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Biomedical subjects

D Clarke

Publications and source records attributed to D Clarke.

At least 181 records · Page 10Linked to original sources
PubMed1989

Subcellular localization of sterol carrier protein-2 in rat hepatocytes: its primary localization to peroxisomes.

Sterol carrier protein-2 (SCP-2) is a nonenzymatic protein of 13.5 kD which has been shown in in vitro experiments to be required for several stages in cholesterol utilization and biosynthesis. The subcellular localization of SCP-2 has not been definitively established. Using affinity-purified rabbit polyclonal antibodies against electrophoretically pure SCP-2 from rat liver, we demonstrate by immunoelectron microscopic labeling of ultrathin frozen sections of rat liver that the largest concentration of SCP-2 is inside peroxisomes. In addition the immunolabeling indicates that there are significant concentrations of SCP-2 inside mitochondria, and associated with the endoplasmic reticulum and the cytosol, but not inside the Golgi apparatus, lysosomes, or the nucleus. These results were confirmed by immunoblotting experiments with proteins from purified subcellular fractions of the rat liver cells carried out with the anti-SCP-2 antibodies. The large concentration of SCP-2 inside peroxisomes strongly supports the proposal that peroxisomes are critical sites of cholesterol utilization and biosynthesis. The presence of SCP-2 inside peroxisomes and mitochondria raises questions about the mechanisms involved in the differential targeting of SCP-2 to these organelles.

Animals↗
PubMed1989

Virus mutation frequencies can be greatly underestimated by monoclonal antibody neutralization of virions.

Monoclonal antibody-resistant mutants have been widely used to estimate virus mutation frequencies. We demonstrate that standard virion neutralization inevitably underestimates monoclonal antibody-resistant mutant genome frequencies of vesicular stomatitis virus, due to phenotypic masking-mixing when wild-type (wt) virions are present in thousandsfold greater numbers. We show that incorporation of antibody into the plaque overlay medium (after virus penetration at 37 degrees C) can provide accurate estimates of genome frequencies of neutral monoclonal antibody-resistant mutant viruses in wt clones. By using this method, we have observed two adjacent G----A base transition frequencies in the I3 epitope to be of the order of 10(-4) in a wt glycine codon. This appears to be slightly lower than the frequencies observed at other sites for total (viable and nonviable) virus genomes when using a direct sequence approach.

Animals↗
PubMed1989

The use of adjunctive hyperbaric oxygen in treatment of orthopedic infections and problem wounds: an overview and case reports.

We summarize indications, contraindications, and therapeutic guidelines for the use of adjunctive hyperbaric oxygen therapy (HBO) in problem wounds and selected orthopaedic infections. Three typical cases that all were successfully treated with HBO are presented: a chronic osteomyelitis which was a sequela to an open tibia fracture, a second- and third-degree burn injury of the entire lower extremity, and a case of chronic osteomyelitis in an insulin-dependent diabetic.

Adult↗
PubMed1989

Sodium valproate interactions with the HPNS: EEG and behavioral observations.

A new baboon model was used to investigate the therapuetic effect of sodium valproate on the high pressure neurologic syndrome (HPNS). A hyperbaric chamber was used to achieve environmental pressures of 61 ATA, over a 5-h period. Eight animals underwent two compressions, a control and a valproate-treated compression (half the animals had valproate on the first compression). Mild signs of HPNS (e.g., paw and limb tremor) were first observed at approximately 20 ATA. More severe signs (e.g., whole body tremor, myoclonus, and vomiting) were observed above 40 ATA. Sodium valproate was administered during the compression phase and for 2 wk previously. It was effective at the higher pressures above 41 ATA in reducing the severity of the signs of HPNS. The major effect of pressure on the EEG was to increase alpha and theta wave amplitude in a linear manner. Alpha wave amplitude was reduced by sodium valproate.

Animals↗
PubMed1989

Growth of xenografted human bone marrow: comparison with hemopoietic reconstitution in patients after allogeneic bone marrow transplant and response to granulocyte macrophage colony stimulating factor.

Normal human bone marrow was grown as xenografts in mice immune-suppressed by thymectomy and total body irradiation. Mononuclear cell fractions isolated from marrow harvests from 17 donors all gave rise to subcutaneous nodules which grew to a variable maximum size and then regressed. Human granulocyte/macrophage progenitors (CFU-GM) were recovered from xenografts up to 20 days postimplantation. Xenograft growth, measured by maximum nodule volume, area under the growth curve, and rate of regression, did not correlate with the speed of neutrophil or platelet recovery in bone marrow transplant patients infused with the same marrow. Assay of numbers of stromal fibroblastoid colony forming cells (CFU-F) in donor marrow was also not predictive of subsequent hemopoietic recovery in recipients. Treatment of host animals with daily intraperitoneal injections of 100 micrograms/kg human recombinant granulocyte/macrophage colony stimulating factor produced a more rapid growth of subcutaneous nodules. This technique may therefore be of use in determining the in vivo efficacy of human hemopoietic regulatory factors.

Animals↗
PubMed1989

An in vitro model for the production of committed haemopoietic progenitor cells stimulated by exposure to single and combined recombinant growth factors.

A plastic-adherent mononuclear cell population in human bone marrow produces non-plastic-adherent nucleated cells in liquid cultures. These cells can be harvested from the culture medium and a proportion of them can be identified as granulocyte-macrophage colony-forming cells (GM-CFC) by plating them in semi-solid cultures with granulocyte-macrophage colony-stimulating factor (GM-CSF). The generation of GM-CFC from their plastic-adherent precursors can be amplified considerably by adding 5637 conditioned medium (CM) to the liquid phase of the adherent cell cultures. This effect of 5637 CM cannot be reproduced by recombinant (r) GM-CSF or interleukins (ILs) 1, 3 or 6 if they are added singly to the culture medium. In contrast, the combination of GM-CSF + IL-1 equalled or surpassed the activity of 5637 CM. The combinations of rGM-CSF + rIL-3 and rGM-CSF + IL-6 also mimicked the activity of 5637 CM but less effectively than GM-CSF + IL-1.

Bone Marrow↗
PubMed1988

Prevention by a platelet-derived factor (platelet factor 4) of induction of low dose tolerance to pneumococcal polysaccharides.

It was previously shown that human or mouse serum, and platelet factor 4 (PF4) prepared from human platelet releasate, counteracts nonspecific immunosuppression induced in mice by injection of concanavalin A or syngeneic gamma-irradiated lymphoma cells. The present studies show that PF4 prepared from normal mouse or human serum by absorption to heparin-agarose and elution between 0.5 and 1.5 M NaCl is also active in this respect. The ability of PF4 to counteract antigen-specific suppression of the antibody response to pneumococcal polysaccharide (pps) was now studied. PF4 derived from human or mouse serum as well as recombinant PF4 interferes with induction of antigen-specific low dose tolerance when they are injected at the same time as a low dose (0.2 microgram) of type 14 pps 3 days before an optimal immunizing dose (25 micrograms). Furthermore, injection of platelet releasate at the time of an optimal primary immunizing dose of pps type 14 enhances the secondary response to killed bacteria injected 2 weeks later, but not the primary response itself. Both effects are interpreted as due to interference with antigen-specific suppressor cell induction during primary immunization. Injection of PF4 is much less effective in reversing low dose tolerance to an optimal immunizing dose (0.1 microgram) of type 3 pps induced by injection of 0.005 microgram of this antigen. Differences in the mechanism of tolerance induction for the two pps types that might be responsible for this are discussed.

Animals↗
PubMed1988

Psychological predictors of future suicidal behaviour in hospitalized suicide attempters.

Rates of attempted suicide have increased in most Western countries over the past 20 years. This level of deliberate self-harm is made more significant by the fact that a large proportion of attempted suicides will reattempt in the six months following their attempt and as a group they have a high risk of eventually killing themselves. While much of the research on prediction of suicidal behaviour has concentrated on demographic factors, little is known about the psychological variables that precede the suicidal act. This study examined 67 New Zealand suicide attempters on a range of psychological variables including self-esteem, hopelessness, depression, cognitive rigidity and social skills. Subjects were followed up after six months to determine their level of suicidal behaviour and ideation. Results suggest hopelessness and self-esteem are important indicators of future suicidal behaviour, with differential effects over short- and long-term periods. The implications for psychological assessment and suicidal risk assignment are discussed.

Adult↗
PubMed1988

Heparan sulfate is necessary for adhesive interactions between human early hemopoietic progenitor cells and the extracellular matrix of the marrow microenvironment.

Human hemopoietic blast colony-forming cells (BI-CFCs) recognize and adhere to the extracellular matrix (ECM) produced by marrow-derived stromal cells in vitro. We have investigated the requirements for this interaction by testing the capacity of BI-CFCs to adhere to ECM components under a variety of conditions. Binding was prevented completely by prior treatment of stromal ECM with nitrous acid, in large part by treatment with heparitinase or hyaluronidase, and slightly by treatment with chondroitinases. Whereas heparan sulfate isolated from marrow stromal cultures effectively blocked binding, heparan sulfate from bovine kidney did not. Chondroitin sulfate and hyaluronic acid did not have any effect in this test. In contrast, collagen was not sufficient for the interaction because dishes coated with collagen type I or IV did not act as adhesive surfaces for BI-CFCs. Ligands for integrin receptors (e.g., fibronectin) did not participate in BI-CFC binding because the synthetic pentapeptide glycine-arginine-glycine-asparagine-serine did not compete with stroma in binding BI-CFCs. These findings indicate that heparan sulfate in the bone marrow microenvironment is necessary for BI-CFC binding to ECM and may contribute to localizing hemopoietic stem cells in hemopoietic tissue.

Antigens, Surface↗
PubMed1987

Phorbol esters stimulate 2-deoxyglucose uptake in glia, but not neurons.

Activation of protein kinase C by phorbol esters caused a time- and dose-dependent stimulation (270% of control) of glucose uptake in cultured glia, but not in neurons from rat brain. The phorbol ester stimulation of Vmax of glial glucose uptake occurred only in glia despite nearly 2.5-fold greater phorbol ester binding in neurons. These differences in cellular responses to protein kinase C activation may be the key to understanding brain glucose regulation.

Animals↗
PubMed1986

Protease-induced immunoregulatory activity of platelet factor 4.

Intravenous injection of human or mouse serum or platelet material secreted from appropriately stimulated platelets ("releasate") together with antigen alleviates the immunosuppression in SJL/J mice induced by injection of irradiated lymphoma cells or in (CB6)F1 mice induced by injection of concanavalin A. We now report that injection of releasate from 10(6) human platelets restores plaque-forming cells to the unsuppressed number; greater amounts increase responses further. Immunoregulatory activity is released from platelets exposed to thrombin in parallel with other alpha-granule components. Heparin-agarose absorbs activity. Purified platelet factor 4 (PF4) has activity; beta-thromboglobulin and platelet-derived growth factor have little or none. Activity in serum is neutralized by goat anti-human PF4. An enzymatic step is necessary for production of immunoregulatory activity. Releasates boiled immediately after platelet aggregation with 250 nM A23187 or those produced by adding A23187 in the presence of 100 microM serine protease inhibitor (p-amidinophenyl)methanesulfonyl fluoride (APMSF) are ineffective, whereas releasates boiled or mixed with APMSF after incubation for 60 min are active. Activity is generated by incubating a mixture of heparin-absorbed releasate (as enzyme source) and heparin-agarose eluate of releasate made in the presence of APMSF (as substrate source). The enzymatic step does not alter the heparin-neutralizing activity of PF4. Apparently a secreted platelet protease converts PF4 to a form with immunoregulatory activity.

Animals↗
PubMed1986

Binding of thrombin-activated human factor VIII to platelets.

To study association of platelets with factor VIII, the purified protein was 125I-labelled with Bolton-Hunter reagent to a specific activity of 243,000-360,000 cpm/U. Autoradiographs of SDS polyacrylamide gels revealed polypeptides of VIII at Mr about 240 kDa, 90 kDa and intermediate values, as well as some radioactive contaminants, but the light chain (Mr 78/76) seen with silver stain was not labelled. After 2.5-5-fold activation with thrombin, the higher radioactive Mr band disappeared, the band at 90 kDa became more intense, and a band appeared at about 45 kDa. The radioactivity associated with platelets, studied in the presence of haemophilic BaSO4-treated plasma, was maximal after 6-8 min and increased 3-15-fold on activation with thrombin. With activated VIII, autoradiographs of platelet pellets showed only VIIIa but results are expressed as units of unactivated VIII bound. At 0.3-0-0.7 U/ml, 10(8) platelets bound 0.0008-0.004 U VIIIa. The amount bound was not affected by the ratio of unlabelled VIII to VIII labelled in the presence of a 50-fold molar excess of unlabelled Bolton-Hunter reagent. Binding increased to 1.5 U VIIIa/10(8) platelets (about 13,600 molecules per platelet) at 140 U/ml, with no evidence of saturation. Binding was not affected by monoclonal antibodies to platelet glycoproteins IIb/IIIa or Ib, quenching the thrombin before adding platelets, or aggregating the platelets with A23187 in the presence of thrombin. Qualitatively, binding of labelled VIIIa and factor Va studied by others are similar. Binding of 125I-VIIIa to aggregated and unaggregated platelets was normal in patient M.S. whose platelets were shown by others to be deficient in their ability to bind radiolabelled factor Xa and generate coagulant activity. This difference. and the fact that platelet coagulant activity is increased by platelet activation and/or aggregation, suggest that binding of Va or VIIIa alone does not determine the assembly of active proteolytic complexes on the platelet surface.

Autoradiography↗
PubMed1986

Computer aided diagnosis of acute abdominal pain at Middlesbrough General Hospital.

This presentation reports the experience of the surgical house staff and registrars at Middlesbrough General Hospital who used a desk-top computer system to support their clinical diagnosis of acute abdominal pain. The results cover a two year period and are compared with a baseline period of one year. Substantial benefits followed the introduction of the computer-aided diagnostic support system; increased diagnostic accuracy of the whole surgical team, reduction in negative laparotomy rates, earlier surgical intervention for acute appendicitis, reduction in the number of cases of perforated appendicitis and more efficient use of resources. The reasons for these improvements include more thorough collection of data, use of common terminology and an educational element provided by detailed feedback from the computer system.

Abdomen, Acute↗