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Biomedical subjects

H Sies

Publications and source records attributed to H Sies.

At least 127 records · Page 7Linked to original sources
PubMed1996

Protein S-thiolation and regulation of microsomal glutathione transferase activity by the glutathione redox couple.

Microsomal glutathione transferase (GSTm) is activated up to fivefold by incubation with glutathione disulfide (GSSG). The process is reversed by the addition of an NADPH-regenerating system consisting of glutathione reductase and glucose 6-phosphate/glucose-6-phosphate dehydrogenase. By treating the microsomes at different GSH/GSSG ratios a Kox value of 0.047 is found, i.e., 21 times more GSSG than GSH is necessary to produce half-maximal activation. The Kox is independent of the total glutathione concentration, indicating that S-thiolation by GSH rather than interchain or intrachain disulfide bridge formation is responsible for activation. Further evidence for S-thiolation of GSTm comes from SDS-PAGE under nonreducing conditions and Western blotting. Treating microsomes with GSSG or with GSH and t-butyl hydroperoxide or cumene hydroperoxide results in the appearance of a second GSTm band at approximately 17.7 kDa in addition to the native band at 17.3 kDa, the size difference approximately corresponding to the molecular mass of glutathione. The 17.7-kDa band is not seen in the presence of mercaptoethanol. Microsomal preparations from rat livers perfused with t-butyl hydroperoxide or cumene hydroperoxide also contain both GSTm forms. We suggest that under oxidative stress the microsomal GST in the cell can be activated through direct hydroperoxide-mediated S-thiolation of the enzyme with GSH, its reversal occurring via a thiol exchange-mediated dethiolation imposed by the intracellular glutathione redox state.

Amino Acid Sequence↗
PubMed1996

Analysis of carotenoids and carotenol fatty acid esters by matrix-assisted laser desorption ionization (MALDI) and MALDI-post-source-decay mass spectrometry.

Matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry and MALDI post-source-decay (PSD) fragment ion mass analysis were employed to examine various model carotenoids and some of their fatty acid esters. It was demonstrated that the use of MALDI helps overcome problems resulting from the inherent thermal instability and lack of solubility which render this group of compounds rather difficult for conventional ionization techniques. Detection limits were in the subpicomolar range. Rather abundant metastable fragmentation occurred under conditions of prompt extraction, but was significantly restricted under conditions of delayed extraction (DE). In DE the quasimolecular ion signals (mainly as odd electron radical ions) became the base peak even in spectra of the most delicate fatty acid esters. PSD fragment ion patterns were similar to those recorded under linked B/E scanning in conventional fast atom bombardment-MS and contained information on structural end group substituents such as hydroxyl, epoxide, or carbonyl functions and on the extent of double bond conjugations. The mono- and bis-fatty acid esters fragmented mainly by simple fatty acid cleavage but also furnished some of the end group-specific fragments.

Carotenoids↗
PubMed1996

Selenium-containing compounds protect DNA from single-strand breaks caused by peroxynitrite.

Peroxynitrite (ONOO-/ONOOH) is a powerful oxidant that can induce mutations and cause single-strand breaks in plasmid supercoiled DNA. The selenium-containing compound ebselen rapidly reacts with peroxynitrite. Therefore, ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one] and other selenium-containing molecules, selenomethionine and selenocystine, were studied as agents protecting DNA from single-strand breaks induced by peroxynitrite. Selenomethionine and selenocystine protected DNA from single-strand breaks more effectively than their sulfur analogs, methionine and cystine, and they also were more effective than glutathione or the hydroxyl radical scavenger mannitol. These results suggest that selenium-containing compounds can protect DNA from damage caused by peroxynitrite.

Cystine↗
PubMed1996

Microsomal formation of S-nitrosoglutathione from organic nitrites: possible role of membrane-bound glutathione transferase.

The formation of S-nitrosoglutathione (GSNO) from amyl nitrite and n-butyl nitrite was studied in rat liver microsomes, employing N-ethylmaleimide (MalNEt) as an activator and indomethacin as an inhibitor of microsomal glutathione S-transferase (GST). Rates were compared with GST activity measured with 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. MalNEt stimulated GST activity and the formation of GSNO from amyl nitrite and n-butyl nitrite about 10-fold. Increasing concentrations of indomethacin inhibited both reactions in parallel. N-Acetyl-L-cysteine but not L-cysteine could substitute for GSH. It is concluded that rat liver microsomal GST catalyses the formation of GSNO from amyl nitrite and n-butyl nitrite. The activity of the MalNEt-stimulated microsomal GST is calculated to be about 17 units/mg of enzyme with the alkyl nitrites and about 16 units/mg of enzyme with CDNB as a substrate, assuming that 3% of microsomal protein is GST. These rates are comparable with those obtained for cytosolic GSTs. Thus microsomal GST may play a significant role in the metabolism of alkyl nitrites in biological membranes.

Animals↗
PubMed1996

Assessment of the C-525 laser dye as a chemiluminescence sensitizer for lipid peroxidation in biological membranes: a comparison with chlorophyll-a.

The C-525 laser dye at micromolar concentration range is shown to enhance up to two to three orders of magnitude the chemiluminescence (CL) accompanying tert-butyl hydroperoxide (t-BHP)-induced rat liver microsome oxidation and Fe(2+)-induced lipid peroxidation (LPO) in liposomes. C-525 is shown to be a more efficient sensitizer of CL accompanying LPO in membrane systems than the known low-energy excited triplet carbonyl sensitizer, chlorophyll-alpha, (Cl-a). Regarding the sensitization mechanism, C-525 and Cl-a were compared in (a) a peroxyl radical-producing system (2,2'-azobis(2-dimethylvaleronitrile) (AMVN); (b) excited carbonyl-producing systems (3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane (HTMD) thermal decomposition and horseradish peroxidase (HRP)-catalyzed isobutanal oxidation); and (c) excited singlet oxygen-producing system [endoperoxide of 3,3-(1,4-naphthylidene)-dipropionate (NDPO2)]. C-525 sensitized CL only in the systems where peroxyl radical and/or triplet excited carbonyls are produced, the mechanism of CL sensitization apparently is energy transfer from the excited triplet carbonyls formed in the peroxyl radical self-reaction via Russell's mechanism or by dioxetane decomposition. Cl-a was found to considerably sensitize CL related to NDPO2 thermal decomposition, a source of singlet oxygen, in addition to acting as a sensitizer of triplet carbonyl CL. The chemical stability of the C-525 laser dye in excited state-generating systems was shown to be appropriate for its application as a sensitizer of CL related to LPO reactions in membranes, but not in the HRP-catalyzed peroxidation system.

Aldehydes↗
PubMed1996

Adjacent guanines as preferred sites for strand breaks in plasmid DNA irradiated with 193 nm and 248 nm UV laser light.

193 nm UV laser light induces single strand breaks as well as double strand breaks in plasmid DNA. The frequency of strand breaks is increased at sites where at least two guanine or, less frequently, a guanine and an adenine are adjacent to each other. 248 nm UV laser light induces predominantly single strand breaks with a less pronounced preference for guanine clusters. At both wavelengths, the presence of oxygen does not change the pattern of strand breaks, but in the presence of nitrous oxide, selectivity is lost; this is attributable to the production of the hydroxyl radical. These findings can be explained by a model in which the propagation of a radical or an electron hole along the DNA helix competes kinetically with the strand cleavage reaction. The difference in selectivity at the two different wavelengths is ascribed to the preferential light absorption by the purine bases at 193 nm.

Base Sequence↗
PubMed1996

Beta-carotene serum levels in patients with erythropoietic protoporphyria on treatment with the synthetic all-trans isomer or a natural isomeric mixture of beta-carotene.

The all-trans-beta-carotene serum level of patients suffering from erythropoietic protoporphyria increases substantially during continuous treatment with beta-carotene (either with the synthetic all-trans compound or with beta-carotene from a natural source consisting of a cis-trans isomeric mixture). On continuous daily ingestion, the beta-carotene serum level rose from day 0 to day 30, and no further increase was observed between day 30 and day 150. Slightly lower beta-carotene steady state serum levels were observed with the natural isomeric mixture than with synthetic beta-carotene. Higher levels of 13-cis-beta-carotene, in some cases up to 10% of the total beta-carotene, were detected after ingestion of the synthetic compound. The level of 9-cis-beta-carotene was below or close to the limit of quantification in all samples, even when the isomeric mixture containing high amounts of 9-cis-beta-carotene was applied.

Administration, Oral↗
PubMed1996

Induction of gap junctional intercellular communication by vitamin D in human skin fibroblasts is dependent on the nuclear Induction of gap junctional intercellular communication by vitamin D in human skin fibroblasts is dependent on the nuclear vitamin D receptor.

The physiologically active metabolite of vitamin D, 1alpha,25-dihydroxyvitamin D3 (calcitriol), induces gap junctional intercellular communication in human skin fibroblasts 161BR at a concentration of 10(-7) M. In human skin fibroblasts, FIB5, devoid of a functional nuclear vitamin D receptor (VDR), there is no effect on gap junctional intercellular communication. Parallel to the increase in cell-cell communication, we observed a VDR-dependent increase in connexin43 protein and connexin43 mRNA levels. These results suggest that 1alpha,25-dihydroxyvitamin D3 affects gap junctional intercellular communication at the level of transcription or of mRNA stability via the nuclear VDR.

Calcitriol↗
PubMed1996

Ultraviolet B wavelength dependence for the regulation of two major matrix-metalloproteinases and their inhibitor TIMP-1 in human dermal fibroblasts.

The wavelength dependence for the regulation of two major matrix-metalloproteinases, interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3), and their major inhibitor, tissue inhibitor of metalloproteinases (TIMP-1), was studied in human dermal fibroblasts in vitro. Monochromatic irradiation at 302, 307, 312 and 317 nm with intensities ranging from 20 to 300 J/m2 increased MMP-1 and MMP-3 mRNA steady-state levels and the secretion of the corresponding proteins up to 4.4-fold, whereas almost no increase was observed at wavelengths < 290 nm. In contrast, the synthesis of TIMP-1 increased only marginally. This imbalance may contribute to the severe connective tissue damage related to photoaging of the skin. The wavelengths responsible for MMP-1 and MMP-3 induction reported here are distinct from the absorption spectrum of DNA and are different from results previously reported in the literature. Importantly, they overlap with wavelengths whose intensity is predicted to increase on the earth's surface upon ozone depletion. Intensities and particular wavelengths used in our studies in vitro can be absorbed readily by fibroblasts within the skin in vivo and, thus, are relevant for risk assessment and development of protective agents.

Cells, Cultured↗
PubMed1996

Ultraviolet B wavelength dependence for the regulation of two major matrix-metalloproteinases and their inhibitor TIMP-1 in human dermal fibroblasts.

The wavelength dependence for the regulation of two major matrix-metalloproteinases, interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3), and their major inhibitor, tissue inhibitor of metalloproteinases (TIMP-1), was studied in human dermal fibroblasts in vitro. Monochromatic irradiation at 302, 307, 312 and 317 nm with intensities ranging from 20 to 300 J/m2 increased MMP-1 and MMP-3 mRNA steady-state levels and the secretion of the corresponding proteins up to 4.4-fold, whereas almost no increase was observed at wavelengths < 290 nm. In contrast, the synthesis of TIMP-1 increased only marginally. This imbalance may contribute to the severe connective tissue damage related to photoaging of the skin. The wavelengths responsible for MMP-1 and MMP-3 induction reported here are distinct from the absorption spectrum of DNA and are different from results previously reported in the literature. Importantly, they overlap with wavelengths whose intensity is predicted to increase on the earth's surface upon ozone depletion. Intensities and particular wavelengths used in our studies in vitro can be absorbed readily by fibroblasts within the skin in vivo and, thus, are relevant for risk assessment and development of protective agents.

Child, Preschool↗
PubMed1996

Vitamin E in humans: demand and delivery.

How much vitamin E is enough? An established use of supplemental vitamin E in humans is in the prevention and therapy of deficiency symptoms. The cause of vitamin E deficiency, characterized by peripheral neuropathy and ataxia, is usually malabsorption-a result of fat malabsorption or genetic abnormalities in lipoprotein metabolism. Genetic abnormalities in the hepatic alpha-tocopherol transfer protein also cause vitamin E deficiency-defects in this protein cause an impairment in plasma vitamin E transport. Impaired delivery of vitamin E to tissues, thereby, results in deficiency symptoms. Also discussed is the use of supplemental vitamin E in chronic diseases such as ischemic heart disease, atherosclerosis, diabetes, cataracts, Parkinson's disease, Alzheimer's disease, and impared immune function, as well as in subjects receiving total parenterol nutrition. In healthy individuals, a daily intake of about 15-30 mg of alpha-tocopherol is recommended to obtain "optimal plasma alpha-tocopherol concentrations" (30 microM or greater).

Diet↗
PubMed1996

Positive and negative regulatory regions in promoters of human glutathione transferase alpha genes.

The human glutathione transferase (GST, EC 2.5.1.18) alpha class locus comprises several genes and pseudogenes. Genomic DNA encoding several human alpha-class-related genes and pseudogenes was cloned and characterized. Three distinct but highly similar 5'-flanking regions of GST alpha genes as well as a series of 5'-deletions were investigated for promoter activity by fusion to the luciferase reporter gene. Transient transfection of these luciferase constructs into human hepatoblastoma, kidney carcinoma, nephroblastoma or bladder carcinoma cells revealed that the promoters are active and contain both positive and negative regulatory regions that behave in a cell-type specific fashion. The 150 bp proximal promoter regions of the three sequences retained the same relative activities as the full length promoters. Two of them were equally active, whereas the third one showed only 20% of the activity of the two stronger promoters. Site-directed mutagenesis indicated that a conspicuous insertion of three nucleotides (TTT) in the weak promoter is not responsible for the different activities.

Cloning, Molecular↗
PubMed1996

Induction of mouse embryonal carcinoma cell differentiation and activation of the retinoic acid receptor beta 2 promoter by 1,25-dihydroxyvitamin D3.

1,25-Dihydroxyvitamin D3 (calcitriol) at 100 nmol/l elicited morphological differentiation and expression of collagen IV in mouse F9 embryonal carcinoma cells, and its effect was enhanced and accelerated by dibutyryl-cAMP (db-cAMP). The RAR beta 2 promoter was also activated, as evidenced by an increase in beta-galactosidase activity in an F9 reporter cell line with a stably integrated RAR beta 2-lacZ construct. All three effects were slower and less extensive with calcitriol than with retinoic acid, even in the presence of db-cAMP. Activation of the RAR beta 2 promoter by calcitriol required its TRE sequence, whereas db-cAMP required the CRE. TPA also activated the RAR beta 2 promoter, requiring a functional TRE. Thus, in the RAR beta 2 promoter the TRE sequence, whose function has so far been unidentified, mediates the effects of calcitriol and TPA. RAR beta 2 promoter activation by calcitriol was blocked by inhibitors of protein kinase C indicating that calcitriol elicits its effect via protein kinase C. Therefore, calcitriol induces differentiation of F9 mouse embryonal carcinoma cells at least in part by a pathway different from the classical one operative with retinoic acids.

Animals↗
PubMed1996

Diminished plasma levels of vitamin E in patients with severe viral hepatitis.

RRR-alpha-Tocopherol (Vitamin E) was assayed in plasma of 48 patients with viral hepatitis and of 32 healthy controls. In patients with highly elevated serum transaminases (ALT > 100 U/L) vitamin E plasma levels were significantly lower (17.5 +/- 4.8 mumol/L) than in controls (22.7 +/- 4.2 mumol/L, p < 0.01). The vitamin E/lipid ratios (3.12 +/- 0.63 mumol/g) in these patients were 33% lower than those of the controls (4.68 +/- 0.54 mumol/g). The lowered vitamin E levels in patients with acute or chronic viral hepatitis with high activity of disease may be due to free radical-mediated liver injury.

Acute Disease↗
PubMed1996

Formation of singlet oxygen in the thermal decomposition of 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane, a chemical source of triplet-excited ketones.

Triplet-excited carbonyl species can be generated by photoexcitation of carbonyl compounds or alternatively by thermal decomposition of 1,2-dioxetanes. Such electronically excited species are involved in the oxidative modification of biologically important molecules such as DNA. This study demonstrates that thermal decomposition of 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane (HTMD), which is a chemical source of triplet-excited ketones, is accompanied by infrared photoemission at 1270 nm characteristic for singlet oxygen monomol emission. The intensity of infrared photoemission at 1270 nm was solvent-dependent, in agreement with the reported lifetime of singlet oxygen in the employed solvents. Calibration of monomol emission with the endoperoxide of 1,4-dimethylnaphthalene (DMNO2) showed that HTMD (30 mM) produced 25 microMs of singlet oxygen. Thus, the yield of singlet oxygen production in the thermal decomposition of HTMD in carbon tetrachloride is about 0.1%.

Heating↗