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Biomedical subjects

T M Wright

Publications and source records attributed to T M Wright.

At least 19 recordsLinked to original sources
PubMed1992

Activation of phospholipase D by alpha-thrombin or epidermal growth factor contributes to the formation of phosphatidic acid, but not to observed increases in 1,2-diacylglycerol.

The receptor-mediated activation of a phosphatidylcholine-hydrolysing phospholipase D (PLD) has recently been described. We investigated the effect of alpha-thrombin and epidermal growth factor (EGF) on cellular PLD activity in order to determine the role of this enzyme in mitogen-induced increases in phosphatidic acid and sn-1,2-diacylglycerol. In the presence of ethanol, stimulation of [3H]myristic acid-labelled quiescent IIC9 cells with alpha-thrombin or EGF resulted in a rapid increase in radiolabelled phosphatidyl-ethanol which reached a plateau at 1 min, indicating the rapid and transient activation of PLD. We observed a concomitant decrease in the mitogen-stimulated increase of radiolabelled phosphatidic acid. In contrast, ethanol did not significantly effect the elevation of sn-1,2-diacylglycerol levels stimulated by alpha-thrombin or EGF as determined by measurement of sn-1,2-diacylglycerol mass or the appearance of [3H]1,2-diacylglycerol. A novel lipid, detected by two-dimensional t.l.c. analysis, was generated in [3H]myristic acid-labelled cells stimulated with alpha-thrombin, but not EGF, in the presence of ethanol. Treatment in vitro of cellular lipids isolated from [3H]myristic acid-labelled cultures with PLD in the presence of ethanol also resulted in the generation of this novel lipid species, supporting the role of this enzyme in its production. These data indicate that in quiescent IIC9 cells: (a) alpha-thrombin or EGF rapidly and transiently activates a PLD; (b) although this activation is responsible for part of the mitogen-induced increases in phosphatidic acid, it does not contribute to induced increases in sn-1,2-diacylglycerol; and (c) activation of this enzyme appears to be involved in the formation of a novel lipid generated in response to alpha-thrombin, but not EGF, in IIC9 fibroblasts.

Animals↗
PubMed1992

Wear of polyethylene in total joint replacements. Observations from retrieved PCA knee implants.

Observations of wear damage were performed on 12 retrieved porous coated anatomic (PCA) tibial components, all of which were removed because of excessive polyethylene wear. Density measurements of the remaining polyethylene were obtained as a function of depth from the surface of the components. Comparison to previous results from similar studies of total condylar type knee components and total hip acetabular components revealed distinct differences between the types and severity of damage, emphasizing the influence of design factors on the corresponding wear damage. These results confirm previous conclusions that nonconforming articulating surfaces on thin polyethylene components will be at higher risk of damage than more conforming surfaces on thicker components. It also appears that the high cyclic loads to which polyethylene implants are subjected in vivo are most responsible for the degradation in properties of the material near the articulating surface, although the heat pressing of the articulating surface of the PCA components may contribute to the problem.

Aged↗
PubMed1991

Dissociation of protein kinase C activation and sn-1,2-diacylglycerol formation. Comparison of phosphatidylinositol- and phosphatidylcholine-derived diglycerides in alpha-thrombin-stimulated fibroblasts.

Diacylglycerols (DAGs) derived from phosphatidylcholine (PC) hydrolysis have been shown to activate protein kinase C (PKC) in vitro, but it is not known whether this event occurs in response to DAGs generated via agonist-induced PC hydrolysis in intact cells. In this report we have addressed this question directly, using alpha-thrombin stimulation of IIC9 fibroblasts. PKC activation in intact cells was assessed in two ways, by measuring: 1) PKC membrane association as determined by kinase activity and Western blot analysis and 2) the phosphorylation of an endogenous PKC substrate, an 80-kDa protein. Treatment with 500 ng/ml alpha-thrombin has been shown to stimulate both phosphoinositide and PC hydrolysis, whereas treatment with 100 pg/ml alpha-thrombin stimulates only PC breakdown. Using these two conditions, we show that DAG produced from phosphoinositide, but not PC hydrolysis, is associated with the activation of PKC.

Animals↗
PubMed1991

5' regulatory region of a novel cytokine gene mediates selective activation by interferon gamma.

A newly described member of the platelet factor 4 family of cytokine genes, mig, is selectively induced by interferon gamma (IFN-gamma), and not IFN-alpha, in the mouse macrophage-like cell line RAW 264.7. Treatment of RAW 264.7 cells with IFN-gamma activated mig gene transcription as determined by nuclear run-on assays. mig genomic clones were isolated, and constructs containing genomic fragments that included the mig promoter region and the CAT reporter gene were prepared. In RAW 264.7 cells transfected with these constructs, CAT activity was found to be selectively induced by IFN-gamma. A 278-bp genomic fragment containing 235 nucleotides 5' of the transcription start site was sufficient for IFN-gamma-selective induction of CAT activity. Analysis of 5' deletion mutants localized a region essential for activation by IFN-gamma to within 64 nucleotides extending from -235 to -172. A genomic fragment containing this sequence was capable of conferring IFN-gamma inducibility to constructs with a heterologous promoter.

Animals↗
PubMed1991

The biologic effects of implant materials.

The interaction between implant materials and the surrounding biological environment continues to be an area of intense research and clinical interest. This article presents the information presented in a symposium, held during the 36th Annual Meeting of the Orthopaedic Research Society, in which several important issues concerning the biologic effects of implant materials were discussed. These issues included the mechanisms by which implant materials are released to the surrounding tissues and the ways in which these tissues respond to implant materials. The problem of bone loss around cementless implants was discussed as a specific example of a biologic effect resulting in both bone remodelling and endosteal erosion.

Biocompatible Materials↗
PubMed1991

Mechanical characteristics of the stem-cement interface.

The mechanical characteristics of the interface between a metallic stem and the surrounding poly(methyl methacrylate) bone cement were determined from experimental tests and finite element analyses. Push-through-stem tests of straight and tapered titanium alloy stems, surrounded by cement columns, were performed and the resulting load-displacement behavior and strain distribution on the surface of the cement column were measured for loading, unloading, and reloading. Test geometries were modelled using nonlinear, axisymmetric, finite element analyses, which incorporated Coulomb friction elements at the titanium alloy-cement interface. Initial residual stresses, due to curing of the cement column, were modeled by thermal contraction of the cement. Good agreement was obtained between load-displacement curves and surface strains predicted from the nonlinear analysis and those obtained from experiments, when a coefficient of friction of 0.3 was assumed for the stem-cement interface. These results show that, in the absence of chemical adhesion, the load-displacement behavior of a stem-cement composite can be described completely in terms of the friction at the interface and the residual stresses normal to the interface.

Animals↗
PubMed1991

Differential dependence of early and late increases in 1,2-diacylglycerol on the presence of catalytically active alpha-thrombin: evidence for regulation at the level of 1,2-diacylglycerol generation.

alpha-Thrombin stimulates a biphasic increase in cellular 1,2-diacylglycerol mass in quiescent IIC9 fibroblasts. This report describes the use of hirudin, a high-affinity inhibitor of alpha-thrombin that renders it catalytically inactive, to investigate the dependence of elevated 1,2-diacylglycerol levels on the presence of catalytically active alpha-thrombin. When cultures were incubated in the presence of alpha-thrombin, 1,2-diacylglycerol levels remained elevated for greater than or equal to 4 h. Inactivation of alpha-thrombin after 15 s did not alter the kinetics of 1,2-diacylglycerol formation occurring over the next 1 h. However, sustained (1-4 h) increases in this lipid were eliminated. Inactivation of alpha-thrombin after 1 h of stimulation resulted in 1) an immediate and reversible decline in 1,2-diacylglycerol levels, 2) elimination of the sustained phase of 1,2-diacylglycerol production, 3) inhibition of the alpha-thrombin-stimulated generation of choline metabolites, and 4) a blunted mitogenic response to alpha-thrombin. These data indicate that early (0-1 h) and late (1-4 h) increases in 1,2-diacylglycerol are differentially dependent on the presence of catalytically active alpha-thrombin. Furthermore, sustained increases in 1,2-diacylglycerol in response to alpha-thrombin are regulated at least in part at the level of generation (via phosphatidylcholine hydrolysis). Our results also support a role for sustained 1,2-diacylglycerol levels in the mitogenic response.

Animals↗
PubMed1991

Demonstration that phosphorescent 6-bromo-2-naphthyl sulfate can be used to probe heme accessibility in heme proteins.

The phosphorescence properties of 6-bromo-2-naphthyl sulfate (BNS) in aqueous solution were studied. The phosphorescence lifetime is several hundred microseconds and is self-quenched. Although a fluorescent photoproduct is formed from BNS, it does not interfere with the decay properties of triplet-state BNS and its utility as a probe of the accessibility of the heme group in heme proteins. Quenching of BNS phosphorescence does not occur for the non-heme protein lysozyme and apomyoglobin but occurs by a dynamic mechanism with a quenching constant of 1-2 x 10(9) M-1 s-1 for cytochrome c and myoglobin and with a quenching constant of 6.2 x 10(9) M-1 s-1 for protoporphyrin IX. The phosphorescence of an inclusion complex of 1-bromonaphthalene and beta-cyclodextrin is not quenched by heme-containing proteins. The temperature and viscosity dependencies of the rate with which BNS phosphorescence is quenched by microperoxidase-11 are consistent with unit quenching efficiency. These results indicate that quenching of BNS phosphorescence occurs only upon contact with the quencher, and the quenching constant can be used to assess the degree of accessibility of the heme group.

Fluorescent Dyes↗
PubMed1990

Sustained increase in 1,2-diacylglycerol precedes DNA synthesis in epidermal-growth-factor-stimulated fibroblasts. Evidence for stimulated phosphatidylcholine hydrolysis.

A property common to many growth factors is that they must be present for several hours before the commitment to DNA synthesis and cell division occurs. The intracellular signals that are relevant during this period are poorly defined. We examined the formation of 1,2-diacylglycerol in IIC9 fibroblasts after stimulation with epidermal growth factor (EGF), and found that the mass of this lipid remained elevated for at least four hours. The concentration-dependence of EGF-stimulated 1,2-diacylglycerol production and [3H]thymidine incorporation were similar. Studies of phospholipid metabolism strongly suggested that phosphatidylcholine was the source of the 1,2-diacylglycerol generated in response to EGF. EGF did not stimulate the hydrolysis of other phospholipids, including the phosphoinositides, nor did it increase synthesis de novo of 1,2-diacylglycerol. This pattern of sustained 1,2-diacylglycerol formation from phosphatidylcholine may be important in the mitogenic signalling of EGF and potentially other growth factors.

Animals↗
PubMed1990

Kinetic and molecular species analyses of mitogen-induced increases in diglycerides: evidence for stimulated hydrolysis of phosphoinositides and phosphatidylcholine.

A wide variety of agonist-induced events appear to be mediated through an increase in cellular diglyceride levels. With regard to the ability of diglycerides to mediate these events, three important parameters must be considered: a) the kinetics of diglyceride generation, b) the absolute mass levels, and c) their molecular species. While this increase is often due to a stimulated hydrolysis of phosphoinositides, there is increasing evidence that the stimulated hydrolysis of phosphatidylcholine also contributes to agonist-induced increases in diglyceride levels. The kinetics of mass increases in diglyceride levels stimulated in cultured fibroblasts are agonist-dependent. High concentrations of alpha-thrombin stimulate a biphasic increase in diglyceride levels with the first phase peaking at 15 s and the second phase peaking at 5 min. In contrast, stimulation with epidermal growth factor, or platelet-derived growth factor, results in a monophasic increase in cellular diglyceride levels. Furthermore, the molecular species and phospholipid source of the stimulated diglycerides are also agonist-dependent. While the hydrolysis of phosphoinositides is major source of diglycerides initially generated in response to some agonists (15 s with alpha-thrombin at 500 ng/ml), phosphatidylcholine is hydrolyzed as well. Following longer incubations, or at all times following stimulation by epidermal growth factor or platelet-derived growth factor, phosphatidylcholine hydrolysis is the principal source of the stimulated diglycerides.

Cells, Cultured↗
PubMed1989

Lysophosphatidylcholine metabolism to 1,2-diacylglycerol in lymphoblasts: involvement of a phosphatidylcholine-hydrolyzing phospholipase C.

We have previously described the chemoattraction of lymphoblasts by lysophosphatidylcholine [Hoffman, R. D., et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3285-3289]. In studying the mechanism of chemoattraction it was found that lysophosphatidylcholine was metabolized to 1,2-diacylglycerol by the lymphoblastic cell line 6C3HED. One route of metabolism involves the acylation of lysophosphatidylcholine to phosphatidylcholine with subsequent hydrolysis to 1,2-diacylglycerol and phosphocholine by the action of phospholipase C. The increase in cellular 1,2-diacylglycerol was established by metabolic experiments using [14C]glycerol-labeled lysophosphatidylcholine and by mass measurements of 1,2-diacylglycerol. The presence of a phosphatidylcholine-hydrolyzing phospholipase C was confirmed in 6C3HED cell homogenates. In intact cells, lysophosphatidylcholine induced a pattern of protein phosphorylation similar to those of 1,2-dioctanoylglycerol and phorbol 12-myristate 13-acetate, two known activators of protein kinase C. This pathway of lysophosphatidylcholine metabolism, which involves a phosphatidylcholine-hydrolyzing phospholipase C, may be important in the activation of protein kinase C independent of inositol phospholipid hydrolysis.

Animals↗
PubMed1988

Kinetic analysis of 1,2-diacylglycerol mass levels in cultured fibroblasts. Comparison of stimulation by alpha-thrombin and epidermal growth factor.

We have examined the kinetics of 1,2-diacylglycerol production in quiescent IIC9 fibroblasts. alpha-Thrombin and epidermal growth factor (EGF) both stimulate an increase in the mass of cellular 1,2-diacylglycerol. The generation of 1,2-diacylglycerol is biphasic when stimulated by a high concentration of alpha-thrombin (500 ng/ml), with an early phase peaking at 15 s and a late phase peaking at 5 min. Production of 1,2-diacylglycerol is monophasic when stimulated by: (a) a low concentration of alpha-thrombin (100 pg/ml); (b) a high concentration of alpha-thrombin added to cultures which had been pretreated with chymotrypsin; or (c) EGF. In all cases the stimulation of 1,2-diacylglycerol was sustained for at least 30 min. In a previous report (Raben, D. M., Yasuda, K., and Cunningham, D. D. (1987) Biochemistry 26, 2759-2765), it was demonstrated that alpha-thrombin stimulates lipid metabolism in fibroblasts via two coupling mechanisms designated R1 and R2. We now present evidence that the early phase of alpha-thrombin-stimulated 1,2-diacylglycerol production is related to R1, which is characterized by: 1) increased release of arachidonic acid, 2) hydrolysis of polyphosphoinositides, and 3) inhibition by pretreating cultures with chymotrypsin. The late phase is related to R2 which is characterized by 1,2-diacylglycerol production in the absence of stimulated phosphoinositide hydrolysis and arachidonic acid release. In addition, EGF activates an R2-like mechanism in that it does not stimulate the release of arachidonic acid or hydrolysis of polyphosphoinositides but does stimulate a 2-fold increase in 1,2-diacylglycerol mass.

Animals↗
PubMed1988

Leukocyte chemoattraction by 1,2-diacylglycerol.

Previous reports have demonstrated the hydrolysis of inositol phospholipids in polymorphonuclear leukocytes (PMN) in response to chemoattractants and in lymphocytes in response to the mitogen phytohemagglutinin. We investigated the role of 1,2-diacylglycerol, one of the products of receptor-linked phosphatidylinositol hydrolysis, in mediating the migratory response of leukocytes. In an under-agarose migration system, we found 1,2-dioctanoylglycerol to be a strong chemoattractant for human PMN, 6C3HED (a mouse thymic lymphoma), and Jurkat (a human T-cell leukemia). By using a modified Boyden chamber assay, the migratory response of PMN to 1,2-dioctanoylglycerol was determined to be primarily chemotactic. Analysis of structural analogs indicated that both the position and number of acyl chains are important in determining chemoattractant activity. These studies demonstrate that exogenous 1,2-diacylglycerol can stimulate the directed migration of leukocytes. They further suggest that the formation of 1,2-diacylglycerol following receptor-mediated stimulation may represent a common step in the migratory responses of myeloid and lymphoid cells.

Animals↗
PubMed1988

Analysis of surface damage in retrieved carbon fiber-reinforced and plain polyethylene tibial components from posterior stabilized total knee replacements.

The performance of carbon fiber-reinforced ultra-high molecular weight polyethylene was compared with that of plain (non-reinforced) polyethylene on the basis of the damage that was observed on the articulating surfaces of retrieved tibial components of total knee prostheses. Established microscopy techniques for subjectively grading the presence and extent of surface damage and the histological structure of the surrounding tissues were used to evaluate twenty-six carbon fiber-reinforced and twenty plain polyethylene components that had been retrieved after an average of twenty-one months of implantation. All of the tibial components were from the same design of total knee replacement. The two groups of patients from whom the components were retrieved did not differ with regard to weight, the length of time that the component had been implanted, the radiographic position and angular alignment of the component, the original diagnosis, or the reason for removal of the component. The amounts and types of damage that were observed did not differ for the two materials. For both materials, the amount of damage was directly related to the length of time that the component had been implanted. The histological appearance of tissues from the area around the component did not differ for the two materials, except for the presence of fragments of carbon fiber in many of the samples from the areas around carbon fiber-reinforced components.

Carbon↗
PubMed1988

Acetabular cup wear in total hip arthroplasty.

Clinical, pathologic, radiographic, and biomechanical factors of 10 severely worn retrieved Charnley acetabular cups were examined to determine whether these factors influenced cup wear. Change in cup thickness was found to be linear with time. It was found that the actual change in cup thickness was not significantly different from the radiographic change in cup thickness. No correlation was found with the other clinical or radiographic factors.

Acetabulum↗
PubMed1987

Role of complement C9 and calcium in the generation of arachidonic acid and its metabolites from rat polymorphonuclear leukocytes.

We have previously shown that antibody-sensitized mouse peritoneal macrophages release arachidonic acid (C20:4) and its oxygenated derivatives when treated with complement, and that the major part of the release depended on the terminal complement complexes (TCC). To further delineate the process(es) responsible for this release we have extended our studies to rat peritoneal polymorphonuclear leukocytes (PMNs). Experiments were performed with antibody-sensitized rat PMNs labeled with [3H]C20:4 and carrying the TCC, C5b-7, C5b-8 or C5b-9. In contrast to the results of other studies, production of leukotriene B4 (LTB4), the major radiolabeled derivative, was strictly dependent on the presence of C9. However, low levels of C20:4 and prostaglandins (PGs) were produced prior to the C5b-9 stage. Kinetic studies demonstrated that release of LTB4 was rapid; the initial release occurred within 4-6 min and a second rise in release coincided with cell death. Virtually all the LTB4 produced was released as we found no evidence of retention of intracellular LTB4 at either the C5b-8 or C5b-9 stages. In the absence of extracellular calcium, the release of LTB4 was completely abolished and the release of C20:4 and PGs was drastically reduced. [3H]C20:4-labeled PMNs carrying C5b-9 did release substantial amounts of radiolabeled material in the presence of EGTA; however, the majority of this lipid was in the form of intact phospholipid and triglyceride. These results indicate that release of C20:4 and its oxygenated derivatives from rat PMNs is (1) dependent on the participation of C9 in the preexisting C5b-8 complex in the cell membrane, and (2) largely dependent on the presence of calcium.

Animals↗