Search PubMed⌕ Search

Biomedical subjects

R T Dean

Publications and source records attributed to R T Dean.

At least 127 records · Page 7Linked to original sources
PubMed1988

Free-radical generation by copper ions and hydrogen peroxide. Stimulation by Hepes buffer.

Hydroxyl radicals (OH.), generated by a phosphate-buffered Cu2+/H2O2 system, were detected by lucigenin-amplified chemiluminescence, deoxyribose degradation and benzoate hydroxylation. In each system the buffer, Hepes, was found to stimulate radical generation significantly. There are two main reasons for this effect: Hepes increases Cu2+ solubility in phosphate-buffered systems, and forms a complex with Cu2+ that is effective in generating OH. from H2O2. Pipes, a structurally similar buffer, and histidine, a known Cu2+ chelator, were found to have a similar effect. These data suggest that the crucial factor in such free-radical-generating systems is the availability of Cu2+, and that these actions of Hepes should be considered in the design of studies utilizing such systems.

Benzoates↗
PubMed1988

Hydroperoxide-mediated fragmentation of proteins.

1. Chemiluminescence and benzoic acid hydroxylation were used to detect oxygen-centred free-radical production by 2.5 mM-H2O2 and 100 microM-Cu2+. Free radicals could not be detected by these methods when H2O2 was replaced with 10 mM-t-butyl hydroperoxide (TBH) or 10 mM-cumene hydroperoxide (CH). The inclusion of the thiol compound dithioerythritol (DTET; 100 microM) increased radical production by H2O2 and Cu2+ as judged by both assays. Mannitol scavenged radicals in the chemiluminescence system in a dose-dependent manner. 2. H2O2, TBH and CH, each with Cu2+, gave rise to substantial fragmentation of the protein bovine serum albumin (BSA). This fragmentation could be increased by the inclusion of DTET. Omission of Cu2+ or the addition of the chelator DETAPAC (diethylenetriaminepenta-acetic acid; 1 mM) lead to virtual abolition of fragmentation. Autoxidized lipid in the presence of Cu2+ caused protein fragmentation by reactions of lipid hydroperoxides. 3. Polyacrylamide-gel electrophoresis in the presence of SDS confirmed that production of fragments had occurred. 4. Susceptibility of BSA to enzymic hydrolysis by two different proteinases acting at pH 5 and pH 7.2 was increased after a limited exposure to hydroperoxides in the presence of Cu2+. 5. These results may have biological significance, particularly for proteins in lipid environments (e.g. membrane proteins and lipoproteins).

Benzoates↗
PubMed1988

Trypanosoma brucei: susceptibility to hydrogen peroxide and related products of activated macrophages.

The susceptibility of procyclic, trypsinized, or bloodstream forms of Trypanosoma brucei to lysis by hydrogen peroxide and by activated mouse macrophages was investigated in vitro using the release of biosynthetically labeled proteins as an assay. Uncoated parasites were more resistant than coated bloodstream forms in both cases. Macrophage trypanolysis upon triggering with phorbol myristate acetate occurred extracellularly and seemed to depend on the release of hydrogen peroxide, as it was prevented by catalase. However, when presented trypanosomes in presence of fresh immune serum which was itself lytic, macrophages did not show any additional lytic effect, and phagocytosis was related to already damaged parasites.

Animals↗
PubMed1988

Alginate inhibition of the uptake of Pseudomonas aeruginosa by macrophages.

Pseudomonas aeruginosa alginate was purified and characterized in terms of uronic acid, carbohydrate and protein content, as well as by infra-red spectroscopy and gel electrophoresis. Added exogenous bacterial alginate inhibited the uptake and degradation of both viable and non-viable radiolabelled non-mucoid P. aeruginosa by resident mouse peritoneal macrophages. Alginic acid (from seaweed) inhibited the same parameters to almost the same degree. Bacterial alginate also inhibited the uptake of fluorescent-labelled zymosan and latex particles. Starch, at equivalent viscosity to the alginate, inhibited the uptake and degradation of radiolabelled nonviable P. aeruginosa to a greater extent, but Dextran T500 had no effect. This suggests that the viscous nature of alginate exerts a non-specific inhibitory effect on the uptake and subsequent degradation of phagocytosible particles.

Alginates↗
PubMed1987

Vitamin E protects proteins against free radical damage in lipid environments.

The fragmentation of the membrane protein monoamine oxidase in submitochondrial particles was induced by defined free radicals during radiolysis and by a system dependent on hydrogen peroxide and a transition metal. By injection of alpha-tocopherol in vivo, the levels of this physiological antioxidant in the mitochondrial preparations could be elevated more than ten-fold. In both radical-generating systems the presence of high levels of alpha-tocopherol in the membrane substantially retarded the protein fragmentation, in parallel with lipid peroxidation. It is suggested that membrane-bound proteins are damaged during lipid peroxidation and that alpha-tocopherol protects cells against both types of damage.

Animals↗
PubMed1987

A mechanism for accelerated degradation of intracellular proteins after limited damage by free radicals.

I propose that limited free radical attack upon proteins, occurring continuously in cells, creates new N-termini (notably aspartate and glutamate) which render the proteins more susceptible to proteolysis by the ubiquitin conjugation system. I suggest that these reactions are a significant part of the previously described 'N-end' and 'PEST' rules, which indicate amino acid termini or sequences which tend to dictate short protein half-lives. I also argue that the N-end rule may apply to sequestered intracellular sites, such as mitochondria, these also being sites of radical generation.

Amino Acid Sequence↗
PubMed1987

Glucose autoxidation and protein modification. The potential role of 'autoxidative glycosylation' in diabetes.

Monosaccharide autoxidation (a transition metal-catalysed process that generates H2O2 and ketoaldehydes) appears to contribute to protein modification by glucose in vitro. The metal-chelating agent diethylenetriaminepenta-acetic acid (DETAPAC), which inhibits glucose autoxidation, also reduces the covalent attachment of glucose to bovine serum albumin. A maximal 45% inhibition of covalent attachment was observed, but this varied with glucose and DETAPAC concentrations in a complex fashion, suggesting at least two modes of attachment. The extent of inhibition of the metal-catalysed pathway correlated with the extent of inhibition of glycosylation-associated chromo- and fluorophore development. DETAPAC also inhibited tryptophan fluorescence quenching associated with glycosylation. Conversely, ketoaldehydes analogous to those produced by glucose autoxidation, but generated by 60Co irradiation, bound avidly to albumin and accelerated browning reactions. It is therefore suggested that a component of protein glycosylation is dependent upon glucose autoxidation and subsequent covalent attachment of ketoaldehydes. The process of glucose autoxidation, or ketoaldehydes derived therefrom, appear to be important in chromophoric and fluorophoric alterations. It is noted, consistent with these observations, that the chemical evidence for the currently accepted 'Amadori' product derived from the reaction of glucose with protein amino groups is consistent also with the structure expected for the attachment of a glucose-derived ketoaldehyde to protein. The concept of 'autoxidative glycosylation' is briefly discussed in relation to oxidative stress in diabetes mellitus.

Aldehydes↗
PubMed1987

Is enhanced free radical flux associated with increased intracellular proteolysis?

Intracellular proteolysis was measured in cultured cells during and after free radical attack. Radicals were generated firstly, throughout the aqueous phase by gamma irradiation and secondly, selectively, either extracellularly or intracellularly by chemical and enzymic methods. With both approaches, stimulation of proteolysis was observed in certain circumstances. Phenylhydrazine stimulated proteolysis at low concentration but inhibited at higher. Depletion of the antioxidant glutathione and inhibition of catalase also increased proteolysis.

Amitrole↗
PubMed1987

Macrophage procoagulant activity in experimental African trypanosomiasis.

Peritoneal macrophages from mice infected with Trypanosoma brucei brucei expressed a greatly elevated procoagulant activity (PCA) which could be reversed to normal levels after trypanocidal therapy. Comparison with infection caused by the non-pathogenic T. musculi suggested that the level of PCA related to parasite pathogenicity. Unstimulated macrophages, which generate only slight PCA upon stimulation with zymosan, become hyperresponsive to this stimulus during the course of infection. Hyperresponsiveness is not a generalized feature of these cells during infection, as they become progressively hyporesponsive to the same stimulus in terms of lysosomal enzyme secretion. We were unable to demonstrate a direct role of the trypanosomes in macrophage activation; however, artificial removal of the glycoprotein coat rendered the parasites highly stimulatory for macrophages even in the absence of opsonins. These results suggest that the parasites may activate macrophages indirectly, and that the resulting elevated PCA may play a role in the abnormal blood coagulation known to occur in this disease.

Animals↗
PubMed1987

T-lymphocyte requirement for the induction of mouse macrophage procoagulant activity by Trypanosoma brucei.

We have previously shown that peritoneal macrophages from Trypanosoma brucei infected mice, but not from uninfected mice, expressed high levels of procoagulant activity that could not be produced in vitro by incubation of unstimulated macrophages with bloodstream forms of trypanosomes. In the present study we demonstrate that trypanosome-induced macrophage activation can be achieved in vitro by providing either sensitized (day 7 of infection) lymphocytes and trypanosomes or the supernatant fluid from this interaction. The ability of lymphocytes to secrete macrophage-activating lymphokines is enhanced up to day 12 of infection but was absent in the later stages. Although enhancement of the procoagulant activity occurred in infected nude mice, it seems that macrophage function in African trypanosomiasis, as regards the expression of procoagulant activity, is regulated by T-lymphocytes.

Animals↗
PubMed1986

Free-radical-mediated fragmentation of monoamine oxidase in the mitochondrial membrane. Roles for lipid radicals.

A flux of hydroxyl radicals generated by gamma-irradiation can fragment monoamine oxidase in the membrane of submitochondrial particles. This fragmentation can be inhibited by mannitol and in addition is more extensive in monoamine oxidase preparations that have been depleted of lipid. This latter observation is consistent with the higher yields of fragmentation induced by hydroxyl radicals in soluble proteins in the absence of added lipids. In the absence of oxygen, gamma-irradiation of submitochondrial particles leads to cross-linking reactions. A flux of hydroperoxyl radicals also causes fragmentation, whereas one of superoxide is virtually inactive in this respect. The irradiation of submitochondrial particles leads in addition to the accumulation of products of lipid peroxidation. When these irradiated preparations are exposed to ferrous or cupric salts a further fragmentation of monoamine oxidase ensues, especially at acid pH. These transition-metal-catalysed reactions do not occur with irradiated preparations depleted of lipid, and the post-irradiation protein modifications are concomitant with further lipid peroxidation. The data indicate roles for lipid radicals in both fragmentation and cross-linking reactions of proteins in biological membranes. These reactions may have an important bearing on control of protein activity and of protein turnover in membranes.

Animals↗
PubMed1986

Fragmentation of proteins by free radicals and its effect on their susceptibility to enzymic hydrolysis.

Defined radical species generated radiolytically were allowed to attack proteins in solution. The hydroxyl radical (OH.) in the presence of O2 degraded bovine serum albumin (BSA) to specific fragments detectable by SDS/polyacrylamide-gel electrophoresis; fragmentation was not obvious when the products were analysed by h.p.l.c. In the absence of O2 the OH. cross-linked the protein with bonds stable to SDS and reducing conditions. The superoxide (O2-.) and hydroperoxyl (HO2.) radicals were virtually inactive in these respects, as were several other peroxyl radicals. Fragmentation and cross-linking could also be observed when a mixture of biosynthetically labelled cellular proteins was used as substrate. Carbonyl and amino groups were generated during the reaction of OH. with BSA in the presence of O2. Changes in fluorescence during OH. attack in the absence of O2 revealed both loss of tryptophan and changes in conformation during OH. attack in the presence of O2. Increased susceptibility to enzymic proteolysis was observed when BSA was attacked by most radical systems, with the sole exception of O2-.. The transition-metal cations Cu2+ and Fe3+, in the presence of H2O2, could also fragment BSA. The reactions were inhibited by EDTA, or by desferal and diethylenetriaminepenta-acetic acid ('DETAPAC') respectively. The increased susceptibility to enzymic hydrolysis of radical-damaged proteins may have biological significance.

Chromatography, High Pressure Liquid↗