The efficiencies of NADPH-dependent phenol hydroxylase (EC 1.14.13.7) and catechol 1,2-dioxygenase (EC.1.13.11.1) in biodegradation of phenol in the cytosolic fraction isolated from yeast Candida tropicalis were investigated. Enzymatic activities of both NADPH-dependent phenol hydroxylase and catechol 1,2-dioxygenase were detected in the cytosolic fraction of C. tropicalis grown on medium containing phenol. Using the procedure consisting of chromatography on DEAE-Sepharose, fractionation by polyethylene glycol 6000 and gel permeation chromatography on Sepharose 4B the enzyme responsible for phenol hydroxylation in cytosol, NADPH-dependent phenol hydroxylase, was isolated from the cytosolic fraction of C. tropicalis close to homogeneity. However, fractionation with polyethylene glycol 6000 lead to a decrease in catechol 1,2-dioxygenase activity. Therefore, another procedure was tested to purify this enzyme. Gel permeation chromatography of proteins of the eluate obtained by chromatography on a DEAE-Sepharose column was utilized to separate phenol hydroxylase and catechol 1,2-dioxygenase. Among gel permeation chromatography on columns of Sephadex G-100, Sephacryl S-300 and Sepharose 4B tested for their efficiencies to isolate phenol hydroxylase and catechol 1,2-dioxygenase, that on Sephacryl S-300 was found to be suitable for such a procedure. Nevertheless, even this chromatographic method did not lead to obtain catechol 1,2-dioxygenase in sufficient amounts and purity for its further characterization. The data demonstrate the progress in resolving the enzymes responsible for the first two steps of phenol degradation by the C. tropicalis strain.
Assessment of humoral response to inhalation antigens is currently the most frequently used method to confirm exposure. It may be useful in extrinsic allergic alveolitis (EAA) patients, especially in cases that are unaware of the source of exposure. However, commercially available test may not include relevant antigens, which may lead to false negativity of the test. We proposed that testing patient serological responses to antigens from respectively environments might be useful in showing the relevance of these exposures. Ten patients diagnosed with EAA were included in the case-control study. Samples from potentially harmful environments were collected, and antigenic extracts were prepared and used for enzyme-linked immunosorbent assays (ELISA) to investigate serological responses to suspected antigens. Plasma samples of unexposed volunteers were used as controls. The results were interpreted in the context of other clinical findings when known (e.g., radiologic patterns, bronchoalveolar lavage fluid findings, histology results) and patient history. We suggest that environmental sampling may provide more information than previous history assessment and commercially available specific IgGs tests and helps to either reveal hidden exposures or find relevant exposure in cases with multiple potential sources. The results of these tests must be interpreted carefully in the context of other clinical data.
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