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A Stern

Publications and source records attributed to A Stern.

At least 181 records · Page 10Linked to original sources
PubMed1987

The control mechanism of opacity protein expression in the pathogenic Neisseriae.

The expression of Neisseria gonorrhoeae opacity protein shows frequent phase transitions. The genome contains at least twelve copies of the opa gene. Each copy is complete and different from most of the others in certain hypervariable regions. All opa genes are constitutively transcribed. Part of the leader peptide of all Op's is encoded by repetitive CTTCT pentameric units, the so-called coding repeat (CR). The number of repeat units found in the genes and mRNA is subject to frequent and precise changes. Such changes affect the expression of individual opa genes at the translational level. This control mechanism is common also to the class 5 proteins of N. meningitidis and the Op-related proteins of N. lactamica.

Antigens, Bacterial↗
PubMed1987

Common mechanism controlling phase and antigenic variation in pathogenic neisseriae.

The expression of the Neisseria gonorrhoeae opacity protein (Op, protein II), a major antigenic determinant of the outer membrane, is subject to frequent phase transitions. At least nine expression loci (opaE) are involved in the production of a large number of serologically distinct Op types. Using opa-specific oligonucleotides as probes in genomic blots, we detect Op-related gene sequences (opr) in N. meningitidis as well as in N. lactamica. DNA sequence analysis of such opr genes derived from N. meningitidis reveals distinct regions of homology with gonococcal opa E genes. As shown in the immunoblot, the proteins encoded by opa and opr are serologically related. Like the opaE genes, the 5'-coding sequences of the opr genes include a repetitive sequence composed of pentameric CTCTT units. The number of these coding repeat (CR) units is variable. This finding, together with the observation that all opr genes are constitutively transcribed, regardless of the status of protein production, suggests a translational control mechanism identical to that of the opa genes in gonococci. The related structures and control mechanisms of opa and opr genes imply a general significance of their gene products for the pathogenic character of the investigated Neisseria species.

Amino Acid Sequence↗
PubMed1987

Mechanism of kainate toxicity to cerebellar neurons in vitro is analogous to reperfusion tissue injury.

The neuroexcitotoxin kainate has been used as a selective lesioning agent to model the etiology of a number of neurodegenerative disorders. Although excitotoxins cause susceptible neurons to undergo prolonged or repeated depolarization, the proximate metabolic pathology responsible for neuronal necrosis has remained elusive. We report here that kainate-induced death of cerebellar neurons in culture is prevented by inhibiting the enzyme xanthine oxidase, a cellular source of cytotoxic superoxide radicals (O2-.). Moreover, neurons are also protected from excitotoxin-induced death by the addition to the culture medium of either superoxide dismutase or mannitol, which scavenge superoxide and hydroxyl radicals, respectively, or serine protease inhibitor, which forestalls formation of xanthine oxidase. These findings indicate that excitotoxin-induced neuronal degeneration is mediated by superoxide radicals generated by xanthine oxidase, a mechanism partially analogous to that proposed for tissue damage seen upon reperfusion of ischemic tissues.

Allopurinol↗
PubMed1987

Extraluminal compression of an aortic graft simulating recoarctation.

Restenosis rarely develops after surgical correction of coarctation of the aorta in adults. Late morbidity is usually related to residual hypertension or progressive aortic valve disease. A patient in whom symptoms and signs of recurrent coarctation developed 19 years after initial graft repair is described. Dehiscence of the original silk suture line was found at operation. Extensive thrombus had produced graft compression. Milder hypertension persisted in the postoperative period despite relief of the aortic obstruction.

Adult↗
PubMed1987

Human red cells scavenge extracellular hydrogen peroxide and inhibit formation of hypochlorous acid and hydroxyl radical.

The ability of intact human red cells to scavenge extracellularly generated H2O2 and O2-, and to prevent formation of hydroxyl radicals and hypochlorous acid has been examined. Red cells inhibited oxidation of ferrocytochrome c by H2O2. Cells treated with aminotriazole no longer inhibited, indicating that protection was almost entirely due to intracellular catalase. Contribution by the GSH system was slight, and apparent only with low H2O2 concentrations when catalase was inhibited by aminotriazole. The cells were about a quarter as efficient at inhibiting cytochrome c oxidation as an equivalent concentration of purified catalase. No inhibition of O2(-)-dependent reduction of ferricytochrome c or nitroblue tetrazolium was observed, although extracted red cell superoxide dismutase inhibited nitroblue tetrazolium reduction at one fortieth the concentration of that in the cells. Red cells efficiently inhibited deoxyribose oxidation by hydroxyl radicals generated from H2O2, O2- and Fe(EDTA), and myeloperoxidase-dependent oxidation of methionine to methionine sulfoxide by stimulated neutrophils. Most of the red cell inhibition of hydroxyl radical production, and all the inhibition of methionine oxidation, was prevented by blocking intracellular catalase with aminotriazole. Thus red cells are able to efficiently scavenge H2O2, but not O2-, produced in their environment, and to inhibit formation of hydroxyl radicals and hypochlorous acid. They may therefore have an important role in extracellular antioxidant defense.

Amitrole↗
PubMed1987

Cholesteryl octanoate breath test. Preliminary studies on a new noninvasive test of human pancreatic exocrine function.

A new breath test for noninvasive assessment of pancreatic exocrine function in humans was developed. The test is based on the hydrolysis of cholesteryl-[1-14C]octanoate by pancreatic carboxyl ester lipase (cholesterol esterase) with subsequent absorption and hepatic metabolism of the liberated octanoate to 14CO2. The rate at which 14CO2 appears in breath appeared to be proportional to the rate of hydrolysis. The substrate is administered as a gum acacia stabilized emulsion of vegetable oil (18 g) containing cholesteryl octanoate (2 g; 4.4 microCi) dispersed in a 500-ml isotonic meal. Tests were performed in 6 healthy volunteers and 11 patients with pancreatic disease with varying degrees of steatorrhea. In healthy subjects, 14CO2 output was rapid with peak output occurring at 60-90 min in all subjects; cumulative output in 4 h averaged 30%. Duplicate studies indicated that the time-course of 14CO2 recovery was reproducible. The pattern of 14CO2 output in patients with pancreatic disease varied widely. Patients without steatorrhea (fecal fat less than or equal to 7 g/day) or with mild steatorrhea (fecal fat 7-11 g/day) had normal or near normal patterns of 14CO2 output, whereas patients with moderate or severe steatorrhea (fecal fat greater than 11 g/day) expired 14CO2 at a rate one-third to one-tenth that of the healthy volunteers. Addition of pancreatic enzyme supplementation to the test meal increased 14CO2 output in 6 of 6 patients with moderate or severe steatorrhea, suggesting that the activity of pancreatic carboxyl ester lipase was rate limiting in these patients. In an additional study in a healthy volunteer, 14CO2 and 13CO2 were measured simultaneously in breath after ingestion of a test meal containing cholesteryl-[1-13C]octanoate and 14C-octanoate. 14CO2 was expired more rapidly than 13CO2, suggesting that hydrolysis of the substrate may also be rate limiting in healthy volunteers. These studies indicate that severe pancreatic exocrine dysfunction can be detected with a simple breath test in 60-90 min.

Adult↗
PubMed1986

Phenylhydrazine-induced changes in erythrocyte membrane surface lipid packing.

Phenylhydrazine-induced oxidative damage in red cells results in increased binding of merocyanine 540, a fluorescence probe sensitive to changes in lipid packing. Fluorescence polarization studies with diphenylhexatriene did not reveal major changes in order parameters both in intact red cells and lysates treated with phenylhydrazine. These fluorescence studies indicate that major changes are observed in membrane lipids. Analytical studies of membrane phospholipids revealed a significant decrease in phosphatidylethanolamine. The results of the fluorescence and lipid studies, taken in association with our previously reported findings on spectrin and other cytoskeletal protein degradation in red cells exposed to phenylhydrazine, suggests that degradation of cytoskeleton membrane proteins is also responsible for changes in the lipid bilayer surface of the red cell membrane.

Diphenylhexatriene↗
PubMed1986

Opacity genes in Neisseria gonorrhoeae: control of phase and antigenic variation.

The chromosome of N. gonorrhoeae contains several complete expression genes coding for variant opacity proteins. DNA sequence analysis of two opacity genes derived from the same locus (opaE1) of two isogenic gonococcal variants reveals common and variable regions in these genes. Genomic blotting experiments using synthetic probes suggest gene conversion as a principle for the assembly of variant sequence information in opacity genes. The 5' region of opacity genes is composed of identical pentameric pyrimidine units (CTCTT) encoding the hydrophobic portion of the opacity leader peptide. This coding repeat is variable in a given locus with respect of the number of pentameric units. While all expression loci in a single cell are constitutively transcribed, the production of opacity proteins is determined by the coding repeat sequence on the translational level.

Amino Acid Sequence↗
PubMed1985

The hydrolytic autoxidation of 1,4-naphthoquinone-2-potassium sulphonate: implications for 1,4-naphthoquinone-2-potassium sulphonate-induced oxidative stress in the red blood cell.

1,4-Naphthoquinone-2-potassium sulphonate (NQKS) undergoes an autoxidation reaction in aqueous solution under physiological conditions to produce 3-hydroxy-1,4-naphthoquinone-2-potassium sulphonate (NQKS-OH). Intermediates of dioxygen reduction, superoxide radicals, hydrogen peroxide and hydroxyl radicals, are also detected. The kinetics of the autoxidation of NQKS show a first order dependence on NQKS and hydroxide ion concentration and a zeroth order dependence on oxygen concentration. For the rate equation r = -d[O2]/dt = kappa obs., [NQKS] [-OH], kappa obs. = (6.4 +/- 0.6) X 10(2) M-1s-1 at 37 degrees C. Hydroxide ion attack on NQKS appears to be the rate determining step. The reaction may be conveniently described as a 'hydrolytic autoxidation'. The hydrolytic autoxidation of NQKS occurs in NQKS-treated red blood cells; the hydroxylated quinone NQKS-OH is produced and hydroxyl radical formation is stimulated. The importance of this reaction in NQKS-induced oxidative stress in red blood cells is discussed. The hydrolytic autoxidation of quinones bearing one or more unsubstituted (hydrogen) positions on the quinone centre is a novel mechanism by which such quinones may induce oxidative stress in cellular systems.

Cyclic N-Oxides↗
PubMed1985

Spectrin degradation in intact red blood cells by phenylhydrazine.

The effects of phenylhydrazine on intact red cells and on red cell ghost membrane proteins were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In intact red cells 1 mM phenylhydrazine induced a marked decrease in intensity of the alpha- and beta-bands of spectrin without the formation of high molecular weight materials. Phenylhydrazine was also responsible for cross-linking of hemoglobin, which is apparent by the appearance of two new broad bands on the gel. Membrane glycoproteins were unaffected. Electrophoretic patterns of cytoskeletal proteins from phenylhydrazine-treated red cells obtained on two-dimensional SDS-polyacrylamide gels and stained with Coomassie blue or fluorescently labeled with monobromobimane indicated the presence of a new band between bands 4.2 and 5 at 60-65 kilodaltons (K). An immunoelectrophoretic blotting procedure utilizing polyclonal IgG antibodies for alpha- and beta-spectrin of the red cell cytoskeletal proteins revealed that the band observed at 60-65 K in the two-dimensional SDS-PAGE studies reacted with the antibodies. The presence or absence of glucose in the incubation medium and modification of oxyhemoglobin to met- or carboxyhemoglobin in the red cells did not protect the phenylhydrazine-mediated degradation of the major cytoskeletal proteins. Metal chelators and antioxidants had no effect on membrane protein changes. Ghost red cell proteins did not undergo changes at 1 mM phenylhydrazine in the presence or absence of hemoglobin, although at 5 mM phenylhydrazine the appearance of a faint high molecular weight band was observed. These results indicate that spectrin degradation without significant polymerization can be induced by phenylhydrazine.

Antioxidants↗
PubMed1985

Mammalian fatty acid synthetase is a structurally and functionally symmetrical dimer.

We have explored a comprehensive experimental approach to determine whether the two condensing-enzyme active centers of the mammalian fatty acid synthetase are simultaneously functional. Our strategy involved utilization of trypsinized fatty acid synthetase, which is a nicked homodimer composed of two pairs of 125 + 95-kDa polypeptides. These core polypeptides lack the chain-terminating thioesterase domains but retain all other functional domains of the native enzyme and can assemble long-chain acyl moieties at a rate equal to that of the native enzyme. The 4'-phosphopantetheine content of these enzyme preparations, estimated from the amount of beta-alanine present, from the amount of taurine formed by performic acid oxidation and from the amount of carboxymethylcysteamine formed by alkylation with iodo[2-14C]acetate, was typically 0.86 mol/mol 95-kDa polypeptide. The stoichiometry of long-chain acyl-enzyme synthesis, measured with radiolabeled precursors, indicated that 0.84 mol acyl-chains were assembled/mol 95-kDa polypeptide. When the small amount of apoenzyme present is taken into account, this stoichiometry translates to 1.94 acyl chains per holoenzyme dimer. The 125-kDa polypeptide of one subunit could be cross-linked to the 95-kDa polypeptide of the other subunit by 1,3-dibromo-2-propanone yielding a single molecular species of 220 kDa. Cross-linking was accompanied by a loss of condensing-enzyme activity. This result is consistent with a structurally symmetrical model for the animal fatty acid synthetase [J.K. Stoops and S.J. Wakil (1981) J. Biol. Chem. 256, 5128-5133] in which the juxtaposed 4'-phosphopantetheine and cysteine thiols of opposing subunits that form the two potential catalytic centers for condensing activity are readily susceptible to cross-linking. Both half-maximal cross-linking and 50% inhibition of activity were observed with 1 mol 1,3-dibromo-2-propanone bound/mol enzyme. After assembly of long-chain acyl moieties on the 4'-phosphopantetheine residues, no vacant condensing-enzyme active sites were demonstrable either by cross-linking with 1,3-dibromo-2-propanone or by formation of carboxymethylcysteamine on treatment with iodoacetate. These results are consistent with a structurally and functionally symmetrical model for the mammalian fatty acid synthetase in which the two condensation sites are simultaneously active.

Acetone↗
PubMed1985

Modification of mammalian fatty acid synthetase activity by NADP.

Experiments are described which show that the mammalian fatty acid synthetase, in the presence of NADP, synthesizes stoichiometric amounts of enzyme-bound acetoacetyl moieties. The acetoacetyl moieties can neither undergo the normal transfer reaction to a CoA acceptor, nor participate in the normal reaction sequence once NADPH is made available. Our results indicate that, since it is the product of the condensation reaction which accumulates on the inhibited enzyme, the previously held view that NADP inhibits the condensation step in fatty acid synthesis is probably incorrect.

Acetyl Coenzyme A↗
PubMed1985

Stoichiometry of substrate binding to rat liver fatty acid synthetase.

Two rat liver fatty acid synthetase preparations, containing 1.6 and 2.0 mol of 4'-phosphopantetheine/mol of synthetase, showed specific activity of 2006 and 2140 nmol of NADPH oxidized/min per mg of protein respectively. The two synthetase preparations could be loaded with either 3.3-4.4 mol of [1-14] acetate or 2.9-3.7 mol of [2-14C]malonate, by incubation with either [1-14C] acetyl-CoA or [2-14C]malonyl-CoA. The 4'-phosphopantetheine site could be more than 90% saturated and the serine site about 80% saturated with malonate derived from malonyl-CoA. However, with acetyl-CoA as substrate, binding at both the 4'-phosphopantetheine and cysteine thiol sites did not reach saturation. We interpret these results to indicate that, whereas the equilibrium constant for transfer of substrates between the serine loading site and the 4'-phosphopantetheine site is close to unity, that for transfer of acetyl moieties between the 4'-phosphopantetheine and cysteine sites favours formation of the 4'-phosphopantetheine thioester. Thus, despite the apparent sub-stoichiometric binding of acetate, the results are consistent with a functionally symmetrical model for the fatty acid synthetase which permits simultaneous substrate binding at two separate active centres.

Acetates↗
PubMed1985

Red blood cell oxidative metabolism induced by hydroxypyruvaldehyde.

Hydroxypyruvaldehyde is a substrate for the red cell glyoxalase system. It was metabolized by glyoxalase I with reduced glutathione to S-glyceroyl glutathione which was subsequently enzymatically hydrolyzed to reduced glutathione and glycerate by glyoxalase II. There was a competing spontaneous reaction of hydroxypyruvaldehyde with oxygen, which produced hydrogen peroxide, inducing oxidative metabolism in hydroxypyruvaldehyde-treated red cells. The incubation of red cells with hydroxypyruvaldehyde produced a stimulation in the flux of glucose oxidized through the hexose monophosphate shunt pathway, a stimulation in lactate production with a decrease in pyruvate production in the Embden-Meyerhoff pathway, an oxidation of reduced pyridine nucleotides and reduced glutathione to their oxidized cogeners, and changes in the oxidative status of hemoglobin. Overall, the majority of hydroxypyruvaldehyde consumption in red cell suspensions appeared to occur via non-oxidative routes, e.g. glyoxalase and/or 2-ketoaldehyde dehydrogenase, and non-enzymic protein binding. Although the observed oxidative metabolism induced by hydroxypyruvaldehyde in red cells was not severe (reduced glutathione levels in hydroxypyruvaldehyde-treated red cells were ca. 80% of the control values in untreated cells), the oxidative effects may be important in red cell ageing processes.

Adult↗