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Biomedical subjects

J B Mudd

Publications and source records attributed to J B Mudd.

At least 19 recordsLinked to original sources
PubMed1997

Ozone does not react with human erythrocyte membrane lipids.

Ozone was applied to sealed red cell ghost membranes at the rate of 95 nmol/min for periods up to 20 min. Acetylcholine esterase, on the outer face of the membrane, was inhibited up to 20%. Glyceraldehyde-3-phosphate dehydrogenase, on the inner surface of the membrane, was inhibited up to 87%. These differences reflected the inherent susceptibilities of the two enzymes and the presence or absence of the membrane barrier. Analysis of the total lipids of the ozone-treated ghosts showed no significant change in the distribution of lipid classes and no significant change in the fatty acid composition. There was no significant change in the fatty acid composition of the phosphatidylcholine fraction. There was a slight increase in 18:0 and 20:2 + 20:3 in the phosphatidylethanolamine fraction. There was no change in the molecular species distribution of the phosphatidylcholine or the phosphatidylethanolamine fraction. There was no evidence for the formation of the phospholipid ozonolysis product, 1-acyl-2-(9-oxo-nonanyl) derivatives of glyceryl-phosphoryl choline. There was no decline in the amount of cholesterol in the lipids derived from ozone-treated red cell membranes. Treatment of red cell ghost membranes and, by implication, the plasma membrane of cells by ozone therefore oxidizes peripheral proteins before it oxidizes lipids.

Acetylcholinesterase↗
PubMed1997

Reaction of ozone with protein tryptophans: band III, serum albumin, and cytochrome C.

Treatment of red cell ghosts with ozone inhibited both AChE (marking the outside of the membrane) and G3PDH (marking the inside of the membrane). There was no change in tryptophan fluorescence of the ghosts after the ozone treatment. Band 3 protein was isolated from the ozone-treated ghosts. The protein was digested with trypsin to obtain water soluble peptides from the cytoplasmic N-terminal tail and the interhelical loops. Fluorescent peptides included GWVIHPLGLR from the outer loop between helices 7 and 8, and peptide WMEAAR from the N-terminal cytoplasmic tail. Neither one of these peptides was oxidized by ozone. This was true whether or not the ghosts were sealed. We conclude that the position of these tryptophans either in the membrane structure, or because of binding to other proteins in the cytoplasmic tail, protects them from oxidation by ozone. Treatment of horse heart cytochrome c with ozone did not change the absorbance spectrum in the heme region or the tryptophan absorbing region. HPLC of the ozone-treated cytochrome c showed that cytochrome c was being modified, indicated by a change in the elution time. Treatment of cytochrome c with ozone did not change the activity in the NADH-cytochrome c reductase assay. Digestion of the ozone-treated cytochrome c with trypsin gave peptides which demonstrated normal fluorescence. (Cytochrome c has abnormally low fluorescence, which is not changed by ozone exposure.) The peptides were separated by HPLC. The fluorescence of the tryptophan-containing peptide (GITWK) was not decreased by treatment of the cytochrome c by ozone. Amino acid analysis of the ozone-treated cytochrome c indicated that methionine was oxidized. We conclude that tryptophan in cytochrome c is protected from oxidation by ozone because of the interaction with the porphyrin ring. Bovine serum albumin and human serum albumin were treated with ozone. There was a monotonic decrease in tryptophan fluorescence in both cases. Digestion of BSA with trypsin produced two fluorescent peptides. The peptide FWGK was identified by coelution with the authentic peptide. The putative peptide AWSVAR was not the same as the chemically synthesized peptide. The peptide sequences FWGK and "AWSVAR" were both oxidized in ozone-treated bovine serum albumin, with no detectable discrimination. Tryptic digestion of the ozone-treated human serum albumin produced a single fluorescent peptide, which was oxidized by ozone. The putative peptide AWAVAR in the tryptic digest of HSA was distinct from chemically synthesized peptide. The oxidation of tryptophans in proteins by ozone is markedly influenced by position in tertiary structure, position in membrane structure, and by chemical interactions within the protein.

Animals↗
PubMed1997

Effect of ozone on metabolic activities of Escherichia coli K-12.

Escherichia coli K-12 cell suspensions in buffer were exposed to ozone at a concentration of 600 ppm. Measurements were made of cell viability, glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, lactate dehydrogenase, glutathione disulfide reductase, nonprotein sulfhydryl and total sulfhydryl compounds. Cell viability was not affected when E. coli K-12 was exposed to ozone for less than 10 minutes. The most sensitive parameter was glyceraldehyde-3-phosphate dehydrogenase followed by nonprotein sulfhydryl and total sulfhydryl compounds. Effects on malate dehydrogenase, lactate dehydrogenase and glutathione disulfide reductase were negligible. Cell survival and induction of lipid oxidation were also determined using two strains of E. coli K-12 (rec A, deficient in DNA repair and wild-type). The extent of membrane lipid oxidation correlated with cell viability in a dose-dependent manner and the survival curves of both strains showed similar sensitivity to ozone. The data suggest that the sulfhydryl group in the membrane is the primary target of ozone attack. Rec A DNA repair system does not appear to play a role in ozone resistance.

DNA Repair↗
PubMed1996

Reaction of ozone with enzymes of erythrocyte membranes.

Ozone is a widespread component of polluted air. It is the cause of many adverse effects on the lung such as decreased athletic performance and exacerbation of asthma. Ozone inactivated acetylcholine esterase (AChE) both in intact washed human erythrocytes and in ghosts prepared from the erythrocytes. This is consistent (a) with the location of AChE on the outer face of the membrane and (b) with the change in structure of AChE when amino acids were oxidized. The glyceraldehyde-3-phosphate dehydrogenase (G3PDH) of intact washed erythrocytes was unaffected by ozone. However, ozone severely inactivated G3PDH of ghosts, much more severely than AChE in ghosts. This result raised questions about the relative permeability of intact erythrocytes and ghosts and also about the inherent susceptibility of the two enzymes. Inhibition of the ozone-treated erythrocyte AChE with the competitive inhibitor trimethyl-(p-aminophenyl) ammonium chloride was measured. The inhibited enzyme had a higher K(M) and slightly lower Vmax than the control. Ozone did not affect the K(M) of the uninhibited enzyme but decreased the K(M) of the inhibited enzyme. Ozone decreased the Vmax of both the inhibited and the uninhibited enzyme. The K(I) was unchanged by the treatment with ozone. This suggested that the active site of the enzyme was not affected by ozone, but other features of the protein were changed by ozone. The effects of products of lipid ozonolysis [hydrogen peroxide, nonanal, and 1-palmitoyl-2-(9-oxononanyl)-sn-3-glycerophosphorylcholine (PN1PC)] were tested on the ghost preparations. The ozonolysis products were tested at concentrations equivalent to calculated amounts that could have been produced by ozone. Hydrogen peroxide had no effect on the G3PDH and AChE. Nonanal slightly increased the permeability of the ghost membrane, as judged by the increase in rate of G3PDH in the absence of Triton X-100, but did not inhibit enzyme activity. PN1PC increased the permeability of the ghosts, as judged by the increase in rate of G3PDH in the absence of Triton X-100. There was also an increase in the activity of G3PDH in the presence of Triton X-100. AChE was not inhibited by ozone in the presence or absence of Triton X-100.

Acetylcholinesterase↗
PubMed1993

Effect of ozone on metabolic activities of rat hepatocytes and mouse peritoneal macrophage.

Peritoneal macrophage from mice and isolated hepatocytes from rats were exposed to ozone. Ozone dosages were expressed as 0-5 nmol/10(6) cells. Measurements were made of viability, glucose transport, glutathione, glyceraldehyde-3-phosphate dehydrogenase, Mg-ATPase, Na/K-ATPase, and lipid synthesis. The most sensitive parameter was glyceraldehyde-3-phosphate dehydrogenase in the peritoneal macrophage. In hepatocytes both lipid synthesis and glyceraldehyde-3-phosphate dehydrogenase were sensitive to ozone. Effects on viability, glucose transport, Mg-ATPase, and Na/K-ATPase were small to negligible in both cell types.

Animals↗
PubMed1992

Reaction of ozone with glycophorin in solution and in lipid vesicles.

Glycophorin from human red blood cells was exposed to ozone in aqueous solution. Amino acid analysis of glycophorin exposed to a 10-fold molar excess of ozone showed that the only residue affected was methionine. Both methionine residues of the protein were oxidized to methionine sulfoxide. Exposure of the oxidized protein to cyanogen bromide caused no cleavage of the polypeptide chain. Glycophorin was incorporated into unilamellar lipid vesicles made from phosphatidylcholine. The protein containing vesicles were exposed to ozone in a 10-fold molar excess to the glycophorin. Gas chromatography of the methyl esters showed negligible change in the fatty acid composition. Amino acid analysis of the ozone-treated protein showed the oxidation of only one methionine residue per polypeptide chain to methionine sulfoxide. Ghosts of human erythrocytes were exposed to ozone. Cyanogen bromide treatment of the oxidized glycophorin yielded fragments showing that the only methionine residue oxidized by ozone was residue 8. These results indicate that in this membrane model (a) amino acid is more susceptible to ozone than is the lipid, and (b) amino acids external to the membrane are more susceptible than those in the polypeptide chain spanning the membrane.

Amino Acids↗
PubMed1990

Biosynthesis of sulfoquinovosyldiacylglycerol in higher plants : use of adenosine-5'-phosphosulfate and adenosine-3'-phosphate 5'-phosphosulfate as precursors.

Adenosine-5'-phosphosulfate (APS) and adenosine-3'-phosphate 5'-phosphosulfate (PAPS) have been used as precursors of sulfoquinovosyldiacylglycerol (SQDG) in intact chloroplasts incubated in the dark. Competition studies demonstrated APS was preferred over PAPS and SO(4) (2-). Rates of SQDG synthesis up to 3 nanomoles per milligram of chlorophyll per hour were observed when [(35)S]APS and appropriate cofactors were supplied to chloroplasts incubated in the dark. The pH optimum for utilization of APS was 7.0. The incorporation was linear for at least 30 minutes. ATP and UTP stimulated the incorporation of sulfur from APS into SQDG, but the most stimulatory additions were DHAP and glycerol-3-P. The concentration curve for APS showed a maximum at 20 micromolar in the absence of DHAP and 30 micromolar in the presence of DHAP. The optimum concentration of DHAP for conversion of APS into SQDG was 2 millimolar. Rates of synthesis up to 4 nanomoles per milligram of chlorophyll per hour were observed when [(35)S]PAPS was the sulfur donor and appropriate cofactors were supplied to chloroplasts. Optimal rates for conversion of sulfur from PAPS into SQDG occurred with concentrations of DHAP between 5 and 10 millimolar. DHAP was by far the most effective cofactor, although ATP and UTP also stimulated the utilization of PAPS for SQDG biosynthesis. In general, triose phosphates, including glycerol-3-P were not effective cofactors for SQDG biosynthesis.

Journal Article↗
PubMed1987

Chilling sensitivity of cucumber cotyledon protoplasts and seedlings.

Cucumber (Cucumis sativus L.) seedlings are more sensitive to chilling stress when transferred to low temperature from the night cycle than from the day cycle. However, greater damage occurs when chilling is carried out in light than in dark. Freshly isolated protoplasts are extremely sensitive to damage when chilled at 4 degrees C in light, but suffer significantly less injury when chilled in dark. If freshly isolated protoplasts are pre-chill conditioned at 27 degrees C in either light or dark for a few hours prior to exposure to various chilling stresses, subsequent chilling damage is markedly reduced. Damage to chilled protoplasts also is reduced if cultures are placed in dark instead of light immediately following removal from low temperature. Experiments utilizing the cell wall synthesis inhibitor, dichlorobenzonitrile, showed that cell wall regeneration during the pre-chill conditioning period at 27 degrees C does not appear to be associated with the enhanced chilling tolerance observed in these cultures. The results obtained in this investigation suggest that the physiological properties of cucumber cotyledon protoplasts accurately reflect those of intact seedlings, and hence provide a good system for studies into the mechanism of chilling damage in plants.

Journal Article↗
PubMed1987

Biosynthesis of Sulfoquinovosyldiacylglycerol in Higher Plants: The Incorporation of SO(4) by Intact Chloroplasts in Darkness.

Intact spinach chloroplasts incorporated (35)SO(4) (2-) into sulfoquinovosyldiacylglycerol in the dark at rates equivalent to those previously reported for illuminated chloroplasts provided that either ATP itself or an ATP-generating system was added. No additional reductant was necessary for SQDG synthesis by chloroplasts. The optimal concentration of ATP was between 2 and 3 millimolar. Rates of synthesis up to 2.6 nanomoles per milligram chlorophyll per hour were observed. UTP, GTP, and CTP could not substitute for ATP. Incubation of UTP with ATP (1:1) stimulated synthesis of sulfoquinovosyldiacylglycerol. No additional stimulation of the reaction was observed upon addition of other nucleoside triphosphates with ATP. For the generation of ATP in the chloroplast, addition of dihydroxyacetone phosphate alone did not promote synthesis of sulfoquinovosyldiacylglycerol, but in combination with inorganic phosphate and oxaloacetate, rates of synthesis up to 3.2 nanomoles per milligram chlorophyll per hour were observed. Dark synthesis was optimal in the presence of 2 millimolar dihydroxyacetone phosphate, 2 millimolar oxaloacetate, and 1 millimolar KH(2)PO(4).

Journal Article↗
PubMed1985

Biosynthesis of sulfoquinovosyldiacylglycerol in higher plants: the origin of the diacylglycerol moiety.

The positional distribution of fatty acids in chloroplast polar lipids and phosphatidylcholine from leaves of four plants has been measured in order to determine the origin of the diacylglycerol (DAG) moieties of each lipid. In spinach and tobacco, the DAG of sulfoquinovosyldiacylglycerol (SQDG), monogalactosyldiacylglycerol (MGDG) and digalactosylglycerol (DGDG) were derived partly from the chloroplast and partly from the cytoplasm. The contribution of the chloroplast pathway differed for each lipid, but in both plants the proportion of a lipid derived from that pathway was in the order SQDG greater than MGDG greater than DGDG. In contrast, all the DAG moieties of the three glycolipids of wheat and cucumber were derived from the cytoplasm. The DAG moiety of chloroplast phosphatidylglycerol was synthesized in the chloroplast in all four plants.

Chloroplasts↗
PubMed1985

Biosynthesis of sulfoquinovosyldiacylglycerol in higher plants: the incorporation of 35SO4 by intact chloroplasts.

Isolated spinach chloroplasts have been found to incorporate 35SO4 into the plant sulfolipid, sulfoquinovosyldiacylglycerol, at rates of up to 700 pmol mg chlorophyll-1 h-1. The reaction is light-dependent, requires that the chloroplasts be intact, and is slightly stimulated by ATP and UTP. UDP-galactose inhibits the formation of sulfoquinovosyldiacylglycerol, presumably by competing for the diacylglycerol pool. The rates of synthesis observed are sufficient to conclude that the chloroplast is autonomous with respect to the synthesis of sulfoquinovose, the headgroup moiety of sulfoquinovosyldiacylglycerol. No evidence could be obtained to support the concept that the proposed sulfoglycolytic pathway is the biosynthetic route for sulfoquinovose formation.

Adenosine Triphosphate↗
PubMed1985

Effect of Chilling Temperatures upon Cell Cultures of Tomato.

The effect of chilling temperatures upon cell cultures of tomato (Lycopersicon esculentum Mill cv ;VF36,' and cv ;VFNT Cherry,' and L. hirsutum Humb. & Bonpl.) was tested. Doubling times for L. esculentum were 2 to 3 days at 28 degrees C, and 3 to 8 days at 12 degrees C. No growth was observed at 8 degrees C, indicating an abrupt limit to growth between 8 and 12 degrees C. Fluorescein diacetate staining indicated that 80 to 90% of the cells were alive when cells were maintained at 8 degrees C for up to 2 weeks. When cultures kept at 8 degrees C for up to 30 days were transferred to 28 degrees C, growth resumed quickly, and at a rate virtually identical to that for unchilled cells. Similar results were found for cells maintained at 0 degrees C, and for cells of ;VFNT Cherry' and of L. hirsutum. Under certain conditions, cultures slowly doubled in fresh weight and cell volume at 8 or 9 degrees C but additional growth at 8 degrees C did not occur, nor could growth be maintained by subculture at 8 or 9 degrees C. The results are contrary to reports that cell cultures of tomato die when exposed to temperatures below 10 degrees C for 1 or 2 weeks. Our observations indicate that chilling temperatures quickly inhibit growth of tomato cells, but do not kill them.

Journal Article↗
PubMed1985

Acclimation to low temperature by microsomal membranes from tomato cell cultures.

Sealed vesicles were prepared from microsomal membranes from cell suspension cultures of tomato (Lycopersicon esculentum Mill cv VF36). ATP-dependent proton transport activity by the vesicles was measured as quenching of fluorescence of acridine orange. Measurements of proton transport were correlated with the activity of a nitrate-inhibitable ATPase. The initial rate of proton influx into the vesicles was strongly temperature dependent with a Q(10) of 2 and a maximum rate near 35 degrees C. The data suggest that passive permeability did not increase at chilling temperatures but did increase rapidly with temperatures above 30 degrees C. A comparison was made between membranes from cell cultures grown at 28 degrees C and 9 degrees C. The temperature optimum for proton transport broadened and shifted to a lower temperature range in membranes from cells maintained at 9 degrees C.

Journal Article↗
PubMed1985

Epicuticular Lipid Accumulation on the Leaves of Lycopersicon pennellii (Corr.) D'Arcy and Lycopersicon esculentum Mill.

A comparison was made of epicuticular lipid accumulation on leaves of Lycopersicon pennellii and Lycopersicon esculentum Mill. cv VF36 from 5 to 16 weeks of age. Epicuticular lipids were a small fraction of the leaf dry weight (0.16%) of 5-week-old ;VF36', and increased to only 0.96% of the leaf dry weight after an additional 12 weeks of growth. In contrast, leaves from 5-week-old and 17-week-old L. pennellii plants had, respectively, 0.94% and 19.9% of their total dry weight in epicuticular lipid. Lipid accumulation was not affected by drought stress. Leaf position appears to influence the amount of lipid on the leaf surface. A glycolipid appears to be exuded from the terminal cell of glandular trichomes found on the leaves, stems, peduncles, calyxes, and fruits of L. pennellii.

Journal Article↗
PubMed1985

Phosphatidylglycerol synthesis in pea chloroplasts: pathway and localization.

Isolated intact pea chloroplasts synthesized phosphatidylglycerol from either [(14)C]acetate or [(14)C]glycerol 3-phosphate. Both time-course and pulse-chase labeling studies demonstrated a precursor-product relationship between newly synthesized phosphatidic acid and newly synthesized phosphatidylglycerol.The synthesis both of CDP-diacylglycerol from exogenous phosphatidic acid and CTP, and of phosphatidylglycerol from exogenous CDP-diacylglycerol and glycerol 3-phosphate, could be assayed in fractions obtained from disrupted chloroplasts. Moreover, the enzymes catalyzing these reactions were localized in the inner envelope membrane. Exogenous phosphatidic acid was incorporated into phosphatidylglycerol, but only following its incorporation into CDP-diacylglycerol. Finally, radio-active phosphatidic acid synthesized in the envelope membranes from [(14)C]palmitoyl-ACP and 1-oleoyl-glycerol 3-phosphate was sequentially incorporated into labeled CDP-diacylglycerol and phosphatidylglycerol upon the addition of appropriate substrates and cofactors. Thus, we have demonstrated that (a) the synthesis of phosphatidylglycerol in chloroplasts occurs by the pathway: phosphatidic acid --> CDP-diacylglycerol -->--> phosphatidylglycerol, and (b) phosphatidylglycerol synthesis is located in the inner envelope membrane.

Journal Article↗
PubMed1985

The Biosynthesis of the Pyrenocines in Cultures of Pyrenochaeta terrestris.

[(14)C]Acetate, [(14)C]formate, and methyl[(14)C]methionine all serve as precursors of pyrenocines A, B, and C when added to cultures of Pyrenochaeta terrestris (Hansen) Gorenz, Walker, and Larson, the pathogen responsible for disease known as pink root of onion (Allium cepa L.). This information supports the hypothesis that these metabolites are methyl-substituted polyketides in origin. Pyrenocine A arises from acetate via uncharacterized intermediates and is subsequently converted to pyrenocine B. The biosynthetic role of pyrenocine C remains uncertain.

Journal Article↗
PubMed1984

The reaction of ozone with glyceraldehyde-3-phosphate dehydrogenase.

Inactivation of glyceraldehyde-3-phosphate dehydrogenase (GPDH) by ozone can be correlated with oxidation of the active-site -SH residue. Oxidation of peripheral -SH groups, and tryptophan, methionine, and histidine residues occurs concomitantly, but loss of activity depends solely on active-site oxidation. Inactivation is only slightly reversible by dithiothreitol. Kinetic studies show that inhibition of GPDH by ozone mimics noncompetitive inhibition and is characterized as irreversible enzyme inactivation. Analysis of products resulting from ozone oxidation of glutathione suggests that cysteic acid is the product of protein-SH oxidation. Despite oxidation of the active-site -SH , no significant decrease in the Racker band absorbance occurs. This is explained by the appearance of a new chromophore in this region of the absorbance spectrum. Increased absorbance at 322 nm following ozone treatment indicates that tryptophan is converted quantitatively to N-formylkynurenine. When the active-site -SH is reversibly blocked by tetrathionate, enzyme activity is completely recoverable following reaction of the derivatized enzyme with a 1.3X excess of ozone over enzyme monomer. Activity is fully recovered despite the oxidation of peripheral -SH, tryptophan, and histidine residues. Circular dichroism spectra of ozone-treated enzyme show that reaction of GPDH with up to a threefold excess of ozone over enzyme monomer results in no significant disruption of protein secondary structure. Spectra in the near-uv show distinct changes that reflect tryptophan oxidation.

Amino Acids↗