Multiple forms of lactate dehydrogenase in Staphylococcus aureus

Activities for nicotinamide adenine dinucleotide (NAD)-dependent and NAD-independent forms of lactate dehydrogenase (LDH) were measured in cell-free extracts of Staphylococcus aureus strain PS 6 for the d and l isomers of lactate. Data obtained for the NAD-dependent lactate dehydrogenases indicate that oxidation of both isomers of lactate is due to both an l-lactate-specific LDH and a lactate racemase. After acrylamide gel electrophoresis, two bands exhibiting LDH activity were detected in crude or in partially purified cell-free extracts. The fast band exhibited LDH activity that was not NAD-dependent for both isomers of lactate, whereas, the slow band had very high NAD-dependent LDH activity for the l isomer but just detectable activity or the d isomer. Both bands appeared when d-lactate was used as the substrate, but only the slow band was formed when l-lactate was the substrate. NAD-dependent LDH, in apparent association with a nonspecific tetrazolium-reducing protein, is responsible for the production of the slow band.     

Instrumental assay of microbial lipase at constant pH

A rapid, accurate method with high sensitivity and reproducibility, and having the advantage of a short incubation period under constant pH, has been developed for routine measurement of microbial lipase. Assembled from readily available and economical instrumental components, the apparatus includes a pH meter, a thermoelectric heating and stirring device, a motor-driven burette, and an automatic recorder. The reaction mixture, consisting of 5 ml of a 10% olive oil-gum arabic emulsion, 2 ml of 3 m NaCl, 2 ml of sodium taurocholate (15 mg/ml) of 0.075 m CaCl(2), 5 ml of water, and 1 ml of enzyme solution, was adjusted to pH 8.0 and 37 C. The pH was maintained at a constant value by automatic addition of 0.01 n NaOH during the incubation period, which usually lasted 5 min. A lipase unit, derived from the use of this technique, may be defined as the number of microequivalents of acid liberated per minute under the specified conditions. The method was sensitive to 0.01 units. Various organisms tested produced 0.17 to 1.32 units per ml of the cell filtrate. An Arrhenius plot for staphylococcal lipase yielded 14,500 cal for function A (energy of activation).     

Nephelometric Assay of Bovine Antistaphylocoagulase Serum

A nephelometric assay of staphylococcal coagulase has been utilized to measure coagulase inhibition by bovine anticoagulase serum. Suitably diluted antisera produced maximal inhibition when incubated with purified coagulase at pH 7.3 in phosphate-buffered saline for 15 min at 22 C or 1 hr at 4 C. Neutralization of coagulase activity was measured as the reduction in the clotting rate of a fibrinogen-plasma substrate, and was directly proportional to the concentration of antiserum over a wide range of coagulase activity. A unit of anticoagulase was defined as the amount of inhibitor that neutralized one unit of coagulase. In addition to the heat-stable (56 C, 30 min) antibody contained in the crude gamma-globulin fraction, a heat-labile, nondialyzable coagulase inhibitor was also detected in the sera from 15 of 16 randomly selected dairy cows.     

A Phloxine-Azure-Hematoxylin Sequence for Differential Staining of Cells in Pancreatic Islets

Sections of 6 μ from tissues fixed in Susa or in Bouin's fluid (without acetic acid) and embedded in paraffin were attached to slides with Mayer's albumen, dried at 37 C for 12 hr, deparaffinized and hydrated. The sections fixed in Susa were transferred to a I₂-K₁ solution (1:2:300 ml of water); rinsed in water, decolorized in 5% Na₂S₂O₃; washed in running water, and rinsed in distilled water. Those fixed in Bouin's were transferred to 80% alcohol until decolorized, then rinsed in distilled water. All sections were stained in 1% aqueous phloxine, 10 min; rinsed in distilled water and transferred to 3% aqueous phosphotungstic acid, 1 min; rinsed in distilled water; stained 0.5 min in 0.05 azure II (Merck), washed in water; and finally, nuclear staining in Weigert's hematoxylin for 1 min was followed by a rinse in distilled water, rapid dehydration through alcohols, clearing in xylene and covering in balsam or a synthetic resin. In the completed stain, islet cells appear as follows: A cells, purple; B cells, weakly violet-blue; D cells, light blue with evident granules; exocrine cells, grayish blue with red granules.     

The inhibition of catalase by glutathione

Reduced glutathione (GSH) inhibited catalase activity in a dose-dependent manner. DL-dithiothreitol (DL-DTT) and dithioerythritol (DTE) also inhibited catalase activity. The inhibition of catalase by GSH and DL-DTT could be reduced by NADPH. Polyacrilamide gel electrophoresis demonstrated the inhibition was partially reversible. The inhibition of catalase by GSH appeared to be partly due to superoxide radicals, since it was inhibited by active manganese superoxide dismutase, but not by heat-inactivated enzyme. Other chemical species also appear to take part in the inhibition, but they could not be identified.     

Identification of the membrane component of the anion pump encoded by the arsenical resistance operon of R-factor R773

The arsenical resistance (ars) operon of the conjugative R-factor R773 encodes an ATP-driven anion extrusion pump, producing bacterial resistance to arsenicals. There are three structural genes, of which the product of the middle gene, arsB, has not previously been identified. From nucleotide sequence data, the ArsB protein is predicted to be a 45577 Dalton hydrophobic protein. A mini-Mu transposition procedure was used to construct an arsB-lacZ gene fusion, producing a hybrid ArsB-beta-galactosidase protein which was localized in the inner membrane. The operon was cloned into a T7 RNA polymerase expression vector. In addition to the previously identified ArsA and ArsC proteins, the cells synthesized an inner membrane protein with an apparent mass of 36 kD identified as the ArsB protein.     

A comparison of two plating techniques to estimate plasmid stability of a prolonged chemostat culture

Summary A comparison of two plating techniques to estimate the segregational stability ofEscherichia coli RR1 harboring plasmid pBR322 in a chemostat was studied. No significant differences were observed between the spread and replica plating techniques in the beginning of the experiments. However, a noticeable discrepancy between these two methods appeared after approximately 100 hours. This inconsistency can be shown to be statistically significant.     

Detection of IgG and IgM antibodies with ELISA technique in human trichomoniasis

The direct wet mount examination of vaginal secretion, widely applied for the diagnosis of Trichomonas vaginalis infection in woman patients, is rapid and economical, however, the sensitivity of this technique is not so high. In this study enzyme-linked immunosorbent assay (ELISA) was employed for the detection of serum anti-T. vaginalis IgG and IgM antibodies from 30 vaginal trichomoniasis patients and 30 non-infected healthy persons. The results were as follows: 1. Serum ELISA-IgG value was 0.37 +/- 0.134 (Mean +/- S.D.) in vaginal trichomoniasis patients and 0.21 +/- 0.054 in healthy controls (p less than 0.005), and the sensitivity and specificity of ELISA for serum IgG antibody were 70.0% and 96.7%, respectively. 2. Serum ELISA-IgM value was 0.33 +/- 0.177 (Mean +/- S.D.) in vaginal trichomoniasis patients and 0.11 +/- 0.051 in healthy controls (p less than 0.005), and the sensitivity and specificity of ELISA for serum IgM antibody were 70.0% and 96.7%, respectively. 3. The ELISA-IgG values showed a significant correlation with ELISA-IgM values (r = 0.77, p less than 0.005). With above results, it is assumed that ELISA is a reliable method for the diagnosis of T. vaginalis infection and simultaneous measurement of serum IgG and IgM with this technique is recommended.     

长江中游鱼类寄生棘头虫区系的研究

经过3年10次调查,剖检湖北省宜都、黄冈两处江段所产72种鱼类,共计766尾。收集棘头虫10种,其中包括2新种和1新组合,即蛇鮈新棘吻虫(新种)Neoechinorhynchus saurogobi sp.nov.,长江丽棘虫(新种)Brentisentis yangtzensis sp.nov.(Illiosontidae),鲤丽棘虫(新组合)B.cyprini comb.nov.。对长江中游鱼类寄生棘头虫区系的特点进行了分析和探讨。