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R T Dean

Publications and source records attributed to R T Dean.

At least 109 records · Page 6Linked to original sources
PubMed1991

Restriction of the participation of copper in radical-generating systems by zinc.

Zinc was able to reduce the availability of copper for several radical-generating reactions: lucigenin-amplified 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.

Binding Sites↗
PubMed1990

Use of bacterial expression cloning to localize the epitopes for a series of monoclonal antibodies against apolipoprotein B100.

Bacterial expression of apolipoprotein (apo) B cDNA constructs has been used to map a series of monoclonal antibodies (mAbs) to apoB by immunoblotting. In some cases assignments have been confirmed and refined by (i) semipurification of expressed protein, CNBr digestion, and assignment of the immunoreactive fragments; (ii) controlled digestion of the cDNA with the exonuclease Bal31 and bacterial expression of the truncated proteins that result; or (iii) expression of specific segments of cDNA amplified by the polymerase chain reaction. Forty mAbs were mapped to a minimum of 17 separate determinants on apoB. Tryptic fragments have been used to confirm the epitope assignments. In addition, this approach in conjunction with immunoassay, enables some deductions to be made about the trypsin-accessible regions in low density lipoprotein (LDL). The cleavage pattern obtained predicts retention of structure in the cysteine-rich domain of the amino terminus and also in the LDL receptor binding region. Trypsinized LDL was shown to bind to the LDL receptor by an authentic process, using monoclonal antibodies as competing ligands. In conjunction with the previous paper (Milne, R. W., Theolis, R., Maurice, R., Pease, R. J., Weech, P. K., Rassart, E., Fruchart, J.-C., Scott, J., and Marcel, Y. L. (1989) J. Biol. Chem. 265, 19754-19760) the mapped mAbs have been used to define the receptor-binding domain of apoB100 in LDL.

Antibodies, Monoclonal↗
PubMed1990

Stimulatory and inhibitory actions of proteins and amino acids on copper-catalysed free radical generation in the bulk phase.

The effect of a variety of proteins and amino acids was investigated on oxygen free radical activity as assessed by copper/hydrogen peroxide induced benzoate hydroxylation as well as copper-catalysed ascorbate autoxidation. Serum albumins from a variety of species (human, bovine and dog) had both inhibitory and stimulatory effects depending on the molar copper to protein ratio; low ratios were inhibitory and high stimulatory. Some other proteins tested (lysozyme, soybean trypsin inhibitor and conalbumin) also had dual (inhibitory and stimulatory) effects, as did both histidine and polyhistidine, but all effects occurred at different molar ratios presumably dependent on the relative affinities for the copper ions. In contrast, metallothionein and caeruloplasmin, proteins specialised to bind copper in vivo had no stimulatory effects. In this paper we show that in addition to their fairly well documented inhibitory effects, under certain conditions some proteins also stimulate radical reactions. The possible role of this phenomenon in vivo is discussed.

Amino Acids↗
PubMed1989

The action of defined oxygen-centred free radicals on human low-density lipoprotein.

The effects of defined oxygen-centred free radicals on human low-density lipoprotein (LDL) structure and receptor affinity are discussed in relation to the mechanisms of cell-mediated oxidative modification of LDL. Both hydroxyl (OH.) and hydroperoxyl (HO2.) radicals caused depletion of endogenous alpha-tocopherol and formation of hydroperoxides. Superoxide (O2-.) radicals produced only very limited oxidation, but could potentiate oxidation stimulated by the addition of Cu2+. All these radicals enhanced the net negative charge of intact LDL and induced fragmentation of apolipoprotein B-100 (apo B). OH. also caused cross-linking of apo B. Radical attack decreased the affinity of LDL for the fibroblast apo B/E receptor, but did not enhance its endocytosis by mouse macrophages.

Apolipoproteins B↗
PubMed1989

Control of exogenous proteinases and their inhibitors at the macrophage cell surface.

The actions and availability of human neutrophil elastase and its protein inhibitor, Eglin, when co-incubated with macrophages were investigated. Eglin did not induce radical production by mouse peritoneal macrophages; nor were specific binding sites for Eglin detected on these cells. Mouse peritoneal macrophages could inactivate both elastase and Eglin extensively, when these targets were used at concentrations appropriate to the extravascular fluids. Two methods were used for assessing such inactivation: one, as in previous literature, only took account of molecules remaining in the supernatant after interaction with the cells; the other (lacking from most previous studies) took into account all target molecules, including those associated with the cells. From an analysis of both types of experiment, it was shown that the cell-derived inactivators were stable products, whose quantity was not significantly influenced by the induction of a macrophage oxidative burst and its associated free radicals. They were probably mainly proteinases and proteinase inhibitors. Thus, mouse peritoneal macrophages restrict the activity of proteinases and inhibitors by means of stable molecules, such as proteins. Other mononuclear phagocytes may use free radicals and oxidants more extensively in this respect.

Animals↗
PubMed1989

Free radical-mediated degradation of proteins: the protective and deleterious effects of membranes.

Lipid membranes have been shown to scavenge free radicals generated by various means. However, under oxidative conditions, unsaturated lipids within membranes can produce damaging free radicals. We have determined the relative importance of these two conflicting properties of lipid membranes with the use of liposomal membrane studies. (1) Liposome membranes can protect extra-liposomal albumin from free radicals derived from sources other than peroxidizing lipid. When albumin or copper (an essential component of the free radical generating systems used) were encapsulated, protein damage was further reduced. (2) Using sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) we demonstrate that the exposure of albumin to peroxidizing liposome membranes results in both cross-linking and degradation. Our results indicate that protein damage is substantially less than in the case of other biologically relevant free radical generating systems. We discuss our findings with respect to membrane function and the in vivo exposure of cells to free radicals.

Benzene Derivatives↗
PubMed1989

Radical initiated alpha-tocopherol depletion and lipid peroxidation in mitochondrial membranes.

The relationships between antioxidant status, lipid peroxidation and membrane protein integrity have been studied in an isolated mitochondrial membrane system. Tocopherol was shown to be present in both the outer and inner membrane of normal rat liver mitochondria; 77.3 and 22.3% of the total alpha-tocopherol was present in the outer and inner membranes, respectively. The endogenous alpha-tocopherol was depleted in a time-dependent manner by low levels of ferrous iron and by irradiation in the presence or absence of ferrous iron. This antioxidant depletion was followed by the appearance of lipid hydroperoxides. Fragmentation of monoamine oxidase, an integral outer membrane protein, was observed at irradiation doses that caused by antioxidant depletion and peroxide generation.

Animals↗
PubMed1989

Free radicals inactivate human neutrophil elastase and its inhibitors with comparable efficiency.

Free radicals produced in a Fenton reaction (H202/Cu), modelling some xenobiotic and cell-mediated inflammatory affronts, efficiently inactivated the elastase-inhibitor eglin, but equally, human neutrophil elastase itself. Elastase activity was not regenerated from proteinase/inhibitor complexes during radical attack. Three different elastase inhibitors, eglin, secretory leukocyte proteinase inhibitor and alpha-1-proteinase inhibitor were all similarly sensitive to inactivation. Unlike certain oxidants which can selectively inactivate alpha-1-proteinase inhibitor, free radicals may influence comparably the availability of both proteinase inhibitors and their targets.

Blood Proteins↗
PubMed1989

A continuous-flow automated assay for iodometric estimation of hydroperoxides.

An iodometric method for the analysis of hydroperoxides has been automated to allow analysis of aqueous biological samples (containing less than 20 mg/ml protein) and lipid hydroperoxide extracts. The evolution of triiodide ions is measured spectrophotometrically at 360 nm. Dependent on the type of sample, 30-60 samples can be analyzed per hour and the system allows detection of less than 100 pmol of peroxide. The assay is linear over a range of 100 pmol to 25 nmol. The sample volume used routinely was 80 microliters.

Animals↗
PubMed1989

Antimyosin imaging in acute transmural myocardial infarctions: results of a multicenter clinical trial.

Murine monoclonal antimyosin antibody has been shown experimentally to bind selectively to irreversibly damaged myocytes. To evaluate the safety and efficacy of monoclonal antimyosin for identifying acute transmural infarction, 50 patients with acute Q wave myocardial infarction were entered into a phase I/II multicenter trial involving three clinical sites. Indium-111 antimyosin was prepared from an instant kit formulation containing 0.5 mg of diethylene triamine pentaacetic acid (DTPA)-coupled Fab fragment (R11D10) and 1.2 to 2.4 mCi of indium-111. Average labeling efficiency was 92%. Antimyosin was injected 27 +/- 16 h after the onset of chest pain. Planar or tomographic imaging was performed 27 +/- 9 h after injection in all patients, and repeat imaging was done 24 h later in 39 patients. Of the 50 patients entered, 46 showed myocardial uptake of antimyosin (sensitivity 92%). Thirty-one of 39 planar scans performed at 24 h were diagnostic; 8 showed persistent blood pool activity that cleared by 48 h. Focal myocardial uptake of antimyosin corresponded to electrocardiographic infarct localization. No patient had an adverse reaction to antimyosin. In addition, 125 serum samples, including 21 collected greater than 42 days after injection, were tested for human antimouse antibodies, and all samples were assessed as having undetectable titers. Intensity of antimyosin uptake was correlated with infarct location and the presence or absence of collateral vessels. There was a significant correlation between faint uptake and inferoposterior infarct location. In 21 patients who had coronary angiography close to the time of antimyosin injection, there was a significant correlation between faint tracer uptake and closed infarct-related vessel with absent collateral flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

Scavenging by alginate of free radicals released by macrophages.

Failure to eradicate mucoid forms of P. aeruginosa has implicated bacterial alginate in a local evasion of host defence mechanisms within the lung of Cystic Fibrosis (CF) patients. We have found that purified bacterial alginate scavenges free radicals released by triggered macrophages as detected by lucigenin amplified chemiluminescence (CL) and reduction of cytochrome c. In agreement with this, alginate was also able to scavenge radicals generated by a chemical system (hydrogen peroxide and copper; detected by benzoate hydroxylation and chemiluminescence), and by an enzymatic system (hypoxanthine and xanthine oxidase; detected by chemiluminescence). All inhibitions were dose-related. Oxygen consumption by neutrophils (unlike that of macrophages) could be detected in a Clark electrode, and was not reduced by alginate, confirming that scavenging of radicals was responsible for the earlier observations. These data suggest that bacterial alginate by scavenging free radicals, may favour the survival of mucoid forms of P. aeruginosa, particularly in the CF lung.

Alginates↗
PubMed1989

Practical considerations in the production, purification, and formulation of monoclonal antibodies for immunoscintigraphy and immunotherapy.

Issues associated with the large-scale production of monoclonal antibodies for pharmaceutical applications are examined. The development of a commercial monoclonal antibody production process involves much more than just scaling-up the laboratory process and making it cost-effective. It involves establishing the hybridoma cell bank with cells that are free of adventitious agents such as viruses and mycoplasma, that have stability in continuous culture for antibody-production rate and cell viability, and that do not have unusual or expensive media requirements. The style and mode of operation of the bioreactor used to produce the antibody must be explored. The antibody-based product must be processed to high levels of purity, and specific contaminants such as DNA and endotoxin must be reduced to extremely low levels. Appropriate labeling or drug conjugation chemistries must also be developed. The product must be formulated so that it has performance characteristics that are stable over a reasonable period of time. Adequate test procedures must be developed to assure product purity, activity, stability, and safety on a lot-to-lot-basis. Compliance with federal regulations, guidelines, and procedures must be guaranteed. In the coming decade, it is likely that the two arms of biotechnology, hybridoma technology and recombinant DNA technology, will be used together to generate unique protein molecules. These new reagents will face the same practical considerations summarized in this review.

Animals↗
PubMed1989

Tc-99m antifibrin Fab' fragments for imaging venous thrombi: evaluation in a canine model.

An antifibrin antibody (T2G1s) Fab' fragment labeled with technetium-99m was tested for its ability to produce images of fresh thrombi in dogs. In gamma camera images, all thrombi were evident by 2-4 hours after injection. Mean thrombus-to-blood and thrombus-to-muscle ratios averaged 4.0 and 69 at four hours after injection and increased to 24 and 270, respectively, by 24 hours after injection. When compared with I-125 fibrinogen injected into the same dogs, Tc-99m-antifibrin Fab' had lower absolute uptake in thrombus but higher thrombus-to-blood ratios due to a faster rate of disappearance from the blood. The primary route of excretion was through the kidneys. Tc-99m-antifibrin Fab' was highly stable in vivo, with an average of 82% of the circulating radioactivity able to bind to fibrin at 4 hours after injection. When compared with an In-111-labeled Fab fragment of antifibrin antibody 59D8, thrombus-to-blood and thrombus-to-muscle ratios were slightly higher for the Tc-99m-labeled antibody, and the blood disappearance rate was slightly faster. The absolute uptake in thrombus, however, was not significantly different, and the thrombus was visualized at about the same time after injection. These studies suggest that Tc-99m T2G1s Fab' is a potential agent for detecting thrombi in a clinical setting.

Animals↗
PubMed1989

Histidine and proline are important sites of free radical damage to proteins.

Our hypothesis that proline and histidine are major sites of damage during radical attack upon proteins, becoming respectively glutamate and aspartate, was investigated using proteins biosynthetically labelled with radioactive proline or histidine as targets. Free radicals were generated by copper and H2O2, or by gamma radiolysis. Protein-bound histidine was substantially converted into aspartate. Much proline was modified during radical attack, but it was not converted into glutamate. We conclude that histidine and proline are important sites of protein attack by radicals; protein cleavage may result from these reactions.

Animals↗
PubMed1989

Antioxidants and the influence of free radical damage to proteins on proteolysis in and around mammalian cells.

The hypothesis that free radical damage to proteins accelerates overall protein catabolism in and around mammalian cells is discussed. It is concluded that most cells are in possession of sufficient antioxidants to control protein damage, so that changing radical fluxes do not greatly change intracellular protein turnover rates. Exceptions exist however, and may be relevant to specialised cells, and to certain pathologies, where free radical damage to proteins may occur outside cells. The cases of inflammatory disease and atherosclerosis are mentioned, and it is argued that therapies based on anti-oxidant supply may be valuable in these cases.

Animals↗
PubMed1988

Membrane proteins are critical targets in free radical mediated cytolysis.

The hypothesis that proteins are critical targets in free radical mediated cytolysis was tested using U937 mononuclear phagocytes as targets and iron together with hydrogen peroxide to generate radicals. Those conditions which, after a lag of approx. 30 min, led to drastic lysis were also associated with very rapid membrane depolarisation. Conversely, when the early membrane depolarisation was prevented (by the addition of chelator and catalase), so was lysis. A similar correlation between early membrane depolarisation and subsequent lysis was also observed when the cells were exposed to a toxin from Actinobacillus actinomycetemcomitans. Those conditions of radical attack which led to lysis normally caused substantial lipid peroxidation. However, depolarisation and subsequent lysis were not prevented even when lipid peroxidation was completely suppressed by exogenous antioxidant. ATP levels were not grossly affected within the critical first 30 min period. These data exclude lipids and ATP as the target for lytic damage. We argue therefore that proteins are probably amongst the primary targets in cytolysis by radicals.

Adenosine Triphosphate↗
PubMed1988

Hydroxyl radical production and autoxidative glycosylation. Glucose autoxidation as the cause of protein damage in the experimental glycation model of diabetes mellitus and ageing.

Protein exposed to glucose is cleaved, undergoes conformational change and develops fluorescent adducts ('glycofluorophores'). These changes are presumed to result from the covalent attachment of glucose to amino groups. We have demonstrated, however, that the fragmentation and conformational changes observed are dependent upon hydroxyl radicals produced by glucose autoxidation, or some closely related process, and that antioxidants dissociate structural damage caused by the exposure of glucose to protein from the incorporation of monosaccharide into protein. We have also provided further evidence that glycofluorophore formation is dependent upon metal-catalysed oxidative processes associated with ketoaldehyde formation. If experimental glycation is an adequate model of tissue damage occurring in diabetes mellitus, then these studies indicate a therapeutic role for antioxidants.

Aging↗