Modulation of the 5-lipoxygenase activity of MC-9 mast cells: Activation by hydroperoxides

In order to identify regulatory steps in leukotriene synthesis, the biochemical characteristics of a 5-lipoxygenase activity in the 100,000 xg supernatant from sonicates of cells of an IL-3 dependent murine mast cell clone, MC-9 were determined. Principal products from exogenous ¹⁴C-arachidonic acid were identified as leukotriene B₄, diastereomeric 5,12-dihydroxy-eicossatetraenoic acids (5.12 diHETEs) 5-hydroperoxy and hydroxyeicosatetraenoic acids (5-HPETE and 5-HEYE) as well as a novel metabolite 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). The lipoxygenase activity had a pH optimum of 6.9 and was highly dependent upon added Ca⁺⁺. The effective Ca⁺⁺ concentration for 50 per cent activation (EC₅₀) was 3 uM. Activity was also stimulated by ATP (EC₅₀ = 160 uM). The cytosolic 5-lipoxygenase activity exhibited a biphasic concentration dependence for arachidonic acid with maximum product formation occurring at 35 uM (ca. 20 nmole/mg/4 min). The lipoxygenase activity exhibited apparent lag phase kinetics which were more pronounced at low protein concentrations (0.3 mg/ml). In addition, the lag phase was greatly accentuated by the addition of a hydroperoxide scavenging system consisting of glutathione (1 mM) plus glutathione peroxidase (0.4 unit/ml). In contrast, addition of any several hydroperoxides, i.e. 5-,8-,9- or 15-HPETE (EC₅₀ ca. 1 uM), but not the corresponding alcohols (5-HETE and 15-HETE), shortened the lag phase. These results show that the 5-lipoxygenase requires hydroperoxide for activation and that cellular level of hydroperoxides may be an important factor regulating leukotriene synthesis.     

Studies of cryptococcus nigricans n. sp. I. Identification and taxonomic classification

Summary An asporogenous, non-filamentous, black pigmented yeast-like fungus, isolated from paint scrapings, was characterized and identified as a heretofore undescribed species of the genusCryptococcus. Because of its jet-black pigmentation, the nameCryptococcus nigricans was proposed.This study was supported in part by a grant-in-aid, from the Rutgers Research Council.     

Studies on cryptococcus nigricans,II. The effects of physical,chemical and nutritional factors

Summary 1. A chemically defined, minimal medium was developed for the cultivation ofCryptococcus nigricans (glucose, nitrate, thiamin, biotin, and inorganic salts).2. Optimum temperature was found to be 29° C with upper and lower limits at 34° C and 23° C.3. Optimum pH for growth was found to be pH 5.0–5.6.4. Ammonium nitrogen was not assimilated.5.C. nigricans was inhibited by Mycostatin, Amphotericin A and B, and Polymyxin B.This study was supported in part by a grant-in-aid from the Rutgers Research Council.