Mycobacterium tuberculosis Catalase Inhibits the Formation of Mast Cell Extracellular Traps.

Tuberculosis is one of the leading causes of human morbidity and mortality. Mycobacterium tuberculosis (Mtb) employs different strategies to evade and counterattack immune responses persisting for years. Mast cells are crucial during innate immune responses and help clear infections via inflammation or by direct antibacterial activity through extracellular traps (MCETs). Whether Mtb induce MCETs production is unknown. In this study, we report that viable Mtb did not induce DNA release by mast cells, but heat-killed Mtb (HK-Mtb) did. DNA released by mast cells after stimulation with HK-Mtb was complexed with histone and tryptase. MCETs induced with PMA and HK-Mtb were unable to kill live Mtb bacilli. Mast cells stimulated with HK-Mtb induced hydrogen peroxide production, whereas cells stimulated with viable Mtb did not. Moreover, MCETs induction by HK-Mtb was dependent of NADPH oxidase activity, because its blockade resulted in a diminished DNA release by mast cells. Interestingly, catalase-deficient Mtb induced a significant production of hydrogen peroxide and DNA release by mast cells, indicating that catalase produced by Mtb prevents MCETs release by degrading hydrogen peroxide. Our findings show a new strategy employed by Mtb to overcome the immune response through inhibiting MCETs formation, which could be relevant during early stages of infection.

Effects of Freeze-Thaw and Storage on Enzymatic Activities, Protein Oxidative Damage, and Immunocontent of the Blood, Liver, and Brain of Rats.

Most scientific studies are too long to be conducted in a single day or even in a few days. Thus, there is a need to store samples for subsequent investigations. There is sparse information about specific sample storage protocols that minimize analytical error and variability in evaluations of redox parameters. Therefore, the effects of storage temperature and freezing time on enzymatic activities, protein oxidative damage, and CAT (catalase) and SOD1 (superoxide dismutase) immunocontent of blood, liver, and brain from rats were determined for two different sample forms (frozen homogenized tissue or frozen intact tissue). Superoxide dismutase activity was drastically decreased in blood and liver with an increase in freezing time, but not in brain. Catalase activity showed a decrease only in intact liver at -20 and -80°C. In contrast, in blood it showed an increase in intact tissue at -20 and -80°C. Reduced thiol groups generally decreased with freezing time, but showed an increase in intact blood at -20 and -80°C, probably because of color interference. Carbonyl groups in homogenized liver and brain, and in intact blood (except at 80°C) drastically increased with freezing time. Freezing time did not modulate the immunocontent of CAT and SOD1 levels in any tissue. In conclusion, our results indicate that storage at -20°C affects redox parameters more than storage at -80°C. Storage for a long time may compromise the samples, leading to changing parameters due to oxidative stress. Thus, we suggest processing the samples as soon as possible. However, if this is not possible, then material can be aliquoted into different tubes to prevent the effect of refreezing of samples.

Effect of Prototheca zopfii on neutrophil function from bovine milk.

This study was carried to investigate neutrophil function in the presence of Prototheca zopfii. For this purpose, bovine milk neutrophils were incubated in the absence (control) of and presence of P. zopfii, and then they were examined hydrogen peroxide (H(2)O(2)) production, antioxidant enzyme activities, and phagocytic capacity. Milk was collected from negative "California Mastitis Test" (CMT) quarter from three lactating Holstein cows after induction of leukocytosis with an intramammary infusion of oyster glycogen. H(2)O(2) production was measured using the phenol red method. Catalase activity was measured following H(2)O(2) reduction at 240 nm and the activity of glutathione reductase was determined by measuring the rate of NADPH oxidation at 340 nm. P. zopfii death was assessed by fluorescent microscopy using acridine orange assay and by colony forming units (CFUs). Comparisons between the groups were initially performed by analysis of variance (ANOVA). Significant differences were then compared using Tukey's test with a significance coefficient of 0.05. Hydrogen peroxide production, catalase and glutathione reductase activities by neutrophils incubated in presence of P. zopfii were stimulated five times, 21% and 27% respectively, compared to the unstimulated-neutrophils. Neutrophils did not affect P. zopfii death as shown by microscopy and CFUs. These observations led to the conclusion that the P. zopfii promote a high increase of H(2)O(2) production by neutrophils from bovine milk during algae exposition accompanied by increase of antioxidant enzyme activities; however, this process did not affect P. zopfii death.

Purification, properties and immunological detection of a bromoperoxidase-catalase from Streptomyces venezuelae and from a chloramphenicol-nonproducing mutant.

A new bromoperoxidase-catalase was purified from the chloramphenicol-producing actinomycete Streptomyces venezuelae ISP 5230. The homogeneous enzyme showed brominating activity, catalase activity and a very low peroxidase activity. The spectral properties and pH dependence of the catalase activity showed similarities to conventional catalases. In contrast to other haem-bromoperoxidases, the bromoperoxidase-catalase was stable when treated with an ethanol/chloroform mixture. Gel filtration gave an estimated Mr of 127,000-136,000. SDS-PAGE showed a single band corresponding in mobility to a species with an Mr of 61,000. The pI was estimated to be 4.5. The bromoperoxidase-catalase was not present in active form in a mutant of S. venezuelae ISP 5230, blocked in the chlorination step of chloramphenicol biosynthesis. However, an inactive species of the enzyme was detected in crude extracts of the mutant by using antibodies. From these results it is concluded that this bromoperoxidase participates in the chlorination step during chloramphenicol biosynthesis.