Standardization and demonstration of antibody-coated candida in urine by direct immunofluorescence test

Acetone, carbontetrachloride, ethyl alcohol, mixture of ethyl alcohol and acetone, and heat were assessed for fixative property for direct immunoflurorescent (IF) staining of antibody-coated Candida cells. The results indicated that ethyl alcohol was the most suitable fixative for the test. Antisera containing 16 units of Candida albicans type A agglutinin were found essential to get optimal detectable fluorescence of antibody-coated yeast cells. IF test showed cross reactivity between the yeasts of C. albicans and C. tropicalis. However, there was no cross reactivity with the conidia of A. flavus.The direct IF test could demonstrate antibody-coated yeast cells and pseudomycelia in deposits of urine in the direct smear. It correlated well with microscopy and culture studies. At times, it could demonstrate the antibody-coated yeasts earlier than routine significant culture. It could also differentiate the significant from non-significant fungal isolates from urine.     

Oxidation of catechol in plants. 3. Purification and properties of the diphenylene dioxide 2,3-quinone-forming enzyme system from Tecoma leaves

In attempting to determine the nature of the enzyme system mediating the conversion of catechol to diphenylenedioxide 2,3-quinone, in Tecoma leaves, further purification of the enzyme was undertaken. The crude enzyme from Tecoma leaves was processed further by protamine sulfate precipitation, positive adsorption on tricalcium phosphate gel, and elution and chromatography on DEAE-Sephadex. This procedure yielded a 120-fold purified enzyme which stoichiometrically converted catechol to diphenylenedioxide 2,3-quinone. The purity of the enzyme system was assessed by polyacrylamide gel electrophoresis. The approximate molecular weight of the enzyme was assessed as 200,000 by gel filtration on Sephadex G-150. The enzyme functioned optimally at pH 7.1 and at 35 °C. The K_m for catechol was determined as 4 × 10^?4m. The enzyme did not oxidize o-dihydric phenols other than catechol and it did not exhibit any activity toward monohydric and trihydric phenols and flavonoids. Copper-chelating agents did not inhibit the enzyme activity. Copper could not be detected in the purified enzyme preparations. The purified enzyme was not affected by extensive dialysis against copper-complexing agents. It did not show any peroxidase activity and it was not inhibited by catalase. Hydrogen peroxide formation could not be detected during the catalytic reaction. The enzymatic conversion of catechol to diphenylenedioxide 2,3-quinone by the purified Tecoma leaf enzyme was suppressed by such reducing agents as GSH and cysteamine. The purified enzyme was not sensitive to carbon monoxide. It was not inhibited by thiol inhibitors. The Tecoma leaf was found to be localized in the soluble fraction of the cell. Treatment of the purified enzyme with acid, alkali, and urea led to the progressive denaturation of the enzyme.     

Mandelic acid-4-hydroxylase, a new inducible enzyme from Pseudomonas convexa

A soluble fraction of $\text{Pseudomonas convexa}$ catalyzed the hydroxylation of mandelic acid to $\text{p}$-hydroxymandelic acid. The enzyme had a pH optimum of 5.4 and showed an absolute requirement for Fe²⁺, tetrahydropteridine, NADPH. $\text{p}$-Hydroxymandelate, the product of the enzyme reaction was identified by paper chromatography, thin layer chromatography, UV and IR-spectra.     

Enzymic conversion of 3-hydroxanthranilic acid into cinnabarinic acid by the nuclear fraction of rat liver

1. An enzyme solely localized in the nuclear fraction of rat liver was found to convert 3-hydroxyanthranilic acid into a red product that was isolated and crystallized from the reaction mixture. The product was identified as cinnabarinic acid (2-amino-3-oxo-3H-phenoxazine-1,9-dicarboxylic acid) by comparing its properties with synthetic cinnabarinic acid. 2. The enzyme had optimum pH at 7·2. Heavy-metal ions like Ag⁺, Hg²⁺, MoO₄²⁻, Fe²⁺ and Cu²⁺ were inhibitory; Mn²⁺ activated the reaction to a considerable extent. 3. The reaction was inhibited by mercaptoethanol, GSH and cysteine, and activated by p-hydroxymercuribenzoate and sodium arsenite, which may suggest the involvement of disulphide groups in the reaction.     

Purification, properties and induction of a specific benzoate-4-hydroxylase from Aspergillus niger (UBC 814).

An inducible benzoate-4-hydroxylase has been partially purified from crude extracts of the mycelial felts of Aspergillus niger. This enzyme catalyzes the transformation of benzoate to p-hydroxybenzoate with equimolar consumption of NADPH and O2. It requires tetrahydropteridine as a prosthetic group. The optimum activity was found at pH 6.2 with a Km value at 30 degrees C of 1.6-10-minus 4 for NADPH and 1.3-10-minus 4 M for benzoate. Fe-2+ (iron) is required for the enzyme activity. The enzyme is stabilized by the inclusion of benzoate, EDTA and glutathione in the extracting buffer. The enzyme is specific for benzoate as substrate. Sulfhydryl groups(s) are essential for enzyme activity as indicated by p-chloromercuri-benzoate and N-ethylmaleimide inactivation. Benzoate-4-hydroxylase activity is decreased in the mycelial felts of Aspergillus niger grown in the presence of higher concentrations of benzoate. Maximum activity of the enzyme was observed at 36 h after inoculation.