Diversity of glutathione peroxidases.
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Amino Acid Sequence↗
Biomedical subjects
L Flohé
Publications and source records attributed to L Flohé.
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↗
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↗
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↗
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↗
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↗
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↗
Inhibitory characteristics and oxidant resistance of site specific variants of recombinant human antileukoproteinase (ALP).
Tandem gene plasmids were constructed and used to express inactive proteins equivalent to human antileukoproteinase (ALP) and the variants [Leu73]-ALP and [Leu73, 82, 94, 96]-ALP in E. coli K12. After extraction, refolding, and purification, highly pure and active inhibitors were obtained in good yields. Inhibitory constants for human leukocyte elastase and cathepsin G were found to be similar. The variants in which methionines were exchanged for leucines were shown to be more resistant to inactivation by oxidizing agents than native ALP. As oxidizing conditions exist at sites of inflammation, these ALP variants are promising candidates for therapies involving suppression of elastase-mediated injury.
Escherichia coli↗
Effects of recombinant human superoxide dismutase on increased lung vascular permeability and respiratory disorder in endotoxemic rats.
A 4 hr intravenous infusion of Escherichia coli endotoxin in a total dose of 100 mg/kg produced significant morphological and functional pulmonary alterations in pentobarbitone anesthetized rats. Lung vascular permeability index was increased from 2.11 +/- 0.34 in normal rats to 4.82 +/- 0.65 in untreated endotoxemic rats. Treatment of endotoxemic rats with recombinant human superoxide dismutase (r-HSOD) in doses of 0.1, 0.215, and 0.464 mg/kg.min i.v., infused concomitantly with endotoxin, dose-dependently reduced the permeability index to 3.28 +/- 0.96, 2.83 +/- 0.55 (P less than 0.05), and 2.16 +/- 0.65 (P less than 0.05). The wet lung weight was 523 +/- 15 and 664 +/- 46 mg/100 g bwt in normal and in untreated endotoxemic rats, respectively. r-HSOD dose-dependently inhibited the endotoxin-induced increase in wet lung weight to 617 +/- 40, 577 +/- 31, and 559 +/- 39 (P less than 0.05) mg/100 g bwt. r-HSOD (0.464 mg/kg.min) did not affect permeability index and wet lung weight in normal, nonintoxicated rats. Endotoxin infusion produced a significant increase in respiratory rate (max. +69%) and blood gas alterations, indicating a hyperventilatory hypocapnia in endotoxemic control rats. Infusion of r-HSOD (0.464 mg/kg.min) significantly inhibited the endotoxin-induced tachypnoe (max. +13%) and blunted the alterations in arterial hydrogen carbonate content and carbon dioxide tension. In conclusion, infusion of r-HSOD dose-dependently and significantly inhibited pulmonary edema formation and hyperventilatory dyspnoe in endotoxemic rats.
Animals↗
Additive fibrinolysis by recombinant tissue-type plasminogen activator (r-t-PA) and recombinant single-chain urokinase type plasminogen activator (r-scu-PA) in rabbit pulmonary thrombosis.
The mode of interaction between recombinant tissue-type plasminogen activator (r-t-PA) and recombinant single-chain urokinase-type plasminogen activator (r-scu-PA) has been investigated in in vivo experiments. 125J-fibrin-labeled clots were embolized via a jugular vein into the lungs of anesthetized rabbits. In saline-treated control rabbits the spontaneous lysis, shown as decrease in radioactivity of the retrieved clots, amounted to 10.4 +/- 1.4% at 255 min after the pulmonary embolization. r-t-PA (0.464 - 4.64 micrograms/kg.min) and r-scu-PA (4.64 - 46.4 microns/kg.min), infused for 60 min, produced dose-dependent lytic effects to a similar extent (maximum lysis rate 53.9 +/- 5.8 and 55.4 +/- 7.2%, resp.). When various ratios of submaximal doses of r-t-PA and r-scu-PA were combined the lytic effects of these combinations were not higher than the calculated summation of the lysis rates by the single components. The fractional dose-response curves of r-t-PA and r-scu-PA and the combination of them, fitted by linear regression analysis, are overlaying each other. The results indicate that r-t-PA and r-scu-PA produce in vivo lysis in rabbits with pulmonary embolized clots in a purely additive manner.
Animals↗
Leukotriene formation by human polymorphonuclear leukocytes from endogenous arachidonate. Physiological triggers and modulation by prostanoids.
Human polymorphonuclear leukocytes (PMN) were isolated from freshly drawn venous blood by Dextran sedimentation and discontinuous Percoll gradient centrifugation. The effects of several putative triggers of the leukotriene formation such as C5a, PAF, FMLP, C3a, PMA, LTC4, LTD4, LTB4 or arachidonate were studied by RP-HPLC analysis. 280 nM C5a, 100 nM FMLP, 1 microM PAF or 20 microM arachidonate induced a marginal formation of 1.5-18 ng of LTB4 plus LTB4 metabolites/2 x 10(7) PMN. 560 nM C3a, 100 nM PMA, 1 microM LTC4, 1 microM LTD4 and 1 microM LTB4 each failed to induce any formation of 5-lipoxygenase products. Pretreatment of the cells with 40 microM ethylmercurithiosalicylate (merthiolate) enhanced the leukotriene formation by 100 nM FMLP about 40-fold, by 280 nM C3a about 120-fold and by 1 microM PAF about 14-fold. Merthiolate itself induced no leukotriene formation from human PMN and reduced the leukotriene formation by 20 microM arachidonate. The FMLP/merthiolate-induced activation of the PMN was concentration-dependent in respect to both FMLP and merthiolate. 1 microM LTC4, 1 microM LTD4 or 1 microM LTB4 also failed to trigger any LTB4 formation of merthiolate-treated PMN. 560 nM C3a or 100 nM PMA in combination with 40 microM merthiolate induced a slight formation of 28 ng and 10 ng of LTB4 plus LTB4 metabolites, respectively. The FMLP/merthiolate-induced leukotriene formation was modulated by prostanoids. PGE2, PGE1, PGD2 and 6-keto-PGE1 each evoked a concentration-dependent inhibition of the leukotriene formation with IC50 values of 0.07 microM, 0.18 microM, 0.27 microM and 6 microM respectively. In addition, significant inhibitory effects by PGI2, Iloprost (a carbacyclin analogue of prostacyclin), PGF2a or 6-keto-PGF1a were achieved; the corresponding IC50 values, however, amounted to 19-59 microM. Thus these compounds were about 500-fold less potent in comparison with PGE2 in inhibiting LTB4 formation by human PMN.
Arachidonic Acids↗
Cooperative action of the prostacyclin analogue taprostene and the fibrinolytic prourokinase (r-scu-PA) in experimental artery thrombosis.
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Animals↗
Additional myocardial salvage by coadministration of the epoprostenol analog taprostene to recombinant single-chain urokinase-type plasminogen activator in a canine coronary thrombosis model.
In open chest dogs myocardial ischemia was induced by formation of an occlusive thrombus in the left anterior circumflex artery (LCX). Reperfusion of the LCX was achieved by infusion of the fibrin specific recombinant single-chain urokinase-type plasminogen activator (r-scu-PA). The myocardial salvage by r-scu-PA alone and in combination with the epoprostenol (prostacyclin) analog taprostene (CG 4203) was compared. There were four experimental groups: group 1 (n = 4) did not receive any treatment after LCX thrombosis; in group 2 (n = 9) at 100 min after LCX thrombosis r-scu-PA (20 micrograms.kg-1.min-1 i.v. for 30 min) was infused; in groups 3 and 4 treatment with taprostene started concomitantly with r-scu-PA infusion. The taprostene infusions lasted for 120 min and the doses were 0.1 microgram.kg-1.min-1 in group 3 (n = 6) and 0.215 microgram.kg-1.min-1 in group 4 (n = 6). Time to r-scu-PA-induced recanalisation ranged from 18-22 min with no significant difference between groups 2-4. Percent of left ventricle at risk did not differ between the groups. Infarct size as percent of the risk zone was 48.3 +/- 7.7 in group 1, 25.3 +/- 3.7 in group 2, 21.3 +/- 6.5 in group 3 and 17.1 +/- 3.5 in group 4 (p less than 0.05 groups 2-4 vs group 1). Incidence of ectopic beats increased after r-scu-PA-induced reperfusion in groups 2-4, but was significantly reduced by taprostene.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Inhibition by prostacyclin analogs of .O2- production in human polymorphonuclear leukocytes.
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Humans↗
Superoxide dismutase for therapeutic use: clinical experience, dead ends and hopes.
The clinical trials performed with bovine superoxide dismutase (SOD) are reviewed. SOD, applied intraarticularly at a dosage of 2-16 mg, proved to be effective in osteoarthritis of the knee joint in three placebo-controlled and one steroid-controlled double-blind trials. Its efficacy in other inflammatory joint disorders is documented by uncontrolled trials. Similarly, some controlled and many open studies support the efficacy of locally injected SOD in periarticular inflammation. Systemic treatment of rheumatoid arthritis by SOD at the dosages indicated yielded disappointing results. Well documented, though open uncontrolled studies demonstrated beneficial effects of locally administered SOD in radiation cystitis, interstitial cystitis and Peyronie's disease. Tolerance is good, but allergic reactions at low incidence have to be anticipated. Human SOD derived from recombinant microorganisms is being developed to explore its therapeutic potential particularly in ischemia-reperfusion damage, adult respiratory distress or similar conditions.
Animals↗
Kinetic studies on arachidonate 5-lipoxygenase from rat basophilic leukemia cells.
Arachidonate 5-lipoxygenase of a 10,000 x g supernatant from RBL-1 cell homogenate was studied by a continuous assay measuring enzyme-catalysed oxygen consumption. Parallel HPLC and TLC analysis of arachidonic acid metabolites revealed that the oxygen consumption measured is solely due to 5-lipoxygenation of arachidonic acid. Oxygen consumption by this lipoxygenase was strictly dependent upon Ca2+, ATP and 5-HPETE. Removal of any of these three cofactors caused a complete inhibition of enzyme activity. Addition of the missing cofactor instantly restored the 5-lipoxygenase-dependent consumption of oxygen which remained linear for 10-20 s. Later on the velocity of the reaction decreased and after 2-3 min the enzyme became inactivated. Kinetic data were obtained from the initial velocity of the reaction using constant and saturating concentrations of CaCl2 and ATP. From Lineweaver-Burk plots substrate inhibition is evident for arachidonic acid concentrations greater than 45-50 microM. Km(app) for arachidonic acid is 182 +/- 16 microM (mean +/- SD, n = 5) and Vmax(app) is 425 +/- 140 nmol O2/(min x mg protein) (mean +/- SD, n = 5).
Animals↗
Effects of prostacyclin analogs on cyclic adenosine monophosphate and superoxide formation in human polymorphonuclear leukocytes stimulated by formyl-methionyl-leucyl-phenylalanine.
The modulation of cyclic AMP levels and superoxide release in isolated FMLP-stimulated human PMN by two oxacyclic analogs and one carbacyclic analog of PGI2 and by PGE1 is investigated over a wide range of concentrations of the test compounds. The prostacyclin analogs only marginally increase the cyclic AMP levels in unstimulated PMN but like PGE1 potentiate the FMLP-induced rise in cyclic AMP. The concentration dependency is bell-shaped with a maximum effect at about 10 microM of the prostanoids. In contrast, all prostanoids dose-dependently inhibit FMLP-induced superoxide release almost to completion. The relative inhibitory potency of the prostacyclin analogs corresponds to their prostacyclin-like action in other systems. It is therefore suggested that PMN contain prostacyclin receptors, which, however, have weaker affinities than those in platelets. The lack of correlation between inhibition of superoxide formation and modulation of the cyclic AMP system rules out the possibility that cyclic AMP can simply be considered the second messenger of prostacyclins in PMN. The potential biological relevance of the effects of prostacyclin-like compounds on PMN functions is discussed.
Cyclic AMP↗