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Biomedical subjects

D W Allen

Publications and source records attributed to D W Allen.

At least 19 recordsLinked to original sources
PubMed1992

Organic peroxides inhibit neutrophil leukotriene B4 biosynthesis.

Leukotriene B4, an autacoid metabolite of arachidonic acid produced by polymorphonuclear neutrophils, induces chemokinesis, chemotaxis, and adhesion of these cells at sites of inflammation. Because neutrophil infiltration is a self-limited process, we hypothesized that oxidized lipid products of neutrophil-damaged tissue might inhibit leukotriene B4 biosynthesis, thereby preventing additional neutrophil infiltration and limiting peroxidative tissue damage. Erythrocyte ghosts exposed to a hydrogen peroxide-generating system served as a model of peroxidized tissue in inflammation and inhibited neutrophil leukotriene B4 production by 50% compared with unoxidized ghosts. Organic peroxides, including tert-butylhydroperoxide, peracetic acid, and linoleic hydroperoxide, resembling the product(s) of tissue membrane peroxidation in lipid solubility and catalase resistance, inhibited leukotriene B4 biosynthesis in a dose-dependent manner (50% inhibitory concentration of 3.9 microM compared to 530 microM for H2O2). Biosynthetic steps prior to the 5-lipoxygenase did not appear to be the site of inhibition. Likewise, the step after the 5-lipoxygenase, the leukotriene A4 hydrolase, was not primarily involved. Thus a possible mechanism for controlling the influx of neutrophils and their oxidative damage during inflammation may be inhibition of the 5-lipoxygenase by catalase-resistant lipid peroxides released by tissue membranes.

Acyltransferases↗
PubMed1992

Massive strychnine intoxication: serial blood levels in a fatal case.

A fatal case of strychnine intoxication is reported. The patient expired despite early aggressive management and prevention of metabolic complications. Serial blood levels are reported. In contrast to a previous report describing first order elimination kinetics, our data suggest that strychnine follows Michaelis-Menton elimination kinetics. The case illustrates the rapid, dramatic course of severe strychnine ingestions. A review of the toxicokinetics, mechanism of action and treatment of strychnine intoxication follows.

Autopsy↗
PubMed1991

Inhibition of arachidonic acid incorporation into erythrocyte phospholipids by peracetic acid and other peroxides. Role of arachidonoyl-CoA: 1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase.

To explore possible mechanisms of the arachidonic acid deficiency of the red blood cell membrane in alcoholics, we compared the effect of ethanol and its oxidized products, acetaldehyde and peracetic acid, with other peroxides on the accumulation of [14C]arachidonate into RBC membrane lipids in vitro. Incubation of erythrocytes with 50 mM ethanol or 3 mM acetaldehyde had no effect on arachidonate incorporation. Pretreatment of erythrocytes with 10 mM hydrogen peroxide, 0.1 mM cumene hydroperoxide or 0.1 mM t-butyl hydroperoxide had little effect on [14C]arachidonate incorporation in the absence of azide. However, pretreatment of cells with N-ethylmaleimide, 0.1 mM peracetic acid or performic acid, with or without azide, inhibited arachidonate incorporation into phospholipids but not neutral lipids. In chase experiments, peracetate also inhibited transfer of arachidonate from neutral lipids to phospholipids. To investigate a possible site of this inhibition of arachidonate transfer into phospholipids by percarboxylic acids, we assayed a repair enzyme, arachidonoyl CoA: 1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase (EC 2.3.1.23). As in intact cells, phospholipid biosynthesis was inhibited more by N-ethylmalemide and peracetic acid than by hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide. Peracetic acid was the only active inhibitor among ethanol and its oxidized products studied and may deserve further examination in ethanol toxicity.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1988

Relative susceptibility of lipids to peroxidation in intact erythrocytes.

Factors relating the site and metabolic state of erythrocyte lipids to their susceptibility to oxidative stress are poorly understood. Therefore red cell lipids were labeled with serum albumin-bound (carbon 14 [ul]) fatty acids in timed experiments. Susceptibility of sequentially labeled lipids to peroxidation in intact cells was determined by the percent conversion of the [14C]lipid to [14C]malondialdehyde (MDA). Other effects of alteration of incubation times could be ruled out by the equality of the conversion of total cell lipid to MDA. Polyunsaturated fatty acids but not other fatty acids served as MDA precursors. In 12 normal subjects the percentage of [14C]arachidonate converted to MDA after 15 minutes of labeling (3.08% +/- 0.69%) exceeded the percentage after 3 hours of labeling (2.01% +/- 0.83%) (paired, two-sided t test, p less than 0.0001). This susceptibility was caused by presence of rapidly labeled polyunsaturated fatty acids not extractable with defatted albumin (F2). Depletion of erythrocyte adenosine triphosphate before incubation favored accumulation of F2 and increased percentage conversion to MDA. Peroxidative susceptibility is determined by the metabolic state of the cell and the site of the peroxidized lipid within the intracellular pools.

Chromatography, High Pressure Liquid↗
PubMed1984

Comparison of hemoglobin Köln erythrocyte membranes with malondialdehyde-reacted normal erythrocyte membranes.

Splenectomized patients with hemoglobin (Hb) Köln have rigid RBCs with membrane polypeptide aggregates that are not dissociable with disulfide-reducing agents. Malondialdehyde (MDA) action on normal RBCs produced rigid RBCs with similar nondissociable aggregates. To test the hypothesis that Hb Köln RBC aggregates contained unsaturated MDA-type bonds, we reduced normal control RBC membranes, Hb Köln RBC membranes, and MDA-reacted membranes with [3H]NaBH4. Hb Köln RBC membranes and MDA-reacted membranes both had significantly more 3H incorporation than control membranes. Furthermore, 3H incorporation in both Hb Köln and MDA-treated membranes was located in the membrane polypeptide aggregates, presumably saturating the crosslinking bonds. After reaction of RBCs with [14C]MDA, the MDA label was similarly concentrated in the membrane polypeptide aggregates. Normal RBC membranes incubated with MDA were analyzed with and without reduction by NaBH4 prior to amino acid determination by high-performance liquid chromatography (HPLC). Reduction with NaBH4 after MDA treatment decreased the lysyl residues by 33% and the serine by 7% and increased by 10% the methionyl residues, but did not affect 12 other amino acids. Similar changes could be detected in NaBH4-reduced Hb Köln aggregates in methionine and serine content. MDA may also alter protein configuration, as evidenced by an increase in the protease susceptibility of membrane proteins from MDA-treated and Hb Köln RBCs. We conclude that Hb Köln RBC membranes, like MDA-treated membranes, have similar high molecular weight aggregates conferring decreased membrane deformability, [3H]NaBH4-reducible unsaturated bonds, changes in amino acid composition upon reduction, and protease-sensitive configurational changes.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

Increased adsorption of cytoplasmic proteins to the erythrocyte membrane in ATP-depleted normal and pyruvate kinase-deficient mature cells and reticulocytes.

How the metabolic defect of pyruvate kinase deficiency (PK(-)) accelerates red blood cell (RBC) destruction is not established, but may be related to RBC membrane abnormalities associated with altered cellular metabolism. Furthermore, it has been shown that PK(-) reticulocytes are especially sensitive to metabolic depletion. Therefore, we compared the membranes of reticulocyte-rich PK(-) RBC, both fresh and ATP depleted, with membranes of fresh and ATP depleted normal mature RBC and reticulocytes. There was no difference between the specific gravity (SG) of the membranes of normal mature RBC (SG 1.152 +/- 0.004) and membranes of reticulocyte-rich RBC from several anemias (SG 1.150 +/- 0.002). However, membranes from fresh, reticulocyte-rich PK(-) RBC were dense with SG of 1.165 +/- 0.004 which correlated with a corresponding increase of protein to lipid phosphorus ratio of 66 +/- 8 micrograms protein/micrograms lipid phosphorus (normal 52 +/- 6 micrograms/micrograms). The membrane density of PK(-) RBC was further increased when the PK(-) RBC ATP was depleted by anaerobic incubation (SG 1.188 +/- 0.004) or cyanide inhibition (SG 1.182 +/- 0.001). When ATP was depleted in normal RBC and in non-PK(-) reticulocytes, corresponding increases in membrane SG occurred. A distinctive 50,000 MW peptide is adsorbed from the cytoplasm to the membranes of reticulocytes (both normal and PK(-) when these cells were depleted of ATP. The increased membrane adsorption of cytoplasmic proteins by PK(-) RBC was not associated with increased RBC calcium uptake, sulfhydryl oxidation, or altered membrane protein phosphorylation. All the observed abnormalities of PK(-) RBC membranes could by reproduced by ATP depletion of reticulocyte-enriched non-PK(-) RBC.

Absorption↗
PubMed1983

Oxidant damage of the lipids and proteins of the erythrocyte membranes in unstable hemoglobin disease. Evidence for the role of lipid peroxidation.

Since unstable hemoglobins have been considered a source of reactive oxygen radicals, and oxidative membrane damage a prehemolytic event, we examined the erythrocyte membranes of six patients (three splenectomized) with hemoglobin Köln disease. In the hydrogen peroxide stress test, the patients' erythrocytes generated more than twice the malonyldialdehyde (a lipid peroxidative product) than control erythrocytes. Fluorescence spectra of lipid extracts of the patients' erythrocytes showed an excitation maximum at 400 nm and an emission maximum of 460 nm, characteristic of malonyldialdehyde lipid adducts. Two types of membrane polypeptide aggregates were found in the erythrocytes of the splenectomized patients. The first, which were dissociable by treatment with mercaptoethanol, contained disulfide-linked spectrin, band 3 and globin. The second, not dissociable by mercaptoethanol, had an amino acid composition similar to that of erythrocyte membranes and spectrin (unlike globin) and like that of aggregates produced by the action of malonyldialdehyde on normal erythrocyte membranes. Atomic absorption spectroscopy of hemoglobin Köln erythrocytes showed no increase in calcium content implying that these cross-links were not due to calcium-stimulated transglutaminase. Using a micropipette technique, we demonstrated that erythrocytes containing membrane aggregates from splenectomized patients were less deformable while aggregate-free erythrocytes from non-splenectomized patients had normal deformability. We conclude that the erythrocyte membranes in hemoglobin Köln disease show evidence of lipid peroxidation with production of malonyldialdehyde, and that the nondissociable membrane aggregates formed in this disease are likely cross-linked by malonyldialdehyde. Because the erythrocytes containing membrane aggregates from splenectomized patients with unstable hemoglobin disease show decreased membrane deformability, we hypothesize that this abnormality results in premature erythrocyte destruction in vivo.

Blood Proteins↗
PubMed1982

Erythrocyte membrane protein changes in glucose-6-phosphate dehydrogenase mutants with chronic hemolytic disease: an example of postsynthetic modification of membrane proteins.

1. G6PD mutants with CHD have decreased GSH, despite reticulocytosis, and increased membrane polypeptide aggregates. Aggregates increase logarithmically with decrease in RBC GSH. 2. These aggregates contain spectrin and can be depolymerized by disulfide reducing agents. Disulfide bonds between spectrin molecules and between the cytoskeleton and the cytoplasmic protein rigidify the red cell membrane and decrease RBC survival. 3. Direct oxidative damage of the RBC membrane, not Heinz body formation, explains the hemolytic anemia of G6PD mutants with CHD. This membrane damage may constitute a useful model system of oxidant-induced injury of other cells, and is an example of postsynthetic modification of membrane proteins by a nonmembrane gene.

Anemia, Hemolytic↗
PubMed1981

Amino acid sequence of p15 from avian myeloblastosis virus complex.

The complete amino acid sequence of the p15 gag protein from avian myeloblastosis virus (AMV) complex has been determined by sequential Edman degradation of the intact molecule and of peptide fragments generated by limited tryptic cleavage, cleavage with staphylococcal protease, and cyanogen bromide cleavage. AMV p15 is a single-chain protein containing 124 amino acids. The charged amino acids tend to be clustered in the primary structure. p15 contains a single cysteine at position 113 which may be essential for the p15 associated proteolytic activity. However, p15 shows no appreciable sequence homology with papain or other classical thiol proteases.

Amino Acid Sequence↗
PubMed1981

Polychlorinated biphenyls in clams and oysters from New Bedford Harbor, Massachusetts, March 1978.

Polychlorinated biphenyl (PCB) concentrations in clams (Mercenaria mercenaria) and oysters (Crassostrea virginica) from 17 stations of the western and New Bedford Harbor areas of Buzzards Bay, Massachusetts, clearly show that the New Bedford Harbor area is severely polluted. Up to 5 ppm PCBs (dry weight) were found in shellfish tissue. The most likely sources of the PCBs are chronic releases from two electrical component manufacturers in New Bedford. Close proximity of the shellfish to the source of input is indicated by a high relative abundance of the di-, tri-, and tetrachlorobiphenyls. The data suggest that the New Bedford Harbor area should be considered, along with the Hudson River and Chesapeake Bay, one of the major sources of PCB inputs to the northeastern United States coastal area.

Animals↗
PubMed1979

Red-cell-membrane polypeptide aggregates in glucose-6-phosphate dehydrogenase mutants with chronic hemolytic disease. A clue to the mechanism of hemolysis.

Red-cell membranes from patients with glucose-6-phosphate dehydrogenase deficiency were studied with polyacrylamide gel electrophoresis and gel filtration chromatography in sodium dodecyl sulfate. Membranes from each of five such patients who also had chronic hemolytic disease contained polypeptide aggregates within two molecular-weight ranges (4.4 X 10(5) and greater than 50 X 10(6) daltons). The 4.4 X 10(5) dalton aggregates were not detectable in red-cell membranes of patients with the enzyme deficiency without chronic hemolysis or in membranes from normal subjects, and the greater than 50 X 10(6) dalton aggregates were not found in appreciable amounts in these cells. The aggregates were dissociated by mercaptoethanol or dithiothreitol -- indicating that they were formed by intermolecular disulfide bonds. The polypeptide aggregates contained spectrin but not globin. Red-cell deformability was decreased in aggregate-containing cells. We postulate that the polypeptide aggregates are indicators of oxidant damage to the red-cell membrane, which results in decreased deformability and chronic hemolysis.

Adolescent↗
PubMed1979

Vitamin c status.

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Ascorbic Acid↗