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Biomedical subjects

H Sies

Publications and source records attributed to H Sies.

At least 199 records · Page 11Linked to original sources
PubMed1992

Cloning, sequencing and characterization of the human alpha glutathione S-transferase gene corresponding to the cDNA clone pGTH2.

The human Alpha glutathione S-transferase gene corresponding to the human liver cDNA clone pGTH2 was isolated from a cosmid genome library. The gene, represented by the clone cosGTH2, spans nearly 12 kb and contains seven exons. The intron/exon borders conform to the standard rules, and an open reading frame is present, starting at position 67 in exon 2, the double-stop codon being at position 733 in exon 7. Exons 1, 2 and 7 differ in length from the known rat gene coding for the Ya enzyme. A 209 bp 5'-upstream region contains TATA and CAT boxes and, in addition, motifs for Sp1-, NF1- and HNFI-binding factors. Clone cosGTH2 represents the less basic subunit, alpha y, of two Alpha glutathione S-transferase subunits (alpha x and alpha y) expressed in liver, which is identical with the kidney subunit alpha 2.

Amino Acid Sequence↗
PubMed1992

Antioxidant activity of the pyridoindole stobadine in liposomal and microsomal lipid peroxidation.

Stobadine, a pyridoindole derivative, is an efficient inhibitor of lipid peroxidation in phosphatidylcholine liposomes and in rat liver microsomes treated with iron/ADP/NADPH as pro-oxidant. Accumulation of thiobarbituric acid-reactive substances (TBARS) or low-level chemiluminescence were taken as a measure of lipid peroxidation and 5 microM stobadine doubled the duration of the lag phase preceding the onset of rapidly increasing chemiluminescence. Inhibition of lipid peroxidation was not observed with tocopherol-deficient microsomes, suggesting that the antioxidant effect of stobadine depends on vitamin E in the membrane. The cis(-) isomer was most effective, with the cis(+) and trans(rac) as well as dehydro- or acetyl derivatives being less active. In liposomes, the presence of reductant (NADPH or ascorbate) protects from the loss of stobadine.

Adenosine Diphosphate↗
PubMed1992

De novo methylation of transfected CAT gene plasmid constructs in F9 mouse embryonal carcinoma cells.

To study the formation of DNA methylation patterns, plasmids containing promoters of different strengths in front of the bacterial chloramphenicol acetyltransferase reporter gene were transfected into F9 mouse embryonal carcinoma cells. Methylation of the integrated plasmids as well as copy numbers and activities of the reporter gene were determined for individual cell clones. The methylation pattern of the integrated plasmids was found to be determined by properties of the DNA sequence itself. In contrast, the specific methylation patterns were invariant with respect to integration site, copy number and arrangement of the integrates; methylation did also not correlate with transcriptional activity of the different promoters. Certain promoter regions may therefore contain signals recognized by the de novo methylation activity in embryonal carcinoma cells.

Animals↗
PubMed1992

Low- and high-Km transport of dinitrophenyl glutathione in inside out vesicles from human erythrocytes.

Kinetic studies on the low- and high-Km transport systems for S-2,4-dinitrophenyl glutathione (DNP-SG) present in erythrocyte membranes were performed using inside-out plasma membrane vesicles. The high-affinity system showed a Km of 3.9 microM a Vmax of 6.3 nmol/mg protein per h, and the low-affinity system a Km of 1.6 mM and a Vmax of 131 nmol/mg protein per h. Both uptake components were inhibited by fluoride, vanadate, p-chloromercuribenzoate (pCMB) and bis(4-nitrophenyl)dithio-3,3'-dicarboxylate (DTNB). The low-Km uptake process was less sensitive to the inhibitory action of DTNB as compared to the high-Km process. N-Ethylmaleimide (1 mM) inhibited the high-Km process only. The high-affinity uptake of DNP-SG was competitively inhibited by GSSG (Ki = 88 microM). Vice versa, DNP-SG inhibited competitively the low-Km component of GSSG uptake (Ki = 3.3 microM). The high-Km DNP-SG uptake system was not inhibited by GSSG. The existence of a common high-affinity transporter for DNP-SG and GSSG in erythrocytes is suggested.

Biological Transport↗
PubMed1992

Inhibition of superoxide and nitric oxide release and protection from reoxygenation injury by Ebselen in rat Kupffer cells.

Luminol chemiluminescence in phorbolester-activated cultured rat liver Kupffer cells was strongly inhibited by the selenoorganic compound ebselen (IC50 = 2 mumol/L). Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]one) also diminished reduction of ferricytochrome c (IC50 = 10 mumol/L), indicating a suppression of superoxide anion formation. Likewise, in lipopolysaccharide-pretreated Kupffer cells, ebselen proved to be a potent inhibitor of the conversion of oxyhemoglobin to methemoglobin (IC50 = 3 mumol/L) as a measure of nitric oxide formation. The sulfur-containing analog (2-phenyl-1,2-benzisothiazol-3[2H]one) and the ebselen derivative, methylselenobenzanilide, were inactive. These results indicate that ebselen is a potent inhibitor of NADPH oxidase in Kupffer cells, as has been reported for other macrophages and granulocytes. In addition, they suggest a novel characteristic of ebselen, namely very effective inhibition of nitric oxide synthase of macrophages. In line with its inhibitory effects on the release of reactive oxygen species by macrophages, complemented by its antioxidant properties, ebselen was potent in the prevention of reoxygenation injury of Kupffer cells (IC50 approximately 5 mumol/L).

Amino Acid Oxidoreductases↗
PubMed1992

cis-trans isomers of lycopene and beta-carotene in human serum and tissues.

Since cis or trans isomers of carotenoids may have different biological reactivities, the isomeric composition of lycopene and beta-carotene was measured in serum and seven human tissues. In addition to all-trans lycopene, at least three cis-isomers (9-, 13-, and 15-cis) were present, accounting for more than 50% of total lycopene. 13- and 15-cis-beta-carotene, however, were present at only 5% of the all-trans isomer. In addition, 9-cis-beta-carotene was present in tissue samples but not in serum. There were interindividual differences in carotenoid levels of the different tissue types, but liver, adrenal gland, and testes always contained significantly higher amounts of the carotenoids than kidney, ovary, and fat; carotenoids in brain stem tissue were below the detection limit. beta-Carotene was the major carotenoid in liver, adrenal gland, kidney, ovary, and fat, whereas lycopene was the predominant carotenoid in testes.

Adipose Tissue↗
PubMed1992

Role of reactive oxygen species in cell toxicity.

Several types of compound exert their cytotoxicity by generating reactive oxygen species, notably the superoxide anion radical. These include quinoid and nitroaromatic compounds serving as redox cyclers, i.e. producing superoxide at the expense of NADPH and oxygen catalyzed by cellular reductases. In specialized cell-types employed in defense such as granulocytes, eosinophils and macrophages, myeloperoxidase, NADPH oxidase and nitric oxide synthase have been identified as major sources of reactive oxygen species in cell toxicity. These include hypochlorite, singlet oxygen, superoxide, nitric oxide and hydrogen peroxide. The interaction of superoxide and nitric oxide generates further oxidants such as peroxynitrite. Lumino-amplified chemiluminescence generated by Kupffer cells is partially sensitive to inhibitors of NO synthase. Superoxide dismutase has been found to catalyze a novel reaction, the reversible conversion of nitric oxide to the nitroxyl anion, the latter being viewed as another form of EDRF. In the defense against oxidative damage, there are enzymatic and nonenzymatic antioxidants. Regarding compounds used pharmacologically, we have been interested in ebselen, a seleno-organic compound exhibiting GSH peroxidase activity, which protects against reactive oxygen species generated, for example, at reoxygenation following a period of hypoxia. Further, we have studied lipoate and dihydrolipoate as antioxidant redox system and as singlet oxygen quencher, e.g. protecting against damage of deoxyguanosines in plasmid DNA generated by singlet oxygen.

Animals↗
PubMed1992

Antioxidant depletion in aortic crossclamping ischemia: increase of the plasma alpha-tocopheryl quinone/alpha-tocopherol ratio.

To assess whether oxidative stress contributes to the ischemia/reperfusion injury of aortic surgery, the contents of alpha-tocopherol, alpha-tocopheryl quinone, ascorbate, lipid-derived malondialdehyde, protein thiols, cholesterol, and lactate were analyzed in plasma samples from 24 patients subjected to aortic crossclamping. alpha-Tocopherol, ascorbate, and protein thiols decreased during ischemia, whereas alpha-tocopheryl quinone increased in all but two cases, doubling on average in proportion to alpha-tocopherol. Upon reperfusion alpha-tocopherol, ascorbate, and protein thiols remained low, whereas alpha-tocopheryl quinone returned to the preischemic level. Lipid-derived malondialdehyde (a measure of lipid hydroperoxides) increased significantly only during reperfusion. The results suggest that oxidative stress occurs simultaneously with ischemia/reperfusion during aortic surgery, and that measurement of the tocopheryl quinone/tocopherol ratio may shed new light on the underlying pathological events.

Aged↗
PubMed1992

Singlet oxygen induced DNA damage.

Singlet oxygen generated by photoexcitation and by chemiexcitation selectively reacts with the guanine moiety in nucleosides (kq + kr about 5 x 10(6) M-1s-1) and in DNA. The oxidation products include 8-oxo-7-hydro-deoxyguanosine (8-oxodG; also called 8-hydroxydeoxyguanosine) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). Singlet oxygen also causes alkali-labile sites and single-strand breaks in DNA. The biological consequences include a loss of transforming activity as studied with plasmids and bacteriophage DNA, and mutagenicity and genotoxicity. Employing shuttle vectors, it was shown that double-stranded vectors carrying singlet oxygen induced lesions seem to be processed in mammalian cells by DNA repair mechanisms efficient in preserving the biological activity of the plasmid but highly mutagenic in mammalian cells. Biological protection against singlet oxygen is afforded by quenchers, notably carotenoids and tocopherols. Major repair occurs by excision of the oxidized deoxyguanosine moieties by the Fpg protein, preventing mismatch of 8-oxodG with dA, which would generate G:C to T:A transversions.

Animals↗
PubMed1992

Glutathione S-transferases in human testicular germ cell tumors: changes of expression and activity.

Glutathione S-transferases are involved in the detoxification of carcinogens and xenobiotics and are potentially associated with the development of drug-resistance. Forty-six testicular germ cell tumors and 33 adjacent normal testicular tissue specimens were analyzed at the RNA level for the expression of glutathione S-transferase alpha and pi. Glutathione S-transferase alpha was expressed in 31 of the 33 normal testicular tissues (94%) but in only three of the 46 germ cell tumors (7%). Glutathione S-transferase pi mRNA was detected in all normal and malignant testicular tissue samples. Thirteen testicular germ cell tumors and eight normal testicular tissue samples were analyzed at the protein level. The mean specific activity of total cytosolic glutathione S-transferase in tumor tissue was decreased by about 80% as compared to normal testicular tissue. Protein analysis of the glutathione S-transferase subunits of normal testicular tissue demonstrated the presence of the glutathione S-transferase classes alpha, mu and pi, with a predominance of the mu class. In testicular germ cell tumors the glutathione S-transferase subunit pattern showed a predominance of glutathione S-transferase pi representing 88% +/- 3% of total glutathione S-transferase. Since all three glutathione S-transferase isoenzyme classes contribute to the resistance to antineoplastic drugs, the altered glutathione S-transferase isoenzyme pattern and the decrease of glutathione S-transferase activity may play a role in the high inherent drug sensitivity of human testicular germ cell tumors.

Glutathione Transferase↗
PubMed1992

Uptake of lycopene and its geometrical isomers is greater from heat-processed than from unprocessed tomato juice in humans.

Lycopene and beta-carotene are the most abundant carotenoids in human blood and tissues. Although lacking provitamin A activity, lycopene may be biologically active by contributing to the antioxidative defense system of the organism. We studied the uptake of lycopene from processed (boiled with 1% corn oil for 1 h) and unprocessed tomato juice in humans. Lycopene concentrations in human serum increased only when processed tomato juice was consumed. Lycopene uptake varied with individuals, but peak serum concentrations were always reached between 24 and 48 h. The carotenoid was eliminated from serum with a half-life of 2-3 d. The increase in peak serum concentrations was dose-dependent but not linear with the dose. Repeated doses led to a continual rise of lycopene in human serum. Of the different geometrical isomers (all-trans, 9-cis and 13-cis), the cis isomers seemed to be somewhat better absorbed than the all-trans form.

Absorption↗
PubMed1992

Singlet oxygen induced DNA damage and mutagenicity in a single-stranded SV40-based shuttle vector.

The effects of singlet oxygen (1O2), generated by the thermal decomposition of water soluble NDPO2 (endoperoxide of the disodium 3,3'-(1,4-naphthylidene) dipropionate), on a single-stranded shuttle vector were analysed. 1O2 induces a much higher level of breaks in the phosphodiester backbone of single-stranded than double-stranded DNA. This may be due to a higher accessibility of guanine residue, primarily damaged by 1O2. The damaged vector was transfected into monkey COS7 cells where single-stranded DNA was converted to the double-stranded replicative form DNA. After 3 days, extrachromosomal DNA was extracted and the plasmids rescued in E. coli to study mutagenesis. There is a significant increase in mutation frequency of damaged single-stranded DNA in comparison to untreated DNA. It is concluded that 1O2 induces breaks in the backbone of single-stranded DNA and that the 1O2-damaged molecules are mutated after passage through mammalian cells.

Animals↗
PubMed1992

Synthetic carotenoids, novel polyene polyketones and new capsorubin isomers as efficient quenchers of singlet molecular oxygen.

Novel synthetic polyene polyketones and new synthetic capsorubin isomers were examined for their ability to quench singlet molecular oxygen (1O2) generated by the thermodissociation of the endoperoxide of 3,3'-(1,4-naphthylene) dipropionate (NDPO2). C28-polyene-tetrone (1) exhibits the highest physical quenching rate constant with 1O2 (kq = 16 x 10(9) M-1 s-1). For comparison, the rate constant for the most efficient biological carotenoid, lycopene (3) is kq = 9 x 10(9) M-1 s-1 and that of beta-carotene (5) kq = 5 x 10(9) M-1 s-1. The presence of two oxalyl chromophores at the ends of the polyene chain seems to enhance the 1O2 quenching ability in the C28-polyene-tetrone (1). C28-polyene-tetrone-diacetal (2) (kq = 9 x 10(9) M-1 s-1) and C40-epiisocapsorubin (4) (kq = 8 x 10(9) M-1 s-1) also have high 1O2 quenching abilities. Two carotenoids from plants, phytoene and phytofluene, were much less efficient, kq values being below 10(7) M-1 s-1. Due to the very high singlet oxygen quenching abilities, C28-polyene-tetrone (1), C28-polyene-tetrone-diacetal (2) and C40-epiisocapsorubin (4) may have potential use in preventing 1O2-induced damage in biological and non-biological systems.

Carotenoids↗
PubMed1992

Ascorbate-dependent capacity of dialysed rat liver cytosol to prevent nonenzymatic lipid peroxidation.

The capacity of rat liver cytosol to decrease Fe/ADP ascorbate-induced lipid peroxidation in microsomes was evaluated using the chemiluminescence technique and measuring the formation of thiobarbituric acid-reactive substances (TBARS). The inhibiting effect of dialysed cytosol depended on the ascorbate concentration and was highest in the range of 1.0mM. Precipitation of cytosolic proteins with ammonium sulfate at 53% saturation yielded an active antioxidative fraction. Gel-filtration on Sephadex G-200 led to the separation of at least two cytosolic compounds of approximate molecular masses of 60 kDa and > 400 kDa. Both factors were active at 1.0mM ascorbate in the presence of freshly prepared microsomes. No inhibition of lipid peroxidation was observed using microsomes stored for one month at -70 degrees C.

Animals↗
PubMed1991

Role of reactive oxygen species in biological processes.

The steady-state formation of prooxidants in cells and organs is balanced by a similar rate of their consumption by antioxidants that are enzymatic and/or nonenzymatic. "Oxidative stress" results from imbalance in this prooxidant-antioxidant equilibrium in favor of the prooxidants. A number of diseases are associated with oxidative stress, being the basis of a potential antioxidant therapy. However, current evidence in clinical research does not show unequivocal distinction between causal or associative relationships of proxidants to the disease process.

Animals↗
PubMed1991

Reversible conversion of nitroxyl anion to nitric oxide by superoxide dismutase.

Superoxide dismutase (SOD) rapidly scavenges superoxide (O2-) and also prolongs the vasorelaxant effects of nitric oxide (NO), thought to be the endothelium-derived relaxing factor. This prolongation has been ascribed to prevention of the reaction between O2- with NO. We report that SOD supports a reversible reduction of NO to NO-. When cyanamide and catalase were used to generate NO- in the presence of SOD, NO was measured by the conversion of HbO2 to MetHb. When SOD[Cu(I)] was exposed to NO anaerobically, NO- was trapped by MetHb forming nitrosylmyoglobin. When NO was generated by 3-morpholinosydnonimine hydrochloride in the presence of SOD, NO- or a similar reductant was formed, which reduced catalase compound II and promoted the formation of the catalase [Fe(III)]-NO complex. It is, therefore, conceivable that SOD may protect NO and endothelium-derived relaxing factor by a mechanism in addition to O2- scavenging and that NO- may be a physiologically important form of endothelium-derived relaxing factor.

Animals↗
PubMed1991

Increase of beta-actin mRNA upon hypotonic perfusion of perfused rat liver.

beta-Actin mRNA levels in livers exposed to hypotonic perfusion (from 305 to 225 mosmol/l) for one hour are increased 2-fold relative to albumin mRNA. Like albumin, glyceraldehyde-3-phosphate dehydrogenase and tyrosine aminotransferase mRNAs remain at the levels observed under normotonic conditions. The increase in beta-actin mRNA is interpreted as a cytoskeletal response due to cell swelling.

Actins↗