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Biomedical subjects

L Flohé

Publications and source records attributed to L Flohé.

At least 55 records · Page 3Linked to original sources
PubMed1997

cDNA and deduced polypeptide sequence of a mouse selenoprotein P.

An 11-day embryonic Swiss Webster/NIH mouse cDNA library was screened with a partial murine selenoprotein P cDNA probe and a murine selenoprotein-P-type cDNA clone of 2075 bp length was obtained. The clone contained a 5'-leader sequence of 132 bp length, the selenoprotein P coding frame, and 803 base pairs in the 3' untranslated region. Alignment and RNA folding studies revealed the presence of two well conserved selenocysteine inserting motifs in the 3' flanking region. The deduced polypeptide sequence comprises 380 residues including ten selenocysteines. Identical amino acid residues in homologous positions are 86%, 71%, and 64% when compared to the previously reported selenoprotein P sequences of rat, man, and cattle, respectively. The comparatively low similarity between the selenoprotein P sequences reported so far leaves open the question whether they belong to the same molecular clade.

Amino Acid Sequence↗
PubMed1997

Lipoic acid increases de novo synthesis of cellular glutathione by improving cystine utilization.

Lipoic acid (thiotic acid) is being used as a dietary supplement, and as a therapeutic agent, and is reported to have beneficial effects in disorders associated with oxidative stress, but its mechanism of action remains unclear. We present evidence that lipoic acid induces a substantial increase in cellular reduced glutathione in cultured human Jurkat T cells human erythrocytes, C6 glial cells, NB41A3 neuroblastoma cells, and peripheral blood lymphocytes. The effect depends on metabolic reduction of lipoic acid to dihydrolipoic acid. Dihydrolipoic acid is released into the culture medium where it reduces cystine. Cysteine thus formed is readily taken up by the neutral amino acid transport system and utilized for glutathione synthesis. By this mechanism lipoic acid enables cystine to bypass the xc- transport system, which is weakly expressed in lymphocytes and inhibited by glutamate. Thereby lipoic acid enables the key enzyme of glutathione synthesis, gamma-glutamylcysteine synthetase, which is regulated by uptake-limited cysteine supply, to work at optimum conditions. Flow cytometric analysis of freshly prepared human peripheral blood lymphocytes, using monobromobimane labeling of cellular thiols, reveals that lipoic acid acts mainly to normalize a subpopulation of cells severely compromised in thiol status rather than to increase thiol content beyond physiological levels. Hence lipoic acid may have clinical relevance in restoration of severely glutathione deficient cells.

Animals↗
PubMed1997

Evidence for a functional relevance of the selenocysteine residue in mammalian thioredoxin reductase.

Human thioredoxin reductase was recently shown to contain a TGA encoded selenocysteine residue at the penultimate position of its amino acid chain. Depending on the availability of selenium during biosynthesis, an authentic selenocysteine-containing or a selenium-free enzyme truncated at the penultimate position is expected to be formed. Correspondingly, the enzymatic activity should be altered by selenium restriction, if the selenocysteine residue is functionally important. In order to check the catalytic role of the selenocysteine residue, four different human cell lines were grown in selenium deficient media or with adequate selenium supplementation (40 nM sodium selenite) and thioredoxin reductase activity was measured as NADPH-dependent DTNB reduction or thioredoxin-mediated insulin reduction. Thioredoxin reductase activities, like glutathione peroxidase activities, were consistently higher in selenium supplemented cells, whereas glutathione reductase activity was not affected by the selenium. The dose-response was similar for thioredoxin reductase and glutathione peroxidase, but the recovery of glutathione peroxidase activity upon selenium supplementation was faster than with thioredoxin reductase. Also the increase of glutathione peroxidase activities was substantially higher than that of thioredoxin reductase (400-1200% versus a maximum of 250%). These observations clearly indicate a catalytic role of the selenocysteine residue in the thioredoxin reductase, but suggest either the existence of a selenium-unresponsive isoenzyme or a residual disulfide reductase activity in the selenium-free truncated protein made under conditions of selenium deficiency.

Animals↗
PubMed1997

Redox regulation of NF-kappa B activation.

Cytosolic reactions of the nuclear factor kappa B/inhibitor (NF-kappaB/IkappaB) complex leading to its activation, NF-kappaB translocation into the nucleus, DNA binding, and transactivation have been described with some degree of clarity, but the upstream processes that stimulate those cytosolic reactions remain obscure. These processes definitely involve multiple protein serine/threonine kinases, as proximal modifiers of IkappaB, as well as the corresponding phosphatases, upstream kinases, and phosphatases, including those acting on tyrosine residues. This complex cascade of phosphorylation and dephosphorylation is modulated by redox reactions of unknown nature in the sense that the oxidant status of the cytosol increases the phosphorylation and degradation of IkappaB. NF-kappaB action, however, requires a thioredoxin-dependent reduced status in the nucleus. Upstream kinase(s) and or phosphatase(s) prone to thiolation or oxidation of vicinal SH groups are at present considered the best candidates mediating the redox regulation of NF-kappaB.

Animals↗
PubMed1997

A unique cascade of oxidoreductases catalyses trypanothione-mediated peroxide metabolism in Crithidia fasciculata.

Parasitic trypanosomatids comprise causative agents of debilitating or life-threatening tropical diseases. The limited capacity of these parasites to cope with oxidative stress has been discussed as a target area for therapeutic approaches but success has been hampered by a lack of comprehension of their peculiar oxidant defense system depending on the unique redox metabolite trypanothione. Here we report that trypanothione-dependent hydroperoxide metabolism in Crithidia fasciculata is catalysed by two distinct proteins working in concert. One is Cf16, a unique protein which, apart from a WCPPC sequence that resembles the thioredoxin-type WCG(A)PC motif, only shows low similarity to thioredoxin-like proteins of bacteria and invertebrates. The second component is Cf21, which can be classified as a member of the peroxiredoxin family of proteins. The two proteins have been purified to homogeneity and shown to be essential for the trypanothione-dependent removal of hydroperoxides. By means of selective derivatisation of the substrate-reduced proteins the flux of reduction equivalents from trypanothione to Cf16, Cf21 and finally to the hydroperoxide was elucidated. Cf21 proved to be a moderately efficient peroxidase with broad specificity. The rate constants for the reaction of the reduced protein with H2O2, t-butyl hydroperoxide, linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide were 1.0 x 10(5), 1.2 x 10(5), 1.0 x 10(5) and 0.4 x 10(5) M-1S-1, respectively. The apparent rate constant for the regeneration of reduced Cf21 by Cf16 was in the range of 1.5-3.5 x 10(6) M-1S-1. This newly discovered metabolic pathway adds two further candidates to the list of potential targets for trypanocidal drugs.

Amino Acid Sequence↗
PubMed1997

Glutathione peroxidase revisited--simulation of the catalytic cycle by computer-assisted molecular modelling.

Glutathione peroxidase, the first example of selenoproteins identified in mammals, was subjected to force field calculations and molecular dynamics in order to enable a clearer comprehension of enzymatic selenium catalysis. Starting from the established X-ray structure of bovine GPX, all kinetically defined intermediates and enzyme substrate complexes were modelled. The models thus obtained support the hypothesis that the essential steps of the catalysis are three distinct redox changes of the active site selenium which, in the ground state, presents itself at the surface of selenoperoxidases as the center of a characteristic triad built by selenocysteine, glutamine and tryptophan. In GPX, four arginine residues and a lysine residue provide an electrostatic architecture which, in each reductive step, directs the donor substrate GSH towards the catalytic center in such a way that its sulfhydryl group must react with the selenium moiety. To this end, different equally efficient modes of substrate binding appear possible. The models are consistent with substrate specificity data, kinetic pattern and other functional characteristics of the enzyme. Comparison of molecular models of GPX with those of other members of the GPX superfamily reveals that the cosubstrate binding mechanisms are unique for the classical type of cytosolic glutathione peroxidases but cannot operate e. g. in plasma GPX and phospholipid hydroperoxide GPX. The structural differences between the selenoperoxidases, shown to be relevant to their specificities, are discussed in terms of functional diversification within the GPX superfamily.

Animals↗
PubMed1997

Product of the Schistosoma mansoni glutathione peroxidase gene is a selenium containing phospholipid hydroperoxide glutathione peroxidase (PHGPx) sharing molecular weight and substrate specificity with its mammalian counterpart.

In the blood fluke Schistosoma mansoni a functionally active, monomeric, phospholipid hydroperoxide glutathione peroxidase (PHGPx) has been purified and characterized. This enzyme contains a catalytically active selenocysteine. The protein has been shown to be the product of a cloned gene, previously referred to as a glutathione peroxidase gene. S. mansoni PHGPx has been found 5 times more abundant in female than in male worm extract. As in vertebrate PHGPx, homology alignment indicates that the residues involved in the glutathione binding by the tetrametric cellular glutathione peroxidase are mutated in the S. mansoni enzyme. Thus, this aspect appears a landmark of the PHGPx-type of glutathione peroxidases, which might be of functional relevance.

Amino Acid Sequence↗
PubMed1996

A selenium-containing phospholipid-hydroperoxide glutathione peroxidase in Schistosoma mansoni.

The 100000Xg supernatant parasite platyhelminth Schistosoma mansoni exhibits a glutathione peroxidase activity with the substrate phosphatidylcholine hydroperoxide. Purification yielded a protein of 20 kDa molecular mass both on gel filtration column chromatography and SDS/PAGE, thus suggesting that S. mansoni expresses a protein similar to the mammalian selenoenzynic phospholipid-hydroperoxide glutathione peroxidase. Kinetic analysis and substrate specificity corroborated this assumption, the second-order rate constants for the oxidation of the ground-state enzyme (k+1) being higher with phosphatidylcholine hydroperoxide than with other peroxide substrates, such as cumene liydroperoxide or H2O2, and quantitatively similar to those of mammalian phospholipid-hydroperoxide glutathione peroxidase. Partial sequencing of the protein and selenium measurement by neutron activation analysis established that the purified peroxidase corresponded to the product of the S. mansoni gene previously reported and supposed to encode a selenium-containing glutathione peroxidase [Roche, C., Williams, D. L., Khalife, J., LePresle, T., Capron, A. & Pierce, R. J. (1994) Cloning and characterization of gene encoding Schistosoma mansoni glutathione peroxidase, Gene 138, 149 - 152]. S. mansoni thus contains a scienoperoxidase sharing molecular mass, catalytic efficiency and substrate specificity with phospholipid-hydroperoxide glutathione peroxidase, dismantling the concept that those enzymes are unique to vertebrate organisms.

Amino Acid Sequence↗
PubMed1996

Analysis of eukaryotic mRNA structures directing cotranslational incorporation of selenocysteine.

Translation of an mRNA encoding a selenoprotein requires that at least one UGA codon in the reading frame is recoded as a site for the insertion of selenocysteine. In eukaryotes, the termination codon recoding event is directed by a cis-acting signal element located in the 3' untranslated region of the gene. This 'selenocysteine insertion sequence' (SECIS) comprises conserved sequences in a region of extensive base-pairing. In order to study the structure-function relationships of the SECIS structure, we have applied a newly developed reporter gene system which allows analysis of stop codon suppression in animal cell lines. This system obviates the need for enzymatic or immunological estimation of selenoprotein synthesis, relying instead on the simple quantification of translational readthrough from the lacZ gene into the luciferase gene. The 3'-UTR of the phospholipid hydroperoxide glutathione peroxidase (PHGPx) gene was shown to contain a highly active SECIS element. Mutations in the base-paired sequences of other SECIS elements were used to analyse the significance of primary structure, secondary structure and pairing stability in the stem regions. The results demonstrate that the exact sequences of the paired nucleotides are comparatively unimportant, provided that a consensus combination of length and thermodynamic stability of the base-paired structures is maintained.

Animals↗
PubMed1995

Probing the presumed catalytic triad of selenium-containing peroxidases by mutational analysis of phospholipid hydroperoxide glutathione peroxidase (PHGPx).

Single and double site mutants affecting the presumed catalytic centre of the selenoenzyme PHGPx were subjected to functional analysis. The rate constants k+1 and k'+2, for the oxidation and the regeneration of the ground state enzyme were estimated, respectively. Moreover, the alkylation rate of the reactive centre by iodoacetate (kinact.) was also analysed. The substitution of the catalytically competent selenocysteine 46 by cysteine (PHGPxcys46) decreased k+1 and k'+2 by about three orders of magnitude, although leaving unaffected kinact.. Furthermore, mutations of PHGPxcys46 involving the other residues of the triad decreased both kinact. and k+1, thus highlighting the involvement of Gln 81 and Trp 136 in the dissociation/activation of the nucleophilic cysteine thiol. In general, substitutions of Gln 81 or Trp 136 by acidic residues in PHGPxcys46 most dramatically depressed the k+1 values, because they practically prevented the dissociation of the thiol group, while neutral or positively charged residues in these positions allowed an intermediate dissociation and induced a corresponding reactivity of the thiol. Our data, for the first time, reveal that the presumed triad of selenocysteine, glutamine and tryptophan residues represents a novel type of catalytic centre, whose integrity is essential for the full catalytic function of glutathione peroxidases.

Animals↗
PubMed1993

Taxonomical classification of the guinea pig based on its Cu/Zn superoxide dismutase sequence.

Cu-Zn superoxide dismutase was purified from guinea pig (Cavia porcellus) liver up to electrophoretic homogeneity and its amino acid sequence was elucidated by automated Edman degradation of proteolytic fragments and mass spectrometry. The protein was classified as a typical mammalian cytosolic Cu-Zn superoxide dismutase by molecular mass, specific activity, amino acid sequence and N-terminal acetylation. A dendrogram constructed from previously known vertebrate cytosolic Cu-Zn superoxide dismutase sequences reflects the commonly accepted taxonomy and phylogenetic relationships of the species, whereas the guinea pig sequence is similarly remote form muriform rodents, lagomorphs, equiforms and primates. The data appear incompatible with the assumption that the Caviomorpha with the representative Cavia porcellus form a common phylogenetic clade with the muriform rodents but rather have to be considered a distinct order of mammals. The degree of similarity of the sequences further suggests that the mammalian clade diverged into rodents, primates, lagomorphs and caviomorphs at about the same time.

Amino Acid Sequence↗
PubMed1992

High expression vectors for the production of recombinant single-chain urinary plasminogen activator from Escherichia coli.

An expression cassette containing a synonymous gene for human single-chain urokinase-type plasminogen activator (Rscu-PA) 5'-flanked by a trp promoter and the Shine-Dalgarno sequence of the xyl A operon of Bacillus subtilis and terminated by the terminators trp A and Tn10 was constructed and inserted into a pBR322 derivative to yield pBF160. When compared to pUK54 trp 207-1 containing the natural scu-PA gene without the Shine-Dalgarno sequence and terminator, the expression efficiency of pBF160 in Escherichia coli strains was improved by one order of magnitude. Replacement of the trp by the tac promoter (pBF171) did not affect expression. Inserting the Shine-Dalgarno sequence and Tn10 terminator into pUK54 trp 207-1 (pWH1320) slightly increased the expression level, whereas elimination of the Shine-Dalgarno sequence and the terminators from pBF160 with almost complete conservation of the synonymous structural gene (pBF191) significantly reduced the expression. Variation of the distance between the Shine-Dalgarno sequence and the start codon between 8 and 10 bp (pBF163) proved irrelevant. In conclusion, poor expression of mammalian genes in E. coli may result from both improperly designed regulatory elements and structural features of the coding region and therefore de-novo synthesis of the gene may be required to obtain satisfactory expression.

Amino Acid Sequence↗
PubMed1991

Time course of IL-6 and TNF alpha release during endotoxin-induced endotoxin tolerance in rats.

Development of endotoxin tolerance in rats induced by repeated application of low dosages of endotoxin is associated with repeatable IL-6 formation, reversible drop in white blood cells, pronounced consumption of platelets, gradual formation of alpha 2M as an example of acute phase proteins, and flattening TNF alpha formation. In the status of full tolerance the TNF alpha release is completely eliminated. Inhibition of TNF alpha biosynthesis and induction of acute phase protein formation by IL-6 are discussed as possible factors in the development of endotoxin tolerance.

Acute-Phase Proteins↗
PubMed1991

Immunohistological detection of Saruplase (recombinant single-chain urokinase-type plasminogen activator) in normal rat tissue.

Saruplase--a recombinant single-chain urokinase-type plasminogen activator was identified immunohistochemically in normal rat tissue after intravenous administration by means of a polyclonal antibody. For this purpose, rat tissues were fixed in various ways (liquid nitrogen, ethanol, formaldehyd solution). Saruplase could be detected by the PAP method, streptavidinbiotin system and indirect immunofluorescence in the kidney (proximal tubule), liver (hepatocytes, Kupffer cells) and spleen (reticular cells). Saruplase was not localized in the rat endothelium. It is discussed that the rat-specific receptors for urokinase-type plasminogen activator on endothelial cells cannot bind Saruplase due to the extreme species specificity.

Animals↗
PubMed1991

Phorbol ester cardiotoxicity: are .O2-radicals involved?

The effect of superoxide dismutase (SOD) on cardiotoxic effects induced by phorbol 12-myristate 13-acetate (PMA) was investigated in isolated perfused rat hearts. The negative inotropic effect, vasoconstriction, and mild bradycardia were uneffected by SOD in the perfusion medium. Perfusion of Langendorff hearts with PMA and oxidized cytochrome c +/- SOD gave no evidence for a formation of .O2 radicals in response to PMA. Cytochrome c reduction detected in the perfusate of PMA-treated Langendorff hearts is not inhibited by SOD and has thus to be attributed to .O2-independent myocardial reducing systems.

Animals↗
PubMed1991

Phospholipid hydroperoxide glutathione peroxidase is a selenoenzyme distinct from the classical glutathione peroxidase as evident from cDNA and amino acid sequencing.

The primary structure of phospholipid hydroperoxide glutathione peroxidase (PHGPx) was partially elucidated by sequencing peptides obtained by cyanogen bromide cleavage and tryptic digestion and by isolating and sequencing corresponding cDNA fragments covering about 75% of the total sequence. Based on these data PHGPx can be rated as a selenoprotein homologous, but poorly related to classical glutathione peroxidase (GPx). Peptide loops constituting the active site in GPx are, however, strongly conserved in PHGPx. This suggests that the mechanism of action involving an oxidation/reduction cycle of a selenocysteine residue is essentially identical in PHGPx and GPx.

Amino Acid Sequence↗