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Biomedical subjects

H Sies

Publications and source records attributed to H Sies.

At least 73 records · Page 4Linked to original sources
PubMed1999

Function of thioredoxin reductase as a peroxynitrite reductase using selenocystine or ebselen.

The activity of mammalian thioredoxin reductase as a peroxynitrite reductase was investigated. Peroxynitrite was infused to maintain a 0.2 microM steady-state concentration in potassium phosphate buffer (pH 7.4). Benzoate hydroxylation and nitrite formation were used as indices of oxidation reactions of peroxynitrite and of peroxynitrite reduction, respectively. In the presence of NADPH (10 microM), thioredoxin reductase at 50 nM alone did not significantly scavenge peroxynitrite, as shown by there being no significant effect on benzoate hydroxylation or nitrite formation. However, when selenocystine (1 microM) or ebselen (2 microM) was present in the reaction mixture, there was significant suppression of benzoate hydroxylation and an increase in nitrite formation until all the NADPH was oxidized. The addition of thioredoxin did not enhance these effects. In contrast, peroxynitrite reduction by ebselen complexed with BSA was enhanced by the presence of thioredoxin. In parallel experiments, thioredoxin reductase efficiently reduced ebselen selenoxide back to ebselen.

Animals↗
PubMed1999

Optimum porphyrin accumulation in epithelial skin tumours and psoriatic lesions after topical application of delta-aminolaevulinic acid.

Photodynamic therapy with topically applied delta-aminolaevulinic acid is used to treat skin tumours by employing endogenously formed porphyrins as photosensitizers. This study examines the time course of porphyrin metabolite formation after topical application of delta-aminolaevulinic acid. Porphyrin biosynthesis in human skin tumours (basal cell carcinoma, squamous cell carcinoma), in psoriatic lesions, and in normal skin was investigated. Skin areas were treated with delta-aminolaevulinic acid, and levels of total porphyrins, porphyrin metabolites and proteins were measured in samples excised after 1, 2, 4, 6, 9, 12 and 24 h. There was an increase in porphyrin biosynthesis in all tissues with maximum porphyrin levels in tumours between 2 and 6 h and in psoriatic lesions 6 h after treatment. The pattern of porphyrins showed no significant difference between normal and neoplastic skin, protoporphyrin being the predominant metabolite. The results suggest that optimum irradiation time for superficial epithelial skin tumours may be as soon as 2 h after application of delta-aminolaevulinic acid, whereas for treatment of psoriatic lesions an application time of 6 h is more suitable.

Administration, Topical↗
PubMed1999

Mitogen-activated protein kinase (p38-, JNK-, ERK-) activation pattern induced by extracellular and intracellular singlet oxygen and UVA.

Ultraviolet A (UVA; 320-400 nm) radiation in human skin fibroblasts induces a pattern of mitogen-activated protein kinase (MAPK) activation consisting of a rapid and transient induction of p38 and c-Jun-N-terminal kinase (JNK) activity but not extracellular signal-regulated kinases (ERK). UVA activation of p38 can be inhibited by the singlet oxygen (1O2) quenchers azide and imidazole, but not by the hydroxyl radical scavengers mannitol or dimethylsulfoxide, pointing to the involvement of 1O2. The same effect has been shown for JNK. Like UVA, 1O2 generated intracellularly upon photoexcitation of Rose Bengal activates p38 and JNK but not ERK. p38 and JNK activation was also elicited by chemiexcitation for the intracellular generation of 1O2 by the lipophilic 1,4-endoperoxide of N,N'-di(2,3-dihydroxypropyl)-1, 4-naphthalene dipropionamide. In contrast, extracellular generation of 1O2, by irradiation of Rose Bengal immobilized on agarose beads or by chemiexcitation employing the hydrophilic 1,4-endoperoxide of disodium 3,3'-(1,4-naphthylidene) dipropionate, was ineffective in activating p38 or JNK. These data suggest that the activation of p38 and JNK by 1O2 occurs only when the electronically excited molecule is generated intracellularly.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1999

Protein oxidation in human stratum corneum: susceptibility of keratins to oxidation in vitro and presence of a keratin oxidation gradient in vivo.

The stratum corneum is located at the interface between body and environment and thus is constantly exposed to a pro-oxidative environment. Previously, we have demonstrated that stratum corneum lipids are targets of oxidative stress induced by ozone and by ultraviolet A and B exposure. Here, we employed an immunoblotting technique to detect protein oxidation in human stratum corneum obtained by tape stripping. After lysis, protein carbonyl groups were measured by derivatization with dinitrophenylhydrazine, separation by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and immunoblotting using antibodies against dinitrophenyl groups. Keratin 10, identified by use of specific antibodies and by microsequencing, was demonstrated in vitro to be oxidizable by ultraviolet A irradiation, hypochlorite, and benzoyl peroxide. In vivo, a keratin 10 oxidation gradient with low levels in the lower stratum corneum layers, and about 3-fold higher contents of carbonyl groups towards the outer layers was demonstrated in forehead stratum corneum of healthy volunteers (n = 6). As protein oxidation can be associated with an increased susceptibility to proteases, this finding may be important for better understanding the process of desquamation.

Adult↗
PubMed1999

Peroxynitrite diminishes gap junctional communication: protection by selenite supplementation.

Loss of intercellular communication via gap junctions has been correlated with progression of cells to a malignant phenotype. Here, we show that peroxynitrite, a mediator of toxicity in inflammatory processes, diminishes gap junctional intercellular communication (GJIC) in WB-F344 rat liver epithelial cells, assayed by the scrapeloading dye-transfer technique as well as by microinjection of a fluorescent dye into single cells. Exposure of cultured cells to a steady-state concentration of peroxynitrite of 1.6 microM for 4 min or to 3-morpholinosydnonimine (SIN-1) at 0.5 mM strongly diminished GJIC. These concentrations of peroxynitrite or SIN-1 were not cytotoxic. When cells were grown in a medium supplemented with sodium selenite (0.1-1 microM) for 72 h, substantial protection was afforded against the decrease in GJIC by peroxynitrite. Thus, peroxynitrite can disrupt GJIC, and selenium-containing proteins protect.

Animals↗
PubMed1999

Association of NAD(P)H:quinone oxidoreductase (NQO1) null with numbers of basal cell carcinomas: use of a multivariate model to rank the relative importance of this polymorphism and those at other relevant loci.

Glutathione S-transferase GSTM1 B and GSTT1 null, and cytochrome P450 CYP2D6 EM have been associated with cutaneous basal cell carcinoma (BCC) numbers, although their quantitative effects show that predisposition to many BCC is determined by an unknown number of further loci. We speculate that other loci that determine response to oxidative stress, such as NAD(H):quinone oxidoreductase (NQO1) are candidates. Accordingly, we assessed the association between NQO1 null and BCC numbers primarily to rank NQO1 null in a model that included genotypes already associated with BCC numbers. We found that only 14 out of 457 cases (3.1%) were NQO1 null. This frequency did not increase in cases with characteristics linked with BCC numbers including gender, skin type, a truncal lesion or more than one new BCC at any presentation (MPP). However, the mean number of BCC in NQO1*0 homozygotes was greater than in wild-type allele homozygotes and heterozygotes, although the difference was not quite significant (P = 0.06). These data reflect the link between NQO1 null and BCC numbers in the 42 MPP cases rather than the whole case group. We identified an interaction between NQO1 null and GSTT1 null that was associated with more BCC (P = 0.04), although only four cases had this combination. The relative influence of NQO1 null was studied in a multivariate model that included: (i) 241 patients in whom GSTM1 B, GSTT1 null and CYP2D6 EM genotype data were available, and (ii) 101 patients in whom these genotypes, as well as data on GSTM3, CYP1A1 and melanocyte-stimulating hormone receptor (MC1R) genotypes were available. NQO1 null (P = 0.001) and MC1R asp294/asp294 (P = 0.03) were linked with BCC numbers, and the association with CYP2D6 EM approached significance (P = 0.08). In a stepwise regression model only these genotypes were significantly associated with BCC numbers with NQO1 null being the most powerful predictor.

Age Factors↗
PubMed1998

Identification of two activating elements in the proximal promoter region of the human glutathione transferase-A1 and -A2 genes.

Promoter regions derived from the human glutathione S-transferase (GST) alpha gene cluster located on chromosome 6p12 were studied: the identical proximal promoters of the GST A1 and GST A2 genes and a proximal promoter of a pseudogene of this class. The sequence of the pseudogene promoter differs in four single nucleotides at positions -86, -66, -41, and -13, and a noncritical TTT insertion at positions -71 to -69 from the GST A1/A2 promoter. Here, it was shown that the GST A1/A2 proximal promoters differed by a factor of 3.4 in their activity from the proximal pseudogene promoter. Therefore, the functional significance of single base exchanges was examined by introducing individual point mutations at the four positions within the proximal GST A1/A2 promoter. In functional tests in transiently transfected human hepatoblastoma HepG2 cells the base exchange at position -13 showed no effect, whereas mutations at position -41 or -86 diminished the promoter activity to a level comparable to the pseudogene promoter. Promoter fragments of both genes spanning over these four sites were analyzed in a heterologous promoter context for their functionality in HepG2 cells. Moreover, gel shift experiments showed specific binding of nuclear proteins to these promoter fragments. The results show that in the proximal GST A1/A2 promoter the sites at position -41 or -86 are essential for the binding of activating transcription factor complexes.

Base Sequence↗
PubMed1998

Activation of JNK and p38 but not ERK MAP kinases in human skin cells by 5-aminolevulinate-photodynamic therapy.

5-Aminolevulinate (ALA) photodynamic therapy (PDT) is being used clinically for the treatment of skin cancers. ALA is applied as a precursor of porphyrins serving as endogenous photosensitizers. Irradiation of HaCaT cells preincubated with 1 mM ALA for 24 h with red light of 570-750 nm at a dose of 4.5 J/cm2 leads to a 6-fold elevation of cellular c-Jun N-terminal kinase activity; phosphorylation of p38 mitogen-activated protein kinase (MAPK) is enhanced to a similar extent. In contrast, neither activation nor increased phosphorylation of the extracellular stimulus-regulated kinase MAPKs is detected. p38 is also phosphorylated by ALA-PDT in the human melanoma cell lines Bro and SkMel-23, applying doses that lead to 80-95% cell death after 24 h. Hence, the effects of ALA-PDT on MAPKs are similar to stresses like UV irradiation or exposure to hydrogen peroxide with respect to activation of JNK and p38 MAPKs. They are different, however, in that extracellular stimulus-regulated kinase activity is not raised by ALA-PDT. Of the 830 pmol porphyrins/mg protein that were present at 24 h in HaCaT cells, 99 pmol/mg were intracellular. When extracellular porphyrins had been removed by washing, p38 responses were retained. Thus, intracellular porphyrins synthesized from ALA are sufficient to elicit activation of p38 on photosensitization.

Aminolevulinic Acid↗
PubMed1998

A 47-amino-acid fragment of SV40 T antigen represses transcription from human GSTalpha promoters.

SV40 T antigen downregulates the expression of an important detoxification enzyme, glutathione S-transferase alpha (GSTalpha). We show here that the target of this repression is a 14-bp element common to the human GSTA1 and GSTA2 promoters. This element, which we have named TAGR, is also critical for high-level, constitutive expression from these promoters. The TAGR element does not appear to contain a binding site for any transcription factor known to be present in fibroblasts, although the TAGR element does resemble the binding site for the Ikaros transcription factor found in hematopoietic cells. We also have identified a 47-amino-acid fragment of T antigen that includes amino acids 83-100 and 119-147, which is sufficient to repress transcription from the GSTalpha promoter in transient transcription assays. Thus, GSTalpha repression does not require binding of T antigen to pRb, p300, or p53, since the domains of T antigen required for binding these cellular proteins are missing from this T antigen fragment. We show, however, that this fragment does bind to three cellular proteins with approximate molecular weights of 54, 59, and 94 kDa.

Animals↗
PubMed1998

Hepatic mercapturic acid formation: involvement of cytosolic cysteinylglycine S-conjugate dipeptidase activity.

The role of cysteinylglycine S-conjugate dipeptidases in the intrahepatic mercapturic acid pathway was investigated in rat liver. Subcellular compartmentation studies and liver perfusions were performed using monochlorobimane and bimane S-conjugates as model compounds. The major part (over 95%) of total hepatic cysteinylglycine S-conjugate dipeptidase activity was located in the cytosol. Lower specific activity appeared in the canalicular plasma membrane fraction. Similar hepatic localization of dipeptidase activity was seen in the guinea pig. In intact rat liver perfused with monochlorobimane, the major products were the glutathione S-conjugate (mBSG) and the cysteinylglycine S-conjugate (mBCG) in bile. Minor amounts of the cysteine S-conjugate (mBCys) and the mercapturic acid (mBNAc) were formed, indicating a limitation in further metabolism of the dipeptide S-conjugate in the biliary space. However, when the dipeptide S-conjugate was offered to the sinusoidal space in liver perfusions, substantial uptake and conversion to mBNAc was observed, and only trace amounts of the infused dipeptide appeared in bile. The data suggest that cytosolic cysteinylglycine S-conjugate dipeptidase as identified here is involved in hepatic mercapturic acid formation from sinusoidal cysteinylglycine S-conjugates. This is especially of significance for species such as guinea pig and human, in which dipeptide S-conjugates are generated in the sinusoidal domain of the liver due to the presence of high gamma-glutamyltranspeptidase activity.

Acetylcysteine↗
PubMed1998

Xanthophyll esters in human skin.

Carotenol fatty acid esters in various fruits and vegetables are sources of carotenoids for humans and are cleaved during intestinal absorption to yield the parent xanthophyll. In the present study we demonstrate that low amounts of carotenol esters are present in human skin. Eighteen different carotenol fatty acid esters were separated in extracts from human skin by means of reversed-phase high-performance liquid chromatography. After saponification of tissue samples, lutein, zeaxanthin, 2',3'-anhydrolutein, alpha-cryptoxanthin and beta-cryptoxanthin were identified as the major parent carotenoids. Based on comparison with synthetic reference compounds, several carotenol mono- and di-fatty acid esters were tentatively assigned, ranging from linoleate to stearate; the levels are low, in the range of pmol/g human skin, i.e. , several orders of magnitude lower than that of beta-carotene. Carotenol esters in human skin may be formed by reesterification of xanthophylls following absorption. Also, very small amounts of esters may circulate in the blood and subsequently accumulate in tissues such as skin.

Adult↗
PubMed1998

Carotenoid mixtures protect multilamellar liposomes against oxidative damage: synergistic effects of lycopene and lutein.

Antioxidant activity of carotenoids in multilamellar liposomes assayed by inhibition of formation of thiobarbituric acid-reactive substances was in the ranking: lycopene> alpha-tocopherol > alpha-carotene > beta-cryptoxanthin > zeaxanthin = beta-carotene > lutein. Mixtures of carotenoids were more effective than the single compounds. This synergistic effect was most pronounced when lycopene or lutein was present. The superior protection of mixtures may be related to specific positioning of different carotenoids in membranes.

Antioxidants↗
PubMed1998

Protein S-thiolation and redox regulation of membrane-bound glutathione transferase.

Membrane-bound GST transferase (GSTm) occurs in hepatic microsomal and plasma membranes as well as in the outer mitochondrial membrane, and it is known to be activated by N-ethylmaleimide. We recently analysed the activation by GSSG in some detail. The approximately 5-fold stimulation is reversed upon reduction of GSSG by GSSG reductase. In steady-state experiments, the Kox value was determined to be 0.05, i.e. 20 times more GSSG than GSH produces half-maximal activation. Kox is independent of the total glutathione concentration, indicating that S-thiolation by mixed disulfide formation, rather than interchain or intrachain disulfide bridge formation, is responsible for activation. In Western blots, a 17.7 kDa band, in addition to the 17.3 kDa band, was detected upon treatment with GSSG or with GSH plus t-butyl hydroperoxide. We suggest that under oxidative stress, GSTm is activated through direct S-thiolation of the enzyme. Dethiolation occurs via thiol disulfide exchange governed by the cellular glutathione redox state.

Animals↗
PubMed1998

Protection by organotellurium compounds against peroxynitrite-mediated oxidation and nitration reactions.

Diaryl tellurides effectively protect against peroxynitrite-mediated oxidation of dihydrorhodamine 123 (DHR), hydroxylation of benzoate, and nitration of 4-hydroxyphenylacetate (HPA). Bis(4-aminophenyl) telluride offered the most efficient protection against oxidation of DHR induced by peroxynitrite. Protection by this compound was approximately 3 times more effective than that afforded by its selenium analog, bis(4-aminophenyl) selenide, and 11 times more effective than selenomethionine. When peroxynitrite was infused to maintain a steady-state concentration, bis(4-aminophenyl) telluride in the presence of GSH, but neither bis(4-aminophenyl) telluride nor GSH alone, effectively inhibited the peroxynitrite-mediated hydroxylation of benzoate. The inhibition of nitration was most pronounced using bis(4-hydroxyphenyl) telluride, and this compound was ca. 3 times more effective than selenomethionine. Bis(4-aminophenyl) telluride also protected proteins in lysates from human skin fibroblasts from peroxynitrite-mediated nitration of tyrosine residues more effectively than selenomethionine. These data establish a potential biological or pharmacological role of organotellurium compounds in the defense against peroxynitrite.

Antioxidants↗
PubMed1998

Central role of Ferrous/Ferric iron in the ultraviolet B irradiation-mediated signaling pathway leading to increased interstitial collagenase (matrix-degrading metalloprotease (MMP)-1) and stromelysin-1 (MMP-3) mRNA levels in cultured human dermal fibroblasts.

Reactive oxygen species (ROS) are important second messengers for the induction of several genes in a variety of physiological and pathological conditions. Ultraviolet B (UVB) irradiation has recently been shown to generate lipid peroxidation products and hydroxyl radicals (HO.) with detrimental long term effects like cancer formation and premature aging of the skin. Here, we addressed the question of whether ferric/ferrous iron via the generation of ROS may mediate the UVB response, finally leading to connective tissue degradation, a hallmark in carcinogenesis and aging. Therefore, we studied the involvement of iron and ROS in the modulation of Jun N-terminal kinase 2 (JNK2) activity, c-jun and c-fos mRNA levels, key signaling steps in the transcriptional control of matrix-degrading metalloprotease (MMP)-1/interstitial collagenase and MMP-3/stromelysin-1 after UVB irradiation of human dermal fibroblasts in vitro. The iron-driven generation of lipid peroxides and hydroxyl radicals were identified as early events in the downstream signaling pathway of the UVB response leading to a 15-fold increase in JNK2 activity, a 3.5-fold increase in c-jun, to a 6-fold increase in MMP-1, and a 3.8-fold increase in MMP-3 mRNA levels, while virtually no alteration of c-fos mRNA levels were observed. Diminished generation of reactive oxygen species resulted in a significant reduction of JNK2 activity, c-jun, MMP-1, and MMP-3 mRNA levels after UVB irradiation compared with UVB-irradiated cells. Collectively, we have identified the iron-driven Fenton reaction and lipid peroxidation as possible central mechanisms underlying signal transduction of the UVB response.

Chelating Agents↗
PubMed1998

Enhancement of gap junctional communication and connexin43 expression by thyroid hormones.

Cells in tissues coordinate their activity by sharing ions, second messengers, and small metabolites through clusters of intercellular channels called gap junctions. The thyroid hormones 3,3',5-triiodo-L-thyronine (T3) and L-thyroxine (T4) are capable of modulating gap junctional communication (GJC) as are 1,25-dihydroxyvitamin D3, retinoic acid, and other nuclear receptor ligands. T3 and T4 were found to stimulate GJC in WB-F344 rat liver epithelial cells dose-dependently at concentrations between 1 nM and 0.1 microM, assayed by the dye transfer method using Lucifer Yellow CH. The stimulation of cell-cell communication was preceded by an increase in connexin43 mRNA levels and was accompanied by an accumulation of connexin43 protein measurable 2 days after incubation with these compounds. These observations establish a novel role of thyroid hormones in the regulation of gap junctional intercellular communication via connexin43 gene expression.

Animals↗