In vitro susceptibility ofPityrosporum ovale (Malassezia furfur) to human androgenic steroids

Cells ofPityrosporum ovale that colonize human pilosebaceous units are constantly exposed to cutaneous androgenic steroids. The aim of our study was to find out whetherP. ovale is susceptible to these hormones. Three strains ofP. ovale were grown in vitro in the presence of various concentrations oftestosterone, dehydroepiandrosterone, androstenedione, androstanedione, 5--dihydrotestosterone andprogesterone (10, 100, and 1000 µg/ml; agar dilution assays). In addition, three strains ofCandida albicans were also exposed to equal concentrations of the same androgens. As a result, allP. ovale strains were suppressed by 1000 µg/mlandrostenedione, which was the strongest inhibitor. The other androgenic steroids also significantly reducedP. ovale growth at different concentrations, depending on the hormone used and the strain tested.Progesterone was inhibitory at the highest concentration for oneP. ovale strain only.Candida albicans was not affected by any of the androgens. These findings demonstrate an in vitro susceptibility ofP. ovale to high concentrations of human androgenic steroids. A relevance of this interaction for the in vivo fungus-host relation is not apparent.     

Plant regeneration from ryegrass ovules cultivated on endosperm-derived feeder cells

An efficient method for the regeneration of zygote-derived plants via ovule culture is desirable for overcoming postzygotic cross incompatibility as well as for the development of certain methods for genetic manipulation. High-frequency plantlet regeneration from ovules of Italian ryegrass (Lolium multiflorum Lam.) and a hybrid Italian/perennial ryegrass excised 1 to 4 days post pollination was obtained by culture on endosperm-derived feeder cells. Ovules excised 3 or 4 days after anthesis and grown on feeder cells generally regenerated about twice as frequently as ovules grown directly on nutrient medium. In one of the genotypes tested, ovules excised 1, 2 and 3 days post pollination developed into plantlets at percentages of 38.1, 52.0 and 52.8, respectively, using the feeder-cell system.Abbreviations EM endosperm multiplications - OC ovule culture - R regeneration - 2,4-d 2,4-dichlorophenoxyacetic acid     

Cloning, sequencing and heterologous expression of a new chitinase gene, chi92, from Streptomyces olivaceoviridis ATCC 11238

A new chitinase gene, chi92, encoding the largest known chitinase from Streptomyces olivaceoviridisATCC 11238 was sequenced by means of different PCR-methods. The cloned gene was expressed in E. coliand the recombinant protein could be detected by Western-blot analysis. The multiplicity of chitinolytic enzymes of this strain is discussed.     

Screening for enzyme activity in turbid suspensions with scattered light

New screening techniques for improved enzyme variants in turbid media are urgently required in many industries such as the detergent and food industry. Here, a new method is presented to measure enzyme activity in different types of substrate suspensions. This method allows a semiquantitative determination of protease activity using native protein substrates. Unlike conventional techniques for measurement of enzyme activity, the BioLector technology enables online monitoring of scattered light intensity and fluorescence signals during the continuous shaking of samples in microtiter plates. The BioLector technique is hereby used to monitor the hydrolysis of an insoluble protein substrate by measuring the decrease of scattered light. The kinetic parameters for the enzyme reaction (V(max,app) and K(m,app)) are determined from the scattered light curves. Moreover, the influence of pH on the protease activity is investigated. The optimal pH value for protease activity was determined to be between pH 8 to 11 and the activities of five subtilisin serine proteases with variations in the amino acid sequence were compared. The presented method enables proteases from genetically modified strains to be easily characterized and compared. Moreover, this method can be applied to other enzyme systems that catalyze various reactions such as cellulose decomposition.     

A Pilot Study to Assess the Feasibility of Transcutaneous Glomerular Filtration Rate Measurement Using Fluorescence-Labelled Sinistrin in Dogs and Cats

In dogs and cats an assessment of renal function is often needed, however, existing methods including urine and plasma clearances are invasive, cumbersome and time consuming. This pilot study evaluated the feasibility of a transcutaneous glomerular filtration rate (GFR) measurement in dogs and cats. Additionally the optimal dose and location for the transcutaneous measurement device were investigated. Renal elimination of fluorescein-isothiocyanate-labelled sinistrin (FITC-S) was measured transcutaneously for 4 hours. The procedures were performed in awake, freely moving animals using escalating doses of FITC-S (10 mg/kg, 30 mg/kg, 50 mg/kg) with a wash-out period of at least 24 h in between. Multiple devices were placed on each animal. The resulting FITC-S disappearance curves were visually assessed to determine the most suitable location and the appropriate dose to reach an adequate transcutaneous peak signal for kinetic analysis. In both species 30 mg/kg were adequate for kinetic calculation. The most suitable place for the device was the lateral thoracic wall in dogs and the ventral abdominal wall in cats, respectively. Transcutaneous FITC-S clearance was then repeated using the optimal dose and location and in parallel with an additional plasma sinistrin clearance. Plasma elimination half-lives [min] were 26, 31 and 35, and corresponding transcutaneous elimination half-lives [min] were 26, 34 and 55, respectively in the dogs. Plasma elimination half-lives [min] were 51, 60 and 61, and corresponding transcutaneous elimination half-lives [min] were 75, 96 and 83, respectively in the cats. In conclusion, transcutaneous FITC-S clearance is a feasible method for the assessment of GFR in awake dogs and cats. It is noninvasive, well tolerated and easy to perform even in a clinical setting with results being readily available. A dose of 30 mg/kg of FITC-S seems adequate for kinetic assessment. Further studies are now needed to establish reference values and evaluate transcutaneous renal clearance in various conditions.