Beate Meier

Induction of oxidative stress by okadaic acid is required for activation of transcription factor NF-kappa B.

The widely used phosphatase 1 and 2A inhibitor okadaic acid is one of the many stimuli activating transcription factor NF-κB in cultured cells. Phosphorylation of IκB-α, one of NF-κBs inhibitory subunits, is a prerequisite for IκB degradation and the subsequent liberation of transcriptionally active NF-κB. This observation suggested that the phosphorylation status of IκB is influenced by an okadaic acid-sensitive phosphatase. In this study, we provide evidence that the effect of okadaic acid on NF-κB activation is indirect and dependent on the production of reactive oxygen intermediates rather than the inhibition of an IκB-α phosphatase. Okadaic acid was found to be a strong inducer of cellular H2O2 and superoxide production in two distinct cell lines. The structurally unrelated phosphatase inhibitor calyculin A also induced oxidative stress. The delayed onset of reactive oxygen production in response to okadaic acid correlated with the delayed activation of NF-κB. Moreover, NF-κB induction was optimal at the same okadaic acid concentration that caused optimal H2O2 production. Both reactive oxygen intermediates production and NF-κB activation were inhibited by the antioxidant pyrrolidine dithiocarbamate and 8-(diethylamino)octyl-3,4,5-trimethyoxybenzoate, a Ca2+ chelator. Future experiments using phosphatase inhibitors in intact cells must consider that the compounds can act as strong inducers of oxidative stress, which provides one explanation for their tumor-promoting activity.

HUMAN FIBROBLASTS RELEASE REACTIVE OXYGEN SPECIES IN RESPONSE TO TREATMENT WITH SYNOVIAL FLUIDS FROM PATIENTS SUFFERING FROM ARTHRITIS

Human fibroblasts in primary culture released reactive oxygen species upon exposure to synovial fluid obtained by joint aspiration from twelve patients suffering from rheumatoid arthritis. The primary radical produced was O2- as determined by ESR spin trapping and cytochrome c reduction. In contrast to the oxidative burst in granulocytes and monocytes, radical formation proceeded continuously for at least four hours. Low-level chemiluminescence was increased upon exposure to inflammatory human synovial fluids. Spectral characteristics and effects of azide and 1,4-diazabicyclo-(2,2,2)-octane led to the conclusion that the photoemissive species were excited carbonyls. Radical production and light emission were not altered either by xanthine or allopurinol, nor by azide, cyanide or rotenone. The O2- production increased in the presence of NADH or NADPH, making an NAD(P)H oxidase a likely source. The liberation of reactive oxygen species correlated with the number of leukocytes present in the inflammatory joint fluids, but not with the concentrations of immunoglobulins and complement factor C3.

Evidence for superoxide dismutase and catalase in mollicutes and release of reactive oxygen species

The presence of superoxide dismutase was demonstrated in 21 strains of mollicutes, including achuloplasmas, mycoplasmas, and ureaplasmas. No superoxide dismutase activities or only traces were detectable in fresh prepared cell lysates, whereas activities were evident after dialysis of the cell lysates. A further increase in superoxide dismutase activities was observed after the cell lysates were heated to 65 degrees C for 30 s. This might be due to the destruction of enzymatic reactions interfering with the activity tests. Additionally, catalase activities were demonstrated in nearly 50% of the cell lysates, whereas no peroxidase activities were detectable. The production of O2- and H2O2 with glucose as substrate was demonstrated for 8 of 10 strains tested. No correlation to the pathogenicities of the strains was indicated. Anaerobic mycoplasmas showed the highest amount of radical production, whereas superoxide dismutase and catalase activities were in the range of activities estimated for aerobic mollicutes.

In vivo incorporation of cobalt into Propionibacterium shermanii superoxide dismutase

Propionibacterium shermanii, an aerotolerant anaerobic bacterium, has already been shown to incorporate, depending on the metal supplementation to the medium, either iron or manganese or copper into the same superoxide dismutase protein. The in vivo incorporation of cobalt in the same superoxide dismutase was obtained in an iron‐, manganese‐ and copper‐depleted medium. The protein was isolated and characterized by NMR which offers the possibility to identify the amino acid residues at the active site exploiting isotropically shifted proton resonance.

Regulation of the Superoxide Releasing System in Human Fibroblasts

Fibroblasts release low amounts of reactive oxygen intermediates (ROIs) upon contact with appropriate stimuli, which have regulatory functions in inter- and intracellular signal transduction. Ca2+/calmodulin are involved in the activation process of the ROI-generating system. A rise of intracellular Ca2+ is necessary, but not sufficient to trigger the release of ROIs. As a second signal, protein phosphorylation seems to be involved in the activation process.

Evidence for Superoxide Dismutase and Catalase in Mollicutes and Release of Reactive Oxygen Species

The presence of superoxide dismutase was demonstrated in 21 strains of mollicutes, including achuloplasmas, mycoplasmas and ureaplasmas. Additionally, catalase activities were demonstrated in nearly 50% of the cell lysates, whereas no peroxide activities were detectable. The production of O2- and H2O2 with glucose as substrate was demonstrated for 8 strains of 10 strains tested. Anaerobic mycoplasmas showed the highest amount of radical production, whereas superoxide dismutase and catalase activities were in the range of activities estimated for aerobic mollicutes. Some pathogenic strains additionally released compounds into the culture medium, which stimulated O2- production by PMNs.

Comparative Studies on a Superoxide Dismutase Exhibiting Enzymatic Activity with Iron and Manganese as Active Cofactor

The SOD of Propionibacterium freudenreichii, ssp. shermanii belongs to a new group of SODs capable of retaining activity with either Fe or Mn as active metal cofactor. Both enzymes exhibit identical secondary structure and immunological determinants. Hydrogen peroxide irreversibly inhibits both enzymes. The protein moiety of the Fe- and Mn-SOD could be digested with trypsin to a single active fragment.