Purification and some properties of steryl β-d-glucoside hydrolase from Sinapis alba seedlings

Homogenates of 7-day-old S. alba seedlings hydrolysed cholesteryl[4-H¹⁴C] β-d-glucoside or sitosteryl β-d-glucoside-[6-³H]. Activity was located predominantly in the cell membrane structures sedimenting at 1000–15 000 g and was solubilized by acetone treatment. Partially purified enzyme preparation, with an about 1500 times higher specific activity with respect to the crude homogenate, was obtained by repeated acetone precipitation and subsequent chromatography on DEAE-Sephadex and Sephadex G-100. During this procedure a considerable separation from other enzymes with β-glucosidase activity was achieved. The enzyme had MW 65 000 daltons, pH optimum at 5.2–5.6. Two observations suggested that the enzyme was a specific steryl β-d-glucoside hydrolase. Firstly, there was no substrate competition between steryl glucosides and several other β-d-glucosides. Secondly, enzyme activity wasstrongly inhibited by low concentrations of various 3β-OH sterols with a planar ring system and an intact side chain.     

Specificity of the agar gel microimmunodiffusion test in rabies diagnostics.

The agar-gel microimmunodiffusion test (MIDT) with commercially available antirabies sera from different sources was applied to evaluation of rabies infection at about 500 brains from suspected animals. The high nonspecificity of the test and false positive results with nonvirulent materials were stated when compared with the histopathological, biological and FA tests. For evaluation of the nonspecificity and its cause, different antirabies sera and brain antigens from noninfected and rabid animals were used. Absorption of sera with tissue powders or immuno absorbent had a little influence on test specificity. The main causes of nonspecificity was the presence of antibrain antibodies in sera of producers-animals hiperimmunized by brain and spinal cord tissue vaccines. Application of the test to rabies diagnostics without purified control antigens and highly specific sera seems to be unjustified.     

Metabolic quenching of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>: efficiency of methods and impact of cold shock

Representative and valid cytoplasmic concentrations are essential for ensuring the significance of results in the field of metabolome analysis. One of the most crucial points in this respect is the sampling itself. A rapid and sudden stopping of the metabolism on a timescale that is much faster than the conversion rates of investigated metabolites is worthwhile. This can be achieved by applying of cold methanol quenching combined with reproducible, fast, and automated sampling. Unfortunately, quenching the metabolism by a sharp temperature shift leads to what is known as cold shock or the cell-leakage effect. In the present work, we applied a microstructure heat exchanger to analyze the cold shock effect using Corynebacterium glutamicum as a model microorganism. Using this apparatus together with a silicon pipe, it was possible to assay the leakage effect on a timescale starting at 1 s after cooling cell suspension. The high turnover rates not only require a rapid quenching technique, but also the correct application. Moreover, we succeeded in showing that even when the required appropriate setup of methanol quenching is not used, the metabolism is not stopped within the required timescale. By applying robust techniques like rapid sampling in combination with reproducible sample processing, we ensured fast and reliable metabolic inactivation during all steps.     

MreC and MreD balance the interaction between the elongasome proteins PBP2 and RodA

Rod-shape of most bacteria is maintained by the elongasome, which mediates the synthesis and insertion of peptidoglycan into the cylindrical part of the cell wall. The elongasome contains several essential proteins, such as RodA, PBP2, and the MreBCD proteins, but how its activities are regulated remains poorly understood. Using E. coli as a model system, we investigated the interactions between core elongasome proteins in vivo. Our results show that PBP2 and RodA form a complex mediated by their transmembrane and periplasmic parts and independent of their catalytic activity. MreC and MreD also interact directly with PBP2. MreC elicits a change in the interaction between PBP2 and RodA, which is suppressed by MreD. The cytoplasmic domain of PBP2 is required for this suppression. We hypothesize that the in vivo measured PBP2-RodA interaction change induced by MreC corresponds to the conformational change in PBP2 as observed in the MreC-PBP2 crystal structure, which was suggested to be the “on state” of PBP2. Our results indicate that the balance between MreC and MreD determines the activity of PBP2, which could open new strategies for antibiotic drug development.