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Biomedical subjects

T M Wright

Publications and source records attributed to T M Wright.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1986

The problem of surface damage in polyethylene total knee components.

Observations of surface damage on retrieved total knee polyethylene components have been combined with experimental and analytical studies of the contact problem to identify important clinical and design factors. The amount and severity of damage occurring on the articulating surfaces of knee components increases significantly with patient weight and with the length of time the component is implanted. The design variables that affect the amount of damage are component thickness, the conformity of the articulating surfaces, and the type of polyethylene material used. Surface damage will be more severe in thin (less than 4-6 mm) components and in components with relatively flat tibial articulating surfaces. Surface damage is also expected to be more extreme for carbon-reinforced polyethylene components than for components made from plain polyethylene.

Body Weight↗
PubMed1986

Retrieval analysis of porous-coated components for total knee arthroplasty. A report of two cases.

Retrieval analysis of two total knee replacements incorporating porous-coated components for biologic fixation was performed. The significant finding in a 63-year-old woman was fracture of the porous-coated metal backing of the tibial component, secondary to aseptic loosening. The significant findings at the time of revision surgery in an 82-year-old woman were nonuniform bone ingrowth (primarily around fixation pegs) and fracture of metal beads from the metal backing of the patellar component.

Aged↗
PubMed1986

The effect of conformity, thickness, and material on stresses in ultra-high molecular weight components for total joint replacement.

Debris resulting from damage to the surface of polyethylene components of total joint replacements has previously been shown to contribute to long-term problems such as loosening and infection. Surface damage has been associated with fatigue processes due to stresses arising from contact between the metal and polyethylene components in these prostheses. In the present study, we used elasticity and finite-element solutions to determine these stresses for total hip replacements with head diameters of twenty-two and twenty-eight millimeters and for a condylar total knee replacement. We also examined the effect on these stresses of using carbon-fiber-reinforced polyethylene instead of plain polyethylene. Stresses associated with surface damage in the tibial component of the total knee replacement were much larger than those in the hip replacements. The analysis of contact stress as a function of thickness of the polyethylene insert for tibial components showed that a thickness of more than eight to ten millimeters should be maintained when possible. The contact stress in the tibial components was reduced most when the articulating surfaces were more conforming in the medial-lateral direction. Contact stresses were much less sensitive to changes in geometry in the anterior-posterior direction. For the hip components, the stresses were lower in the acetabular component of the twenty-eight-millimeter hip replacement than in the twenty-two-millimeter replacement. The use of carbon-fiber-reinforced polyethylene resulted in stresses that were higher by as much as 40 per cent. Because the contact area between articulating surfaces moves during flexion, portions of the surface will be subjected to cyclic stresses. The contact area for the knee replacements in flexion was smaller than for the hip replacements, and the range of the maximum principal stress was larger. Consequently, the combination of the higher stress and the moving contact area is more likely to cause surface damage due to fatigue in tibial components than in acetabular components, which is consistent with clinical observations.

Biomechanical Phenomena↗
PubMed1986

The effect of centrifugation on the fracture properties of acrylic bone cements.

In this study, centrifugation did not alter the static or cyclic fracture properties of bone cement. Tests of fracture toughness and fatigue-crack propagation of centrifuged specimens of commercial cements (with and without antibiotic additions) demonstrated no significant difference from control values. Among the cements tested, Palacos (with and without antibiotic) was found to have a significantly higher fracture toughness than either Simplex or Zimmer. We attributed this difference in fracture toughness to the higher molecular weight measured for the Palacos cements. For the tested cements, only Simplex had a significantly greater volume contraction on setting due to centrifugation. The results of our study demonstrate that centrifugation of bone cement does not improve the cement's resistance to fracture in the presence of surface imperfections, such as those found at the bone-cement interface.

Bone Cements↗