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Biomedical subjects

S A Harrison

Publications and source records attributed to S A Harrison.

30 records · Page 2Linked to original sources
PubMed1990

Scleral fibroblasts. Human leukocyte antigen expression and complement production.

The authors investigated the ability of recombinant human gamma-interferon (rhIFN-gamma) to influence production of complement and expression of human leukocyte antigens (HLA) by human scleral fibroblasts in culture. Cell cultures were established by explanting sclera from normal human donor eyes. To study complement production, fibroblasts were treated with 500 units/ml rhIFN-gamma in cell culture, and media were tested for complement components by hemolytic assay after 0, 1, 3, 6, 9, and 11 days. To induce Class II HLAs, fibroblasts were exposed to rhIFN-gamma at concentrations ranging from 10-500 units/ml and incubated for 1, 3, and 6 days. The HLAs were detected by immunofluorescence in conjunction with flow cytometry. Class I antigen was detected using a monoclonal antibody directed against beta 2-microglobulin. Class II histocompatibility antigens were identified using monoclonal antibodies specific for HLA-DR, -DP, and -DQ. Although complement component C1 was produced constitutively in cell culture, the addition of rhIFN-gamma resulted in an increase in production. Complement components C2 and C4 were detected only after treatment with rhIFN-gamma. Complement production was completely inhibited by cycloheximide, and C3, C5, C6, and C7 were not present in cell culture media with or without rhIFN-gamma. Class I antigen was present on all cells before induction, and an increase in expression was noted after exposure to rhIFN-gamma. Class II antigens were absent before induction with rhIFN-gamma. After treatment with rhIFN-gamma, scleral fibroblasts expressed HLA-DR, -DP, and -DQ in a dose-dependent, time-related fashion. These findings suggest that rhIFN-gamma has multiple effects on scleral fibroblasts: (1) increased production of C1, (2) production of C2 and C4, (3) up-regulation of Class I antigen expression, and (4) expression of Class II antigens. They also suggest that scleral fibroblasts have the potential to participate in immunologic diseases of the eye.

Adult↗
PubMed1988

Cyclic AMP-dependent and cyclic AMP-independent actions of a novel cardiotonic agent, OPC-8212.

Possible cAMP-dependent and cAMP-independent mechanisms of action for the cardiac effects of OPC-8212, a novel piperazinyl-quinolinone derivative, were evaluated. OPC-8212 was tested for in vitro potency as an inhibitor of soluble bovine cardiac phosphodiesterases using a rapid isolation and assay method involving monoclonal antibodies that distinguish among isozymes. The drug was selective for a low-Km, cGMP-inhibited phosphodiesterase (CGI-PDE) with an IC50 (half-maximal inhibition concentration) of 7.4 mumol/l when measured at a substrate level of 0.35 mumol/l cAMP. Under the conditions used, sulfolane, the solvent for OPC-8212, did not affect CGI-PDE activity. In electrophysiological measurements, OPC-8212 prolonged the action potential duration in canine Purkinje strand preparations up to 148% (APD90) at 10 mumol/l. Concomitantly, OPC-8212 produced a 100% increase in developed force. Both prolongation of the action potential duration and the positive inotropic effect were readily reversed after exposure to tetrodotoxin, 3 mumol/l. Using Na-selective microelectrodes, intracellular Na+ ion activity increased 225% upon exposure to 10 mumol/l OPC-8212. OPC-8212 represents a novel type of positive inotropic agent, possessing both cAMP-dependent (selective PDE isozyme inhibition) and cAMP-independent (activation of intracellular Na+) mechanism of action.

Action Potentials↗
PubMed1987

Age-related defect in signal transduction during lectin activation of murine T lymphocytes.

Interleukin 2 (IL-2) production and recognition are clearly involved in the age-associated proliferative defect of mitogen-stimulated T lymphocytes. The external signal delivered by mitogens is transmitted across the membrane via the release of two messenger molecules, diacylglycerol and inositol 1,4,5-trisphosphate (IP3), involved in the activation of protein kinase C (PK-C) and the elevation of cytosolic free Ca2+. In that Ca2+ mobilization and PK-C activation appear to be crucial events in the production of IL-2 and the expression of IL-2 receptors, a defect in transmembrane signaling would result in decreased synthesis and response to IL-2. We therefore examined PK-C activity and translocation, generation of inositol 1,4,5-trisphosphate, and cytosolic Ca2+ levels as a function of age in murine G0 T lymphocytes before and after exposure to mitogenic doses of concanavalin A (Con A). The basal levels and distribution of PK-C before and after direct activation of the enzyme by 2 or 20 nM phorbol myristate acetate were comparable in both age groups indicating no inherent age-associated functional defect in the enzyme. However, the Con A-induced PK-C translocation was reduced by 50% in cells from 24-mo-old animals. The Con A stimulation of G0 T lymphocytes increased free cytoplasmic Ca2+ concentration ([Ca2+]i) and the production of inositol phosphates to the same level, irrespective of the age of the donor. However, basal levels of both of these second messengers were consistently higher in lymphocytes derived from old mice. As a result, the net increase in inositol phosphates and [Ca2+]i was reduced by approximately the same extent as that observed for the translocation of PK-C. These results clearly point to an age-associated defect in the generation of phosphoinositide-derived second messengers and indicate that an alteration in signal transduction plays a primary role in the age-related impairment of the mitogen-induced, IL-2-mediated proliferative response of T lymphocytes.

Aging↗
PubMed1986

Immunological identification of the major platelet low-Km cAMP phosphodiesterase: probable target for anti-thrombotic agents.

Immunoblot and enzyme-activity analyses, using specific immunological probes, indicated that more than 80% of the total low-Km cAMP phosphodiesterase activity present in bovine and human platelets resided in a single phosphodiesterase isozyme. In the presence of protease inhibitors, the platelet enzyme has an apparent subunit size of 110 kDa and appears immunologically and structurally indistinguishable from a recently purified bovine heart isozyme. When protease inhibitors were absent during homogenization and centrifugation, this platelet phosphodiesterase was susceptible to sequential proteolysis forming 80-kDa and 60-kDa peptides. As a previous report on the purification of the platelet low-Km cAMP phosphodiesterase described a 61-kDa protein, our data would suggest that this was a proteolytic fragment. Moreover, in our study a 40-70% increase in catalytic activity was associated with proteolysis. Further similarities between the platelet and heart phosphodiesterases were demonstrated by pharmacological studies that showed identical inhibitor profiles for both enzymes. Several known phosphodiesterase inhibitor compounds that have been found useful in inhibiting platelet aggregation also inhibited the platelet low-Km cAMP phosphodiesterase with potencies very similar to their antithrombotic effects. Cilostamide, Ro 15-2041, milrinone, papaverine, isobutylmethylxanthine, and theophylline inhibited the 110-kDa platelet enzyme with IC50 values of 0.04, 0.13, 0.46, 1.4, 2.6, and 110 microM, respectively.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Differential inhibition of cardiac cyclic nucleotide phosphodiesterase isozymes by cardiotonic drugs.

Several new cardiotonic drugs are postulated to act as potent inhibitors of cyclic nucleotide phosphodiesterase activity. Unfortunately, the presence of multiple phosphodiesterase isozymes in cardiac muscle makes it difficult to determine whether any of these enzymes are specific targets for the cardiotonic agents. We have developed a method for rapid isolation and assay of bovine cardiac muscle phosphodiesterases using monoclonal antibodies that distinguish between isozymes without inhibiting catalytic activities. By this method, milrinone, amrinone, and MDL 17,043 were tested for potency as inhibitors of soluble bovine heart phosphodiesterases. All three drugs were highly selective for a low-Km, cyclic GMP (cGMP)-inhibited phosphodiesterase. IC50s (half-maximal inhibitory concentrations) for cGMP-inhibited phosphodiesterase were 0.5 microM (milrinone), 30 microM (amrinone), and 2 microM (MDL 17,043) when measured at 0.35 microM cyclic AMP (cAMP). Milrinone and MDL 17,043 had greater than 50-fold lower potencies for the other heart phosphodiesterases (and amrinone 20-fold). These data suggest the cGMP-inhibited phosphodiesterase as a probable site of action for these new cardiotonic drugs. In addition, the method described here should be useful for screening new drugs, and for studying physical and chemical properties of this phosphodiesterase/drug receptor in cardiac and other tissues.

Aminopyridines↗
PubMed1986

Isolation and characterization of bovine cardiac muscle cGMP-inhibited phosphodiesterase: a receptor for new cardiotonic drugs.

We have identified and highly purified a "low Km" cAMP phosphodiesterase from bovine cardiac muscle. This phosphodiesterase was inhibited by low concentrations of cGMP and has, therefore, been temporarily designated as cGMP-inhibited phosphodiesterase. After a 16,000-fold increase in specific activity, the highly purified enzyme had a specific activity of 6 mumol/min-mg and contained three major polypeptides. Initial data indicated that all of these polypeptides were derived from a single common precursor by proteolysis. We used this enzyme preparation to generate polyclonal antisera and monoclonal antibodies directed against the "low Km" phosphodiesterase. Immunoadsorption and immunoblot analysis allowed us to identify and isolate several molecular weight species of phosphodiesterase, including a larger form than previously reported for any purified low Km phosphodiesterase. This large form of the enzyme had a subunit molecular weight of approximately 110,000 and was the only one seen in fresh extracts of cardiac muscle. Full catalytic activity was recovered in the phosphodiesterase-antibody complex and enzyme prepared by immunoprecipitation exhibited Michaelis-Menten kinetics for cAMP hydrolysis and for inhibition by cGMP. The Km for cAMP hydrolysis was 0.15 microM and the Ki for cGMP inhibition of cAMP hydrolysis was 0.06 microM. This immunoprecipitation approach also allowed us to determine that the enzyme was phosphorylated on serine residues by cAMP-dependent protein kinase, and that the low Km, cGMP-inhibited phosphodiesterase was selectively inhibited by several new cardiotonic agents. Milrinone, amrinone, and fenoximone were highly selective inhibitors of this isozyme, and the relative affinities of these inhibitors were consistent with their order of potency as positive inotropic agents. These studies suggest that the cGMP-inhibited phosphodiesterase is a receptor for several new cardiotonic drugs.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

Hallucinations after preoperative baclofen discontinuation in spinal cord injury patients.

Baclofen is a muscle relaxant approved in this country for the treatment of muscle spasms secondary to multiple sclerosis, spinal cord injury, and other spinal diseases. Although baclofen is considered to have fewer side effects than alternative drugs, problems can occur when the dosage is changed abruptly. Two patients are presented who developed hallucinations after discontinuing baclofen as part of routine preoperative procedures. Both patients responded at 23 and 31 hours after reinstituting baclofen. If possible, patients should be withdrawn slowly from baclofen before surgery. Any patient withdrawn abruptly from baclofen should be restarted as soon as possible.

Adult↗
PubMed1984

Immunologic approaches to the study of cyclic nucleotide phosphodiesterases.

The cyclic nucleotide PDE activity in crude tissue extracts is due to the composite activity of several distinct isozymes, each having different kinetic and functional characteristics. Most of these isozymes will hydrolyze the same substrates, that is, cyclic AMP and cyclic GMP. Therefore, it is difficult to conclusively identify and quantitate any given isozyme in crude systems. Since the cyclic nucleotide PDE are present in low concentrations in cells, it is also difficult to isolate these proteins without having specific solid phase affinity probes. This chapter described the use of monoclonal antibodies as probes to specifically identify, measure, characterize, and isolate the PDE isozymes in impure preparations. Monoclonal antibodies have been produced to the cyclic-GMP-stimulated PDE, ROS PDE, and a calcium-calmodulin-dependent PDE of bovine tissues. A polyclonal antiserum has also been produced to the cyclic-GMP-stimulated PDE. Sensitive immunoprecipitation assays for the measurement of PDE activity and cyclic GMP binding have been developed that have allowed the detection of femtomole amounts of each specific PDE isozyme. None of the antibodies appear to recognize antigenic determinants on other isozymes, suggesting that the PDE are antigenically distinct and therefore different proteins. Most of the monoclonal antibodies do not appear to affect kinetic parameters of the enzymes and have been used to specifically identify, measure, and characterize each isozyme in crude systems. A monoclonal antibody to the calcium-calmodulin-dependent PDE requires calcium and calmodulin for high-affinity binding to the enzyme. This apparent conformational requirement has been used to purify the isozyme from both bovine brain and heart. These two tissues contain enzymes with differing subunit MW on SDS-polyacrylamide gel electrophoresis, although no other biochemical differences were observed.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1983

Characterization of Hantaan virions, the prototype virus of hemorrhagic fever with renal syndrome.

Hantaan virus, strain 76-118, was propagated to high titer in a clone of Vero cells, and infectious virions were successfully concentrated and purified. Infectivity and virus antigenic activity were closely associated with a virus particle that exhibited a sedimentation rate indistinguishable from a representative member of the Bunyaviridae. Purified virions sedimented to a density of 1.16-1.17 in sucrose and 1.20-1.21 in cesium chloride. Detergent disruption of virions resulted in a nucleocapsid structure (density, 1.18 in sucrose and 1.25 in cesium chloride) and soluble protein antigens. Three separate nucleocapsids were resolved by rate-zonal centrifugation and contained a single but common polypeptide of 50,000 daltons. Electrophoresis of radiolabeled RNA extracted from purified virions yielded a profile of three RNA species with apparent molecular weights of 2.7, 1.2, and 0.6 X 10(6). These data support earlier electron microscopy reports which suggested that Hantaan virus has characteristics similar to some members of the virus family Bunyaviridae.

Animals↗