Dual mechanism of protein-tyrosine phosphorylation in concanavalin A-stimulated platelets

Treatment of human platelets with the lectin Concanavalin A (Con A) resulted in the tyrosine phosphorylation of several proteins with molecular masses 65, 80, 85, 95, 120, 135, and 150 kDa. These proteins were divided in two groups: the first group included the 65-, 85-, 95-, and 120-kDa bands, which were tyrosine phosphorylated also in thrombin-stimulated platelets; the second group (80-, 135-, and 150-kDa bands) included proteins whose tyrosine phosphorylation was exclusively promoted by Con A, but not by thrombin. Members of the second group were rapidly dephosphorylated when the lectin was displaced from the cell surface by methyl alpha-D-mannopyranoside. Pretreatment of intact platelets with the prostacyclin analog iloprost, inhibited Con A-induced tyrosine phosphorylation of the first group of proteins, but had no effect on the tyrosine phosphorylation of the proteins of the second group. Succinyl-Con A, a dimeric derivative of the lectin, which binds to the platelet surface but does not promote clustering of the receptor, did not induce tyrosine phosphorylation of the second group of proteins, although phosphorylation of some members of the first group was observed. Our results demonstrate the presence of two different mechanisms leading to protein-tyrosine phosphorylation in Con A-stimulated platelets, and identify a new signal transduction pathway, promoted by the clustering of membrane glycoproteins, that produces tyrosine phosphorylation of specific substrates. This new pathway may be activated by platelet interaction with multivalent ligands, such as adhesive proteins, during adhesion, spreading, and aggregation.     

Tumoricidal properties of mouse macrophages activated with mediators from rat lymphocytes stimulated with concanavalin A

Macrophage-activating factor (MAF) was obtained from cultures of normal F344 rat lymphocytes incubated with insoluble concanavalin A. The MAF rendered macrophages from normal C57BL/6 mice cytotoxic against the syngeneic B16 melanoma and the allogeneic AC 15091. At the same time, normal syngeneic or allogeneic embryo cells were unharmed, even in the presence of susceptible tumor cells. Optimal MAF levels followed incubation of lymphocytes for 48 hr with Sepharose-bound concanavalin A. A 2-hr incubation of macrophages with MAF was sufficient to initiate activation, providing that 46 hr were allowed to elapse before tumor cells were added. The MAF activity was enhanced after heating the supernatant to 199 degrees. Control experiments largely excluded the possibility that residual unbound concanavalin A caused the observed macrophage-mediated tumoricidal effects.     

Differences in the in vitro response of lymphocytes from leukotic and normal cattle to concanavalin A

The response of cultured peripheral blood lymphocytes from cattle affected with bovine leukosis and from animals free of detectable leukosis to the nonspecific mitogen Concanavalin A ws determined by the measurement of 3H-thymidine incorporation. The stimulatory effect of the mitogen was significantly decreased in leukotic lymphocytes in comparison with normal lymphocytes. This depression was dependent on the severity of the disease. Leukotic lymphocytes showed high basal levels of spontaneous thymidine uptake possibly due to their low maturity. It is suggested that leukotic lymphocytes are not stimulated by Concanavalin A because of their B cell characteristics. A residual cell population of normal reacting lymphocytes--enriched by nylon wool column fractionation--is probably responsible for the remaining but weak in vitro reaction of leukotic lymphocytes.     

Purification of endogenous digitalis-like factors from normal human urine

We detected two candidates for endogenous digitalis-like factors in human urine based on the inhibition of 3H-ouabain binding to human erythrocytes. Two ouabain-displacing compound(ODC)s were consistently eluted off the C18 reverse phase HPLC column with 18% and 31% acetonitrile. The more-polar ODC-1 was ubiquitously found in mammals, markedly increased after acute and chronic salt loading in humans, and was thought to be a natriuretic factor with vasoactive property. ODC-1 mostly resembled ouabain in biological, physicochemical, and chromatographic properties and may correspond to ouabainlike compound purified by other investigators. The less-polar ODC-2 was indistinguishable from digoxin in proton nuclear magnetic resonance(NMR) and fast atom bombardment(FAB) mass spectrum.     

Characterization of B lymphocytes rescued from apoptosis by platelet-activating factor

B lymphocyte development is characterized by deletion via apoptosis of immature cells that are insufficiently stimulated. We have previously demonstrated that crosslinking of the B cell receptor (BCR) using anti-IgM antibodies (alphaIgM) (2 microg/ml) in Ramos B lymphoblastoid cells causes deletion of 30-40% of cells by apoptosis in 24 h. Addition of the potent lipid mediator platelet-activating factor (10(-7) M) to alphaIgM stimulated Ramos cells significantly decreases the number of apoptotic cells as measured by annexin V labeling. We have characterized the phenotype of Ramos cells that have not become apoptotic following BCR stimulation. In these cells, there is a significant decrease in the surface expression of the VLA-4 adhesion molecule (31% of control expression) and surface IgM expression (sIgM) (53% of control expression). Significantly fewer cells co-incubated with platelet-activating factor (PAF) underwent apoptosis, and the remaining cells maintained control levels of VLA-4 (104% of control expression) and sIgM expression (104% of control). All of these protective effects were inhibited by the specific PAF receptor antagonist, WEB 2170. The action of PAF on alphaIgM induced apoptosis was not inhibited by either cycloheximide or cytochalasin B, suggesting that de novo protein synthesis and F-actin polymerization were not implicated in the rescue of Ramos cells by PAF. In contrast, the ability of PAF to maintain sIgM and VLA-4 expression at control levels was inhibited by cycloheximide (7. 5 microg/ml). Cytochalasin B (5 microg/ml) had no effect on sIgM expression but blocked the decrease in VLA-4 expression mediated by alphaIgM. These data indicate that PAF's effect on rescuing and maintaining alphaIgM stimulated Ramos B cells is mediated via at least two pathways. Abrogation of apoptosis does not require de novo protein synthesis, while maintenance of sIgM and VLA-4 expression requires protein synthesis.     

Activation signals regulate heat shock transcription factor 1 in human B lymphocytes

Since the mid-1980s, worldwide reports confirm that scabies in individuals infected with the human immunodeficiency virus (HIV) result in a wide range of-clinical manifestations which differ from those seen in immunocompetent patients. There is also general agreement that HIV-related scabies is more difficult to treat. Oral ivermectin has been shown in several countries to be a safe and effective therapy. In otherwise healthy persons, one dose of 200 microg/kg is usually curative. In HIV-related scabies, one treatment may be curative but repeated doses may be required. Crusted scabies in these individual requires a combination of oral ivermectin, total body treatments with 5% permethrin cream, and keratolytic agents to hasten removal of crusts.     

Release from alginate enhances the biological activity of vascular endothelial growth factor

A primary factor which limits engineering tissues of substantial size is the lack of nutrients readily available to transplanted cells. One potential solution to this nutrient limitation is to encourage the rapid development of a vascular network within three-dimensional tissue engineering matrices. Vascular endothelial growth factor (VEGF) has been identified as a potent stimulator of angiogenesis in vivo. Though effective at stimulating endothelial cells to form blood vessels VEGF degrades rapidly. Spherical alginate beads (3.3+/-0.1 mm diameter) were examined as a means of delivering biologically functional VEGF at a controlled rate over extended times. The alginate beads demonstrated the ability to incorporate VEGF with an efficiency between 30 and 67%, depending on the processing conditions, and release it at a constant rate (5%/day) for up to 14 days in vitro. The released VEGF, when assayed for its ability to stimulate endothelial cells in culture, was found not only to be functional but more potent (three to five times) than the same mass of VEGF added directly to the culture medium. The release kinetics of freeze dried VEGF containing alginate beads were also examined and found to be comparable to non-freeze dried samples.     

Enhancement of phytohemagglutinin and concanavalin A responses of splenic lymphocytes by adherent cell removal

Removal of glass wool-adherent cells resulted in an enhancing effect on DNA synthesis as measured by increased [3H]-thymidine uptake by normal swine spleen cells stimulated with phytohemagglutinin or concanavalin A (Con A). Depletion of nylon-adherent cells caused decreased DNA synthesis in response to Con A, except at high doses of Con A, whereas similar cultures stimulated with phytohemagglutinin showed enhanced DNA synthesis throughout the dose range employed. Titration experiments revealed that enhancing effects of adherent cell removal are most pronounced at higher cell and mitogen concentrations. Glass wool was more efficient than nylon in the enhancement of mitogen-induced DNA synthesis.     

Radiation sensitivity of lymphocytes from human blood and from the thoracic duct

Rats were cerebellectomized 72-96 hr prior to evaluation (1) during maximum electroshock seizures and (2) for their capacity to respond to pentylenetetrazol-induced clonic seizures. Cerebellectomized rats failed to exhibit tonic hindlimb extension, an endpoint characteristic of maximal electroshock seizures. The dose of pentylenetetrazol required to produce clonic seizures or death was not different in cerebellectomized and sham-operated controls. The anticonvulsant efficacy of diazepam, when assessed as a pentylenetetrazol antagonist, was not influenced by removal of the cerebellum. These data indicate that whereas cerebellar influences may suppress seizure activity which is largely focal, seizures of more diffuse origin are not markedly influenced by cerebellar activity. It is, therefore, essential that the role of the cerebellum in suppressing seizures be characterized for each kind of experimentally induced seizure process.     

Release of granule proteins from human eosinophils stimulated with mast-cell mediators

The distribution of three integrin subunits, alpha3, alpha5 and alpha(v), in immature and mature human oocytes has been examined using immunofluorescence and confocal microscopy. The results demonstrate that both alpha5 and alpha(v) are present at the germinal vesicle stage, while alpha3 was only detected in oocytes after germinal vesicle breakdown, in metaphase I and II stage oocytes. The cortical concentration of integrin subunits alpha3 and alpha5 is consistent with their localization in the oolemma. In contrast, the homogeneous distribution of alpha(v) throughout the oocyte suggests the existence of cytoplasmic reservoirs of this protein in the oocyte.     

Release of ciliary neurotrophic factor from cultured astrocytes and its modulation by cytokines

CNTF rescues various types of lesioned neurons in vivo, and it needs to be released from astrocytes into the extracellular space to have the effect. However, direct evidence for CNTF release has not been unequivocally demonstrated. We hypothesized that the rapid sequestration by CNTF receptor present on cultured astrocytes might be the cause of the inability to detect CNTF released into astrocyte-conditioned medium (ACM). Therefore, we measured CNTF immunoreactivity in medium conditioned by astrocytes treated with phosphatidylinositol-specific phospholipase C (PI-PLC) which was used to prevent released CNTF from binding to the CNTF receptor, since PI-PLC cleaves glycosyl-phosphatidylinositol anchor of CNTFR alpha, the unique component involved in CNTF binding. CNTF was not detectable in untreated ACM, but was detectable in PI-PLC-treated ACM. These results together with the evidence that PI-PLC treatment did not have a toxic effect on astrocytes prove the fact that CNTF can be released from astrocytes without cell lysis. Subsequently, the effect of cytokines such as IL-1 beta, TNF-alpha, and EGF on CNTF release was examined. These cytokines increased CNTF protein levels in ACMs without increasing CNTF protein levels in astrocyte-extracts, indicating that they enhanced CNTF release from astrocytes.     

The accessory cell effects of liver macrophages in concanavalin A stimulation of rat spleen lymphocytes

Liver and peritoneal macrophages under similar test conditions behaved in an identical manner with regard to accessory cell effects in the lymphocyte response to concanavalin A. When present in low concentrations (less than or equal to 3.3%) they stimulate lymphocytes, and when present in high concentrations (greater than or equal to 10%) they inhibit lymphocyte proliferation. These two effects are, however, mediated through totally different mechanisms. Stimulation was an early effect, required viable cells, was not affected by enzymatic treatment of macrophages, and was similar to the effect of 2-mercaptoethanol, allogeneic macrophages, and even non-macrophages. Inhibition occurred at a larger stage of lymphocyte transformation, was sensitive to collagenase and pronase treatment of macrophages, was more specifically due to macrophages, was reduced with allogeneic macrophages, and persisted after freeze-thawing of macrophages. Removal of Fc receptors or related segments of the surface of macrophages greatly reduced their inhibitory capacity, whereas removal of foreign surface receptors apparently had no consequence.     

Detection of IgG rheumatoid factor by concanavalin A treatment and complement fixation with IgG rheumatoid factor

Concanavalin A (Con A) froms precipitates with carbohydrate-rich protein such as IgM, IgD, IgE, and IgA. Since IgG contains little carbohydrate and does not react with Con A, the activity of IgG-rheumatoid factor (RF) can be measured in the supernate of the Con A-treated serum. When the latex fixation test (LFT) and the sensitized sheep cell agglutination test (SSCA) were perfromed in the supernate for the detection of IgG-RF, LFT was positive in 32-1% of sera, out of 137 sera originally positive for LFT, and SSCA was positive in 18-5% of sera, out of 119 sera originally positive for SSCA. IgG-RF exhibited lower complement fixing ability than IgM-RF and correlated with agglutination titres of IgG-RF, while the CH50 of the original serum did not correlate with haemolytic activities of either IgM-RF or IgG-RF.     

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.     

Release of soluble tumor necrosis factor receptors from polymorphonuclear cells of breast cancer patients

The release of soluble tumor necrosis factor receptors (sTNF-Rs): soluble tumor necrosis factor receptor I (sTNF-RI) and soluble tumor necrosis factor receptor II (sTNF-RII) by polymorphonuclear cells (PMNs) derived from patients with breast cancer were measured. Serum levels of sTNF-RII and sTNF-RII in patients group were also determined. Significantly higher values of sTNF-RI and sTNF-RII released by PMNs of patients were observed than those in the healthy subjects. There was no significant differences in the serum levels of sTNF-RI and sTNF-RII between breast cancer patients and healthy control. Presented study demostrate that the tumor bearing state, involving breast cancer, may induce an altered soluble cytokine receptors release from the peripheral blood PMNs characterized by increased concentrations of sTNF-RI and sTNF-RII in the culture supernatants of these cells in vitro.     

A new endogenous natriuretic factor: LLU-alpha

For over three decades, renal physiology has sought a putative natriuretic hormone (third factor) that might control the body's pool of extracellular fluid, an important determinant in hypertension, congestive heart failure, and cirrhosis. In our search for this hormone, we have isolated several pure natriuretic factors from human uremic urine that would appear, alone or in combination, to mark a cluster of phenomena previously presumed to be that of a single "natriuretic hormone." This paper reports the purification, chemical structure, and total synthesis of the first of these compounds, LLU-alpha, which proved to be 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman, presumably a metabolite of gamma-tocopherol. Both natural LLU-alpha and synthetic material are identical (except for optical activity) with respect to structure and biological activity. It appears that the natriuretic activity of LLU-alpha is mediated by inhibition of the 70 pS K+ channel in the apical membrane of the thick ascending limb of the kidney.