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Showing 2021–2040 of 2058 publications.
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Dean, Roger T.; Gebicki, Janusz M.; Gieseg, Steven Paul; Grant, Adrienne J.; Simpson, Jeremy A.This paper discusses our knowledge of protein oxidation and its relationship to aging. It also outlines new observations from our laboratories concerning reactive species produced during protein oxidation, and proposes that these may inflict damage on other molecules, and hence contribute to the progression of aging. Whereas it has previously been difficult to see how relatively inert protein oxidation products could possibly have any causal role in aging, the detection of these novel reactive species implies a potentially significant role. 1992.
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Bowry, Vincent W.; Stanley, Keith K.; Stocker, RolandAnalysis of untreated fresh blood plasma from healthy, fasting donors revealed that high density lipoprotein (HDL) particles carry most (?85%) of the detectable oxidized core lipoprotein lipids. Low density lipoprotein (LDL) lipids are relatively peroxide-free. In vitro the mild oxidation of gel-filtered plasma from fasting donors with a low, steady flux of aqueous peroxyl radicals initially caused preferential oxidation of HDL rather than LDL lipids until most ubiquinol-10 present in LDL was consumed. Thereafter, LDL core lipids were oxidized more rapidly. Isolated lipoproteins behaved similarly. Preferential accumulation of lipid hydroperoxides in HDL reflects the lack of antioxidants in most HDL particles compared to LDL, which contained 8-12 ?-tocopherol and 0.5-1.0 ubiquinol-10 molecules per particle. Cholesteryl ester hydroperoxides (CEOOHs) in HDL and LDL were stable when added to fresh plasma at 37C for up to 20 hr. Transfer of CEOOHs from HDL to LDL was too slow to have influenced the in vitro plasma oxidation data. Incubation of mildly oxidized LDL and HDL with cultured hepatocytes afforded a linear removal of CEOOHs from LDL (40% loss over 1 hr), whereas a fast-then-slow biphasic removal was observed for HDL. Our data show that HDL is the principal vehicle for circulating plasma lipid hydroperoxides and suggest that HDL lipids may be more rapidly oxidized than those in LDL in vivo. The rapid hepatic clearance of CEOOHs in HDL could imply a possible beneficial role of HDL by attenuating the build-up of oxidized lipids in LDL.
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Gieseg, Steven Paul; Forrester, Ian T.; Carne, AlanA modified procedure for the purification of ovine pancreatic lipase (triacylglycerol acyl-hydrolase, EC3.1.1.3) is described. The method is more rapid and more reproducible than that reported previously and results in a pure lipase preparation, that gives a better yield at the same specific activity, free of colipase and uncontaminated by lipid. The procedure involves the preparation of a lipid-free acetone powder from fresh pancreas without the use of chloroform or butanol as was used in the procedure described earlier. The aqueous purification of the lipase from the delipidated powder is similar to that described earlier, but includes the use of ?-mercaptoethanol and uses salt gradient elution from CM-Sepharose. An assay procedure for lipase is reported involving the extraction of released free fatty acids with chloroform/methanol before titrating with sodium hydroxide. A modification of this assay is used for the determination of colipase. The above assay procedure is compared to the potentiometric method reported previously. Polyacrylamide gel, amino acid composition analysis and N-terminal sequence data for the purified ovine lipase are presented. 1992 Raven Press, Ltd., New York.
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Simpson, Jeremy A.; Narita, S.; Gieseg, Steven Paul; Gebicki, Silvia; Gebicki, Janusz M.; Dean, Roger T.We have demonstrated two novel reactive species on radical-modified proteins which are relatively long-lived, one oxidizing and one reducing. The two species are reactive with critical biological components, and so may be of physiological and pathological importance. The oxidizing species, which have been identified as protein hydroperoxides, can consume key cellular reductants, such as ascorbate and glutathione. The reducing species can act on both free and metalloprotein forms of copper and iron ions, which participate in radical generation. These findings suggest that proteins may act as traps for the chemical energy released by free radicals, with the capacity to pass it on to other molecules. The long-lived nature of both the reactive moieties indicates that they may be able to diffuse and transfer damaging reactions to distant sites.
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Walsh, John D.; Wheeler, Helen Ruth; Geczy, Carolyn L.Coagulation disorders have been associated with the use of chemotherapeutic drugs. Pharmacological doses of cisplatin and adriamycin directly induced low levels of procoagulant on normal human blood monocytes and on a human myelomonocytic cell line, RC2a. Activity was maximal after 24 h and was not due to cell lysis as increasing drug doses which decreased cell viability were less effective. Procoagulant induction was markedly enhanced in the presence of bacterial lipopolysaccharide (LPS), with as little as 10100 pg/ml LPS potentiating the cisplatin response by 25?fold and more than doubling the adriamycin response. Greater than 90% of the procoagulant activity was membrane?bound tissue factor as indicated by the factor VII?dependent generation of factor Xa by viable cells and by the neutralization of this activity by a monoclonal antibody to tissue factor. Tissue factor antigen was measured simultaneously by immunohistochemical staining and by cell ELISA. Blood monocytes activated with LPS expressed high levels of tissue factor antigen; by contrast, adriamycin and cisplatin did not appear to induce antigen expression, but to enhance the specific activity of that already present. Results suggest that membrane alterations which occur following treatment with DNA/RNA intercalating drugs, may result in a highly active form of monocyte/macrophage tissue factor which may contribute to the complications caused by activated coagulation. Secondary Gram?negative infection or cyto?kines released by an active immune response to a tumour may contribute to the procoagulant potential of these cytotoxic drugs. Copyright 1992, Wiley Blackwell. All rights reserved
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Lovering, K. Erin; Dean, Roger T.Zinc was able to reduce the availability of copper for several radical-generating reactions: lucigeninamplified chemiluminescence due to copper and hydrogen peroxide; copper-dependent ascorbate oxidation and its concomitant oxygen consumption: and copper-dependent benzoate hydroxylation. This was the case both in the presence of bovine serum albumin (when most zinc was protein-bound) and in its absence (when zinc was 'available' Competition between zinc and copper for binding to the fluorophore calcein was also examined, and this allowed assessment of copper availability in several circumstances. Competition between copper and zinc for binding to biological components seems to be a rather general phenomenon, and thus zinc is commonly a protective entity, restricting free radical generation. 1991 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
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Dawes, Joan; McLaren, Margaret; Forbes, C.; Belch, Jj J.F.; Lane, Da; Bray, B.; McEwen, John; Houin, Georges; Gianese, Francesco1. The pharmacokinetics of dermatan sulphate MF701 were studied in 12 healthy males after administration of single intravenous bolus (200 mg), intramuscular (100 and 300 mg) and oral (1 g) doses. The study was conducted according to a within?subject crossover design in two paired blocks. 2. Plasma drug concentrations were measured using a competitive binding assay and a range of biological activity assays, including a sensitive catalysed thrombin inhibition test. 3. Following intravenous administration, plasma concentrations of dermatan sulphate determined by competitive binding assay were described by a two?compartment open model with an initial t1/2, in of 0.6 h and a t1/2,z of 7.5 h. Biological activity assays were insufficiently sensitive to detect the second phase, and therefore yielded apparent monoexponential kinetics. 4. After intramuscular injection the apparent bioavailability of dermatan sulphate was 16?20%. Plasma drug concentrations increased in proportion to dose when measured by competitive binding assay. Low concentrations persisted for more than 24 h at the higher dose, and these may prove therapeutically relevant on chronic administration. 5. We confirm that dermatan sulphate is the only glycosaminoglycan known to generate significant plasma concentrations following oral administration. Oral bioavailability was estimated to be 7%. 1991 The British Pharmacological Society
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Dean, Roger T.; Hunt, James V.; Grant, Adrienne J.; Yamamoto, Yorihiro; Niki, EtsutoFree radicals were generated at known rates in the aqueous phase (by means of 2,2?-azobis (2-amidinopropane) dihydrochloride [AAPH]) and in a membranous (lipid) phase (by means of 2,2?-azobis (2,4-dimethylvaleronitrile [AMVN]). A soluble protein (bovine serum albumin; BSA), and membranes of lysed mitochondria containing radioactively labeled monoamine oxidase (MAO), were exposed to the resultant radical fluxes. Antioxidants were added to the system, either in the aqueous phase (Trolox) or in a liposomal membrane phase (?-tocopherol). Protein damage was assessed as tryptophan oxidation and conformational changes in tryptophan fluorescence of the soluble protein. BSA, and as framentation of both BSA and monoamine oxidase. Radicals generated in the acquous phase, by AAPH, were affective in damaging BSA and MAO. Radicals generated within the liposome membrane phase (by AMVN) were less effective against BSA than those deriving from AAPH. Liposomal AMVN radicals could damage MAO, present in a separate membranous phase, though again, less effectively than could APPH-derived radicals. BSA could be protected by Trolox, the aqeous soluble antioxidant, but hardly by tocopherol itself. Damage to MAO was limited by Trolox, and also by the hydrophobic antioxidant, tocopherol. Damaging reactions due to radicals generated in a membrane phase were significantly accelerated when the membrane was peroxidizable (soybean phosphatidylcholine) rather than nonperoxidizable (saturated dimyristoyl phosphatidylcholine). Thus lipid radicals also played some role in protein damage in these systems. BSA was attacked similarly in the presence of liposomes by AAPH. Correspondingly, BSA could inhibit the peroxidation of liposomes induced by AAPH and less efficiently that induced by AMVN. It is concluded that the relative localization of radical generation, antioxidants, and protein targets has a major influence on the extent of radical attact on proteins and membranes. 1991.
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Walsh, John D.; Geczy, Carolyn L.The mechanisms underlying the superinduction of procoagulant activity by cycloheximide (CHX) on LPS-activated human monocytes have been investigated. Tissue factor (TF) activity of intact, viable cells was quantitated with a plasma recalcification assay and assays using chromogenic substrates specific for thrombin and factor Xa (FXa). TF antigen was measured simultaneously by immunocytochemical staining and immunoblotting with an anti-TF monoclonal antibody (MAb). Peripheral blood mononuclear cells (PBMC) activated with LPS in the presence of low dose CHX expressed more TF activity (approx. 100% increase) than cells activated with LPS alone. However, TF antigen levels were decreased approximately 70% by CHX. This discordant relationship was due primarily to differences in rates of activation of factor X (FX); LPS/CHX-treated PBMC activated nearly twice as much FX as LPS-treated cells (2.19 0.37 versus 1.10 0.21 ng FXa/106 PBMC/min, respectively). These studies indicate that TF cofactor activity on LPS/CHX-treated monocytes was approximately 7 times greater than that present on LPS-treated cells. Increased TF functional activity may be due to CHX-induced alterations in the type and content of phospholipids (PL) in the cell membrane. Results showed that exogenous mixed PL markedly increased TF activity on LPS-activated monocytes, but not on LPS/CHX-activated cells, without increasing TF antigen levels or altering cell viability. Membrane alterations may occur on monocytes in certain pathological or iatrogenic conditions resulting in a highly active form of TF in vivo.
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Stocker, Roland; Bowry, Vincent W.; Frei, Balz B.The temporal disappearance of natural antioxidants associated with human low density lipoprotein (LDL) in relation to the appearance of various classes of lipid hydroperoxides was investigated under three types of oxidizing conditions. Freshly isolated LDL from plasma of healthy subjects was free of detectable amounts of lipid hydroperoxides as measured by HPLC postcolumn chemiluminescence detection. Exposure of such LDL to a mild, constant flux of aqueous peroxyl radicals led to rapid and complete oxidation of ubiquinol-10, followed by slower partial depletion of lycopene, ?-carotene, and ?-tocopherol. After an initial lag period of complete inhibition of detectable lipid peroxidation, formation of hydroperoxides of cholesterol esters, triglycerides, and phospholipids was observed. The onset of detectable lipid peroxidation corresponded closely with the completion of ubiquinol-10 consumption. However, small amounts of ascorbate, present as a contaminant in the LDL preparation, rather than ubiquinol-10 itself were responsible for the initial lag period. Thus, complete consumption of ubiquinol-10 was preceded by that of ascorbate, and exposure of ascorbate-free LDL to aqueous peroxyl radicals resulted in immediate formation of detectable amounts of lipid hydroperoxides. The rate of radical-mediated formation of lipid hydroperoxides in ascorbate-free LDL was low as long as ubiquinol-10 was present, but increased rapidly after its consumption, even though more than 80% and 95% of endogenous carotenoids and ?-tocopherol, respectively, were still present. Qualitatively similar results were obtained when peroxyl radicals were generated within LDL or when the lipoprotein was exposed to oxidants produced by activated human polymorphonuclear leukocytes. LDL oxidation was reduced significantly by supplementing the lipoprotein preparation with physiological amounts of either ascorbate or ubiquinol-10. Our data show that ubiquinol-10 is much more efficient in inhibiting LDL oxidation than either lycopene, ?-carotene, or ?-tocopherol.
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Wheeler, Helen Ruth; Rockett, Elizabeth J.; Clark, Ian A.; Geczy, Carolyn L.The antitumour antibiotic actinomycin D (Act D) and the aminosugar D?galactosaminc both enhance the sensitivity of animals to bacterial lipopolysaccharide (LPS), Lipopolysaccharide stimulates macrophage membrane?bound procoagulant activity (MPCA) and tumour necrosis factor?alpha (TNF?a) production in vitro. We investigated the cfTects of LPS combined with either Act D or tJ?galactosaniine on procoagtilant and TNF?x production in vitro. Actinomycin D directly induced procoagulant on the malignant monocytoid cell line WEHI 265. and synergized with LPS to enhance MPCA on both WEHI 265 cells and thioglycollate?induccd peritoneal exudate maero?phages. In the presence of Act D, exudate maerophages expressed procoagulant in response to concentrations of LPS lOOOOO?fold lower than normally required. Pulsing experiments demon?strated that LPS primed these cells within 4 h to respond to Act D. whereas 4 h priming with Aet D inhibited subsequent procoagulant induction by LPS. Although itseflects on TNF?a production were less intense, low levels of Act D more than doubled TNF?a produced by LPS?stimulated exudate maerophages, Procoagulant expression and TNF?a production were not always co?ordinately expressed; interferon?gamtna (IFN?))synergi/ed with LPS to enhance both responses but when IFN?) was combined with Act D only procoagulant was upregulated. t)?galactosaminc failed to alTect these macrophage responses. Results indicate dillerent; vivo mechanisms of enhancement of LPS toxicity by these two agents. Copyright 1991, Wiley Blackwell. All rights reserved
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Hoad, Richard B.; Geczy, Carolyn L.Monoclonal antibodies to human plasma factor X (FX) and factor Xa (FXa) have been developed using several modifications of previously described techniques. These include the use of footpad immunisation with a suspension of free and nitrocellulose-bound antigen with subsequent fusion of popliteal lymph node cells. From a panel of 17 reactive hybridomas to FX, 3 were selected for further characterisation. An additional hybridoma reactive to FXa but not FX was also selected. Two monoclonal antibodies designated FX52 and FX64 were specific for FX with no reactivity to FXa, while antibody FXa24 was specific for FXa. Another FX/FXa95 reacted with both FX and FXa. All selected antibodies were of the IgG isotype and reacted both with native antigen and antigen transferred to nitrocellulose by Western blotting. Initial observations suggest that Mab FX52 may be used to quantitate FX levels in plasma. 1991.
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Greer, Ian A.; Dawes, Joan; Johnston, Tracey A.; Calder, Andrew A.The aim of this study was to determine whether neutrophil activation occurs in the fetal circulation in pregnancy-induced hypertension and to correlate this with evidence of neutrophil activation in the maternal circulation. Twenty-one normal pregnancies and 23 complicated by pregnancy-induced hypertension were studied in the third trimester. The mean length of gestation at delivery was significantly shorter (P < .01) and the mean birth weight percentile was significantly lower (P < .05) in the hypertensive group; otherwise the groups were comparable. Blood was obtained before cesarean delivery or established labor in the mothers and immediately after delivery from the umbilical vein. Plasma neutrophil elastase, which is released after neutrophil activation, was measured by radioimmunoassay as a marker for neutrophil activation. The mean ( standard error) concentration of neutrophil elastase in maternal plasma in the hypertensive group (35.9 4.7 ng/mL) was significantly higher than in the normal group (20.8 0.87 ng/mL) (P < .005). The concentration of neutrophil elastase in umbilical venous plasma was not significantly different between the normal and hypertensive groups. However, significantly higher concentrations of neutrophil elastase were found in the umbilical venous plasma of pregnancies delivered vaginally compared with those delivered by cesarean (P < .05) regardless of diagnosis. There was no correlation between maternal venous and umbilical venous plasma neutrophil elastase concentrations, birth weight percentile, plasma urate, or platelet count. These data suggest that neutrophil activation is confined to the maternal circulation in pregnancy-induced hypertension where it may contribute to vascular damage and dysfunction in areas such as the placental bed. In addition, neutrophil activation occurs in the fetal circulation during vaginal delivery. The neutrophil activation in the fetal circulation may reflect disturbance of the placental endothelium during labor or may be a physiologic mechanism involved in parturition. 1991 by The American College of Obstetricians and Gynecologists.
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Manolios, Nicholas; Geczy, Carolyn L.; Schrieber, LeslieLymphocytes are in a dynamic state of flux moving from the blood stream into lymphoid and extralymphoid tissues and eventually returning to the general circulation. This continuous process permits the full repertoire of lymphocyte specificities to be available for immune reactions. The regulation of lymphocyte traffic to specific tissue locations is complex and involves lymphocyte-endothelial interactions at sites of extravasation. Some of the molecules involved in lymphocyte-endothelial adhesion interactions have now been cloned and sequenced using molecular biology techniques. Lymphocyte migration to chronic inflammatory sites such as rheumatoid synovium shares a number of similarities to that involved in normal organized lymphoid tissue. First, at sites of chronic disease there are marked morphological changes to the vascular endothelium resulting in high endothelial venules identical to those found in normal lymphoid tissue. In addition, there are functional changes in vascular endothelial behavior that are receptor-mediated and regulate lymphocyte traffic to inflamed sites. The nature of these molecules on endothelial cells (addressins) and the regulation of their expression is still unclear. The control of lymphocyte migration appears to be influenced by selective lymphocyte-endothelial recognition in both health and inflammatory disease. The factors that modulate these interactions are reviewed. 1991.
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Greer, Ian A.; Leask, Rosemary M.; Hodson, Benjamin A.; Dawes, Joan; Kilpatrick, David Chalmers; Liston, William A.[No abstract available]
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Hodson, Benjamin A.; Pepper, Duncan S.; Dawes, JoanA new system for the isolation and purification of glycosaminoglycans (GAGs) and related molecules from complex systems such as plasma is described. Affinity chromatography which exploits the very high affinity between the polymeric base Polybrene and sulphated polysaccharides was used. A novel volatile buffer system composed of ammonium formate and formic acid, which allows the complete recovery of samples, was developed, and elution conditions were optimised for the separation and purification of GAGs of different charge densities. Using this system the losses associated with dialysis and desalting, frequently necessary preliminaries to further analysis, are avoided. 1991.
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Lackmann, Martin; Geczy, Carolyn L.A radioimmunoassay for recombinant (r.) hirudin was used to study the effect of heparin and other glycosaminoglycans (GAG's) on interactions between hirudin and thrombin. Binding of 125I-r.hirudin to thrombin in the presence or absence of heparin (and other GAG's) was monitored by immunoprecipitation of thrombin-125I-r.hirudin complexes with a monoclonal antibody to human a-thrombin. Heparin and dextran sulfate substantially reduced hirudin binding to thrombin in human plasma. Inhibition by heparin was restored by addition of increasing amounts of antithrombin (AT) to samples containing constant amounts of heparin, thrombin and 125I-r.hirudin and was not neutralized by protamine sulfate. At very low heparin concentrations competitive displacement of 1251-r.hirudin was observed, while in the presence of heparin-AT complexes some 125I-r.hirudin remained bound to thrombin, suggesting that two or more binding sites for hirudin on the thrombin molecule may be occupied simultaneously. 1991.
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Lackmann, Martin; Hoad, Richard B.; Kakakios, Alyson M.; Geczy, Carolyn L.A sensitive radioimmunoassay (RIA) for the quantitation of recombinant (r) hirudin in biological fluids is described. Taking advantage of the highly specific hirudinthrombin interaction, a monoclonal antibody to human ?-thrombin was used to capture hirudin-thrombin complexes in a competitive binding assay. Quantitation of r. hirudin in buffer, plasma or urine at concentrations ranging from 0.17 to 20 ng/ml (1.70-3 to 20-2 antithrombin units/ml) was achieved. In the absence of competing unlabelled r.hirudin the assay also measured ?-thrombin (from 20-4 to 1l0-2 NIH units/ml) in citrated or defibrinated human plasma. A series of peptides corresponding to the carboxyl-terminal region of hirudin and with varying anticoagulant activities did not displace 125I- r.hirudin in the RIA described, confirming published data that these hirudin fragments bind to a site distant to the catalytic site of thrombin. The assay was used to test hirudin clearance after bolus i.v. injections of 0.1mg r.hirudin [Val1-Val2] into human volunteers. The plasma concentrations and elimination kinetics of r. hirudin were in good agreement with published data and a close correlation between hirudin plasma concentration and prolonged clotting time was observed. 1991.
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Skerritt, John H.; Devery, Jannine M.; Penttila, Irmeli A.; LaBrooy, Justin TheodoreThe humoral and cellular immune responses to grain protein extracts from coeliactoxic and non-toxic cereals were compared by use of a number of ELISA and immunoblotting methods and the indirect leucocyte migration inhibition factor (LMIF) assay. Both adult and child coeliacs had elevated levels of serum antibody to proteins from the coeliac-toxic cereals, namely bread wheat, durum wheat, rye and barley and low levels of proteins from other cereals. Using protein blotting techniques, antibody binding was greatest to gliadins/low mol mass glutenin subunits and homologous prolamins from rye and barley, consistent with the ELISA findings. Competition ELISA and preabsorption tests indicated that antibody reaction to maize storage proteins did not simply result from cross-reaction of antigliadin antibodies. In LMIF assays, only the wheat extracts had activity in coeliac patients. This is most likely partly due to loss of some of T-cell epitopes from the extraction technique required for these proteins, as well as the relatively small effects seen for even very active fractions in the LMIF assay. 1991.
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Dean, Roger T.; Simpson, Jeremy A.[No abstract available]
