be less responsive to CD3/T cell receptor (TcR)-mediated activation than CD45R0+ T cells. To analyze the underlying mechanism of the differential responses we compared CD3/TcR-triggered tyrosine phosphorylation in the two subsets and studied the role of co-stimulatory signals provided either by accessory cells or pharmacologic activation of protein kinase C by phorbol ester. Stimulation of purified CD45RA+ and CD45R0+ T cells with CD3/TcR antibodies induced similar patterns and intensities of tyrosine phosphorylation in the two subsets, but no proliferation. If accessory cells were used as the source of co-stimulatory signals, strong expression of the 55-kDa chain of the interleukin-2 (IL-2) receptor (CD25), significant IL-2 production and vigorous proliferation were observed in CD45R0+ cells, whereas CD45RA+ cells responded weakly. However, when CD3/TcR-mediated triggering was combined with activation of protein kinase C by phorbol ester, CD45RA+ cells responded strongly. These data indicate that the transmembrane signaling capacity of the T cell receptor expressed by CD45RA+ and CD45R0+ cells is similar and, therefore, is presumably not responsible for the differential reactivities of the two subsets. It is more likely that co-stimulatory signals determine whether CD3/TcR-initiated activation results in strong or weak responses.

Antigen-Presenting Cells↗
PubMed1994

Ligation of the T cell receptor complex results in activation of the Ras/Raf-1/MEK/MAPK cascade in human T lymphocytes.

Stimulation of T cells with antibodies directed towards the T cell receptor complex results in the activation of mitogen-associated protein kinase (MAPK). Two pathways have been described in other cell types that can lead to MAPK activation. One of these pathways involves the activation of Ras, leading to the activation of Raf-1, and the subsequent activation of MEK (MAPK or ERK kinase). The contribution of this pathway in T cells for anti-CD3 or phorbol myristate acetate (PMA)-mediated MAPK activation was examined. We detected the kinase activities of Raf-1 and MEK towards their substrates (MEK for Raf-1 and MAPK for MEK) in this pathway leading to the activation of MAPK. Stimulation of the T cells with either anti-CD3 antibody or PMA resulted in a rapid activation of both Ras and Raf-1. MEK activity towards kinase-active or -inactive recombinant MAPK also increased upon stimulation. In addition, both MAPK and p90rsk were activated in these cells. We suggest that activation of MAPK and the subsequent activation of ribosomal S6 kinase (p90rsk) occurs by the Ras/Raf-1/MEK cascade in T lymphocytes stimulated by ligation of the T cell receptor complex.

CD3 Complex↗
PubMed1993

Platelet-activating factor triggers the phosphorylation and activation of MAP-2 kinase and S6 peptide kinase activity in human B cell lines.

Platelet activating factor (PAF), a phospholipid mediator produced by a variety of cell types, has potent biological activities in many cellular systems. We report that stimulation of B cell lines with PAF induced tyrosine phosphorylation of a protein, approximately 42 kDa in size. The PAF receptor antagonist WEB 2086 as well as the structural analogue PAF antagonist CV3988 inhibited the response, demonstrating that the tyrosine phosphorylation was attributable to PAF binding to a specific receptor. Immunoblot analysis, using an antibody to microtubule-associated protein-2 kinase, demonstrated the presence of a single protein species in unstimulated cells. PAF treatment induced the appearance of an additional species with a slightly reduced mobility in the gel. This PAF-inducible band had the same mobility on SDS-PAGE gels as the protein that was tyrosine-phosphorylated after PAF stimulation. Tyrosine phosphorylation of the 42-kDa protein was rapid, detectable within 30 s, and evident over a wide range of PAF concentrations (10(-7) to 10(-10) M). Treatment of cells with phorbol myristate acetate induced tyrosine phosphorylation of a protein with the same mobility in SDS-PAGE gels as the protein induced after PAF stimulation. Lysates from PAF-stimulated lymphoblastoid cells exhibited more than twice the ability to phosphorylate myelin basic protein than lysates from untreated cells. In addition, the ribosomal S6 peptide kinase activity (S6PK) was increased after PAF stimulation of the B cells. Taken together, the results suggest that PAF stimulation of B cells induces the tyrosine phosphorylation and activation of microtubule-associated protein-2 kinase.

B-Lymphocytes↗
PubMed1993

Failure of rapamycin to block proliferation once resting cells have entered the cell cycle despite inactivation of p70 S6 kinase.

Rapamycin (RAP) has recently been shown to inhibit the phosphorylation and activity of p70 S6 kinase (p70s6k). In interleukin (IL)-2-induced cell activation of the human IL-2-dependent T-cell line, Kit225, RAP inhibited p70s6k phosphorylation and activation, but not the activation of MAP kinase, p90 S6 kinase (p90rsk), early tyrosine kinases, or the transcription of the c-fos and c-myc genes. Cell cycle progression induced by IL-2 was arrested by RAP prior to p110Rb phosphorylation and the major increase in total RNA synthesis, both of which were initiated around 6 h after addition of IL-2 and 9 h before the beginning of DNA synthesis. Interestingly, RAP could not inhibit DNA synthesis if addition of the drug was delayed for 6 h after addition of IL-2, despite the fact that even at this time, RAP rapidly induced the accumulation of the dephosphorylated form of p70s6k and that p70s6k was inactivated within 1 h of RAP addition. Furthermore, when RAP was added to continuously growing Kit225 cells, cell proliferation was maintained for at least two additional cell cycles, in the absence of apparent p70s6k activity. These results indicate that 1) among the earliest detectable signals after IL-2 treatment, RAP selectively inhibits p70s6k activation, 2) RAP inactivates p70s6k regardless of the stage of the cell cycle in which the drug is added, 3) RAP blocks resting T-cells from entering the cell cycle, but does not directly arrest cell cycle progression once cells have entered the cycle, and 4) inactivation of p70s6k does not cause immediate arrest of cell cycle progression once cells have entered the cycle.

Amino Acid Sequence↗
PubMed1993

Rapamycin blocks cell cycle progression of activated T cells prior to events characteristic of the middle to late G1 phase of the cycle.

The effects of rapamycin (RAP) on cell cycle progression of human T cells stimulated with PHA were examined. Cell cycle analysis showed that the RNA content of cells stimulated with PHA in the presence of RAP was similar to that of control T cells stimulated with PHA for 12-24 hr in the absence of the drug. This level was substantially higher than that seen in cells stimulated in the presence of cyclosporin A (CsA), an immunosuppressant known to block cell cycle progression at an early point in the cycle. However, the point in the cell cycle at which RAP acted appeared to be well before the G1/S transition, which occurs about 30-36 hr after stimulation with PHA. In an attempt to further localize the point in the cell cycle where arrest occurred, a set of key regulatory events leading to the G1/S boundary were examined, including p110Rb phosphorylation, which occurred at least 6 hr prior to DNA synthesis, p34cdc2 synthesis, and cyclin A synthesis. In control cultures, p110Rb phosphorylation was detected within 24 hr of PHA stimulation; p34cdc2 and cyclin A synthesis were detected within 30 hr. Addition of RAP to the cultures inhibited each of these events. In contrast, early events, including c-fos, IL-2, and IL-4 mRNAs expression, and IL-2 receptor (p55) expression, were only marginally affected, if at all, in PHA-stimulated T cells. Furthermore, the inhibition of cell proliferation by RAP could not be overcome by addition of exogenous IL-2. These results indicate that RAP blocks cell cycle progression of activated T cells after IL-2/IL-2 receptor interaction but prior to p110Rb phosphorylation and other key regulatory events signaling G1/S transition.

Base Sequence↗
PubMed1993

Macrophages suppress lectin-induced proliferation of lymphocytes from aged rats.

Splenocytes from old rats exhibit a significant reduction in proliferative responses to Con A. This defect can be overcome by passing the cells over nylon wool, which suggests that nylon wool adherent cells contribute to the reduction in proliferation of mixed cell populations from aged rats. To examine the potential role of macrophages in this process, thioglycollate-elicited peritoneal macrophages were added to Con A-activated spleen cells from young and old rats. Addition of as few as 3% macrophages suppressed proliferation of Con-A-activated splenocytes and this inhibition was significantly greater if either the macrophages or spleen cells were derived from aged rats. Although prostaglandin E2 (PG-E2) inhibited the proliferation of splenocytes and nylon-wool enriched splenocytes from 3-month-old animals, its suppressive effects were much greater on Con A-activated lymphocytes derived from 24-month-old rats. Addition of catalase to deplete macrophage-derived H2O2 resulted in greater augmentation of Con-A-induced proliferative responses of splenocytes from old rats than those from young rats. TGF-beta 2, but not TNF-alpha, also significantly suppressed the proliferation of Con A-activated splenocytes to a greater degree in aged than in young rats. Since macrophages are known to release PG-E2, H2O2 and TGF-beta 2 after stimulation with Con A, these data support the idea that T cells within the splenocyte population become increasingly susceptible during aging to inhibitory products released by macrophages.

Aging↗
PubMed1992

Rapamycin inhibits the phosphorylation of p70 S6 kinase in IL-2 and mitogen-activated human T cells.

Phosphorylation of 40S ribosomal protein S6 is regulated in part by the mitogen-activated p70 S6 kinase (p70s6k). Following the addition of IL-2 to the IL-2 dependent human cell line Kit225, or mitogenic activation of resting human T cells, a rapid phosphorylation of p70s6k was observed by immunoblotting. Rapamycin (RAP), a potent suppressor of T-cell proliferative responses, markedly inhibited the phosphorylation of p70s6k induced by IL-2 in Kit225 cells or by the mitogens added to resting T cells. Other immunosuppressants such as cyclosporin A or an FK506 analogue were without effect. Moreover, the effect of RAP was restricted to p70s6k; it did not inhibit the phosphorylation of p90rsk, another kinase which utilizes the S6 protein as a substrate. These data indicate for the first time that RAP may target the pathway leading to p70s6k phosphorylation during human T-cell proliferation.

Amino Acid Sequence↗