Human fibroblasts in primary culture released reactive oxygen species upon stimulation with cytokines such as interleukin-1 alpha (IL-1) or tumour necrosis factor-alpha (TNF). The primary radical produced was O2.- as determined by e.s.r. spin trapping and cytochrome c reduction. In contrast to the oxidative burst in granulocytes and monocytes, radical formation took place continuously for at least 4 h. Low-level chemiluminescence was increased by stimulation with IL-1 and TNF. Spectral characteristics and tests with azide led to the conclusion that the photoemissive species were excited carbonyls and not singlet oxygen. Further, there was a liberation of ethane from the cells. Radical production and light emission were not altered by either xanthine or allopurinol, nor by azide, cyanide or rotenone. O2.- production increased in the presence of NADH or NADPH, making an NAD(P)H oxidase a likely source.

Adherent human mesangial cells (HMC) were unable to phagocytose serum-treated zymosan (STZ), nevertheless this stimulus (1 mg/ml) induced a marked immediate increase of H2O2 and O2- release at a rate of 3.15 +/- 0.35 and 3.40 +/- 0.12 nmol/10(6) HMC/hr, respectively. Zymosan alone resulted in no release of either H2O2 or O2-. Phorbol myristate acetate (PMA, 2 X 10(-6) M) had only marginal effects on HMC leading to the generation of 0.273 +/- 0.014 nmol O2-/10(6) HMC/hr. After a lag period, human recombinant tumor necrosis factor-alpha (TNF-alpha) and human recombinant interleukin 1-alpha IL-1 alpha) both induced significant O2- production measured as SOD inhibitable reduction of cytochrome c, 5 X 10(-5) M, by adherent HMC for up to five hours, the maximum rates being 3.04 +/- 0.08 and 3.2 +/- 0.08 nmol/10(6) HMC/hr for IL-1 alpha and TNF-alpha, respectively. Significant O2- release was detectable at 0.625 ng/ml (37 pM) IL-1 alpha or 1 ng/ml (59 pM) TNF-alpha (P less than 0.05). Catalase inhibitable H2O2 production was also induced by IL-1 alpha and TNF-alpha in a dose dependent manner. Using scopoletin (40 nM) and 1 microM peroxidase we fluorimetrically measured 1.73 +/- 0.14 and 1.49 +/- 0.19 nmol H2O2/10(6) HMC/hr induced by IL-1 alpha (25 ng/ml) and TNF-alpha (20 ng/ml). Finally, we ascertained the type of radical species produced by HMC stimulated by cytokines employing ESR-spin-trapping with DMPO.2+ These results demonstrated that O2- was the primary radical species formed.(ABSTRACT TRUNCATED AT 250 WORDS)

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Aus dem Hautextrakt des in Panamavorkommenden,auffallend gefarbten Froschs Derrrlrobufes punrilio laBt sich das gut kristallisierende Hydrochlorid einer Base Pumiliotoxin C der Formel C~~H~S N isolieren. Durch Rontgen-Strukturanalyse dieser Base nach den1 ,,symbolischert ,4dditionsverfuhren" (symbolic addition procedure) IaRt sich zeigen, daR es sich urn einen zum ersten Ma1 in der Natur beobachteten, dem Coniin weitllufig verwandten Ciftstofftyp handelt, namlich urn ein 2-n-Propyl-5-methyl-cis-decahydrochinolin, dessen vier asymmetrische Zentren in ihrer relativen stereochemischen Beziehung aufgeklart werden. Neben Pumiliotoxin C leiten sich auf Grund der Massenspektren die Pumiliotoxine A und B sowie noch weitere aus dern verwandten Frosch Dendrobates aiirufus isolierte Basen von einem ahnlichen ris-Deca-oder Octahydrochinolin-System ab.Frog Verronts. Isohtion find Strircture of Punrilioto.uin C1)Pumiliotoxin C, a base of the composition C I~H~~N , is isolated as the crystalline hydrochloride from the skin extracts of Dendrobures pundio, a strikingly colored Panamanian frog. Complete X-ray analysis of this new venom by the so-called symbolic addition procedure establishes the structure as 2-n-propyl-5-rnethyl-cis-decahydroquinoline, a type of base, though related to coniine, is here reported for the first time as a natural product. The relativc configurations of the four asymmetric centers are elucidated. In addition to this venom there are the congeners pumiliotoxins A and B. as well as further homologs and analogs which have also been isolated from the related frog Dendrohufesurrrcr/rru, and characterized by mass spectra. 1) Diese sich uber den Ozean erstreckende Gemeinschaftsarbeit zwischen Washington undDarmstadt ist dem Altmeister der Naturstoffchemie, Clenrerrs Schiip/; zum 70. Geburtstag mit allen guten Wunschen gewidmet.

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