Sporothrix schenckii, mainly in the yeast form of the organism, produced extracelluilar proteinases when cultivated in liquid media containing albumin or collagen as a nitrogen source, but did not do so in brain heart infusion medium. Isolation of two extracellular proteinases from albumin-containing medium was performed by chromatography on DEAE-Sepharose CL-6B and Sephacryl S-200. Proteinase I had a molecular weight of 36,500, an optimal pH at 6.0, and a pI at 4.8. Despite its activities in weakly acidic conditions, proteinase I demonstrated chymotrypsinlike characteristics, these being .indicated by strong inhibitory activity by phenylmethylsulfonyl fluoride and chymostatin and good kinetic constants for a synthetic chymotrypsin substrate, Suc-Ala-Ala-Pro-Phe-MCA. Proteinase II had a molecular weight of 39,000, an optimal pH at 3.5, and a pl at 3.8. Proteinase II showed cathepsin D-like characteristics, these being indicated by strong inhibitory activity by pepstatin, an acidic optimal pH, and good kinetic constants for hemoglobin. These two enzymes hydrolyzed natural substrates such as stratum corneum, type I collagen, and elastin although not type IV collagen. Proteinase production and cell growth in collagen-containing medium and the enzymatic digestion of skin constituents by isolated proteinases suggested that these two proteinases cooperatively enable the organism to invade skin and to obtain peptides from insoluble proteins. proteinases, 200-ml samples of BSA-containing media in 500-ml flasks were incubated under the same conditions as described above for 1 week.Assay of proteinase activity. Proteinase activity was assayed with Azocoll (Sigma) as a substrate. A 5-mg sample of Azocoll in 50 mM citric acid-100 mM disodium phosphate buffer was incubated with 50 to 100 ,ul of enzyme solution (total volume, 1.0 ml) for 1 h at 37°C. Incubation was terminated by the addition of 2 ml of cold distilled water. After centrifugation at 1,500 x g for 10 min, released dye in the supernatant was measured at 520 nm with a Hitachi spectrophotometer (model 100-60). One unit of proteinase activity was defined as the amount of enzyme which produced an increase in absorbance of 0.10 under the abovementioned conditions. Determination of protein concentration. Protein concentration was determined by the method of Lowry et al. (9) (Amicon Corp., Lexington, Mass.). After centrifugation at 1,500 x g for 15 min, the supernatant was applied to a DEAE-Sepharose CL-6B column (1.2 by 11 cm) which had been previously equilibrated with the same 4104
Sporothrix schenckii produces two extracellular proteinases in albuminor collagen-supplemented unbuffered liquid medium. Proteinase I had an optimal pH of 6.0, and its activity was strongly inhibited by chymostatin. Proteinase Il had an optimal pH of 3.5, and its activity was strongly inhibited by pepstatin. Speculating that these two proteinases are key enzymes for fungal growth, we investigated the influences of culture medium pH and either or both of the proteinase inhibitors pepstatin and chymostatin on the cell growth of S. schenckii. In buffered medium at a pH of 3.5, an optimal pH for proteinase 11, only proteinase II activities were observed, while at pH 6.0, an optimal pH for proteinase I, only proteinase I activities were observed. However, there was no cell growth inhibition. These results suggested that the regulation of the production of the two proteinases is dependent on environmental pH. The addition of pepstatin or chymostatin to the culture medium did not inhibit the cell growth of S. schenckii, but the addition of both inhibitors at a concentration of 10 ,Lg/ml strongly inhibited growth. These results suggested that at least one of the two proteinases was expressed to allow fungal growth in albumin-supplemented media. The indirect fungistatic action of the proteinase inhibitors, which inhibit proteolysis, may be applied to a topical therapeutic agent in vivo.
Antibodies against the cytoplasm of normal human epidermal cells were found in both a newborn baby with incontinentia pigmenti and in her normal mother. These antibodies showed no tissue or species specificity as in the case of anti‐nuclear antibodies in SLE. The antibodies were assayed by indirect immunofluorescence. The antibody titer in the serum of the baby gradually decreased over a period of months while the mother's titer remained high. The generalized vesiculo‐bullous eruption noted at birth subsided with the decrease of antibody titer. The immunoglobulin class of the antibodies was determined to be IgG, which passes through the placental barrier. It is suggested that the anti‐cytoplasmic antibodies produced by some unknown cause in pregnant women could be transferred to the baby and induce a variety of disorders.
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