Molecular modeling of the enantioselectivity in lipase-catalyzed transesterification reactions.

Two strategies based on the use of subsets for calculating the enantioselectivity in lipase-catalyzed transesterifications using the CHARMM force field were investigated. Molecular dynamics was used in our search for low energy conformations. Molecular mechanics was used for refining these low energy conformations. A tetrahedral intermediate with a rigid central part was used for mimicking the transition state. The energy differences between the transition states of the diastereomeric enzyme-substrate complexes were calculated. The way of defining the subsets was based on two fundamentally different strategies. The first strategy used predefined parts of the enzyme and the substrate as subsets. The second approach formed energy-based subsets, varying in size with the substrates studied. The selection of residues to be included in these energy-based subsets was based on the energy of the interaction between the specific residue or water molecule and the transition state. The reaction studied was the kinetic resolution of secondary alcohols in transesterifications using the Candida antarctica lipase B as chiral biocatalyst. The secondary alcohols used in the study were 2-butanol, 3-methyl-2-butanol, and 3,3-dimethyl-2-butanol.     

Cholesterol-induced growth stimulation, macromolecule synthesis, and increased phosphoinositide metabolism of ascites tumour cells in culture

Ascites tumour cells have previously been shown by us to require exogenous cholesterol for growth. To investigate further this phenomenon, we have used, in addition to free cholesterol, cholesterol complexed to digitonine, to elaborate the specificity of this growth-controlling process using a chemically defined medium. Our data show that only free cholesterol stimulates cell growth and macromolecule synthesis in a dose-dependent manner, suggesting that the proper embedding of the sterol into the membrane is a prerequisite for its function. Furthermore, studies have been performed on the influence of cholesterol on the phosphoinositide metabolism of our cells, as phosphoinositides furnish important second messenger molecules in the cascade of signal transduction. We could show that cholesterol stimulates a transient release of inositol trisphosphate and other inositol phosphates by inducing the activation of phospholipase C (PLC). PLC activation by a factor of about 3 with phosphatidylinositol 4-phosphate and phosphatidyl inositol 4,5-bisphosphate as substrates could be measured directly by using a partially purified membrane preparation. This enzyme activity was found to be strongly dependent on free Ca2+ ions with optimal concentrations of 100 nM for cholesterol- and 50 nM for cholesterol-digitonide-treated cells. Ca2+ concentration for half-maximum activation, however, was identical under both conditions. Phospholipase C activity could be synergistically increased about 2-fold with 25 microg GTP gammaS in cholesterol-digitonide-treated cells as well, suggesting that the coupling between phospholipase C and the G-protein was not disturbed by the complex. These data demonstrate a functional role of cholesterol on cell growth, macromolecule synthesis, and phosphoinositide metabolism mediating the release of important second messenger molecules.     

Characterisation of<Emphasis Type="Italic">fusarium graminearum</Emphasis> and<Emphasis Type="Italic">F. culmorum</Emphasis> isolates by mycotoxin production and aggressiveness to wheat

A total of 27Fusarium culmorum isolates from Germany and 41F. graminearum isolates from Kenya were investigated for aggressiveness and mycotoxin production on wheat ears. In addition, ergosterol content of the kernels from ears inoculated withF. graminearum was determined and theF. culmorum isolates were tested for mycotoxin productionin vitro. For both pathogens, isolates markedly differed in aggressiveness. 59% and 37% of theF. culmorum isolates produced NIV and DON, respectively,in vivo andin vitro. The DON-producing isolates also produced 3-acDONin vitro. The more aggressive isolates produced mainly DON while the less aggressive isolates produced mainly NIV. 12% and 85% of theF. graminearum isolates produced NIV and DON, respectively. The highly aggressive isolates produced higher amounts of DON, aggressiveness being highly correlated to DON content in the kernels. NIV-producing isolates were less aggressive. Ergosterol content of kernels was moderately correlated to aggressiveness but highly correlated to DON content. Disease severity was associated with kernel weight reduction.     

Difference in plasma membrane structure between two sublines of ehrlich-lettrè ascites tumor cells

The plasma membranes of the glycogen-free and the glycogen-containing subline of Ehrlich-Lettrè ascites cells were purified and compared with respect to their enzyme activity, chemical, lipid and protein composition, and membrane fluidity. Both membrane fractions differed in a number of parameters which are discussed as differences in the expression of malignant transformation of the two sublines. 1. The 5′-nucleotidase activity was 3–5-times higher and the sialic acid content 3-times lower in the glycogen-containing than in the glycogen-free subline. 2. Differences were also observed with respect to the phospholipid composition, that is in the relative proportions of mainly phosphatidylcholine, -inositol and -serine. 3. The fatty acid spectrum of the two sublines differed in the C-18 series and in the percentage of polyunsaturated acids, which was about 6% lower in the glycogen-containing line. 4. Measurements of fluorescence polarization (P) using 1,6-diphenyl-1,3,5-hextriene as probe generally gave higher P values, indicating a decreased membrane fluidity for the plasma membranes of the glycogen-containing subline both below and above the transition temperature at 33°C. 5. Polyacrylamide gel electrophoresis revealed different protein patterns mainly in the molecular weight range of around 90 000 and in the range between 31 000 and 14 000.     

Temporal pattern in swimming activity of two fish species (Danio rerio and Leucaspius delineatus) under chemical stress conditions

Circadian periodicity of swimming activity was investigated in two fish species, the zebrafish (Danio rerio) and the sunbleak (Leucaspius delineatus) under sublethal long-term exposure to the cyanobacteria toxin microcystin-LR (nominal concentrations of 0.5 μg l^- 1, 5 μg l^- 1, 15 μg l^- 1, 50 μg l^- 1) in 15-litre tanks. Swimming activity of fish was monitored continuously by using an automated video-monitoring and object-tracing system over a period of 17 days. Influenced by long-term exposure to microcystin-LR, Leucaspius delineatus reversed their significant diurnal swimming activity and the fish became statistically significant nocturnal. Danio rerio remained diurnal active, but a significant phase shift was registered. In both Danio rerio and Leucaspius delineatus analysis of time series by cosinor regression revealed microcystin-LR induced dose-dependent alterations of the mean of oscillation, amplitude, acrophase and period length in a different extent. For Danio rerio the periodogram analysis revealed a significant circadian component of swimming activity for control as well as exposure groups, whereby the spectral amplitude clearly decreased at microcystin-LR concentrations of 15 and 50 μg l^- 1. For Leucaspius delineatus the amplitude of circadian rhythm was decreased at all exposure concentrations of MC-LR. Furthermore the dominance of circadian rhythm was clearly reduced, whereas the rate of ultradian rhythms increased at elevated MC-LR concentrations of 5 μg l^- 1, 15 μg l^- 1 and 50 μg l^- 1. The studied temporal aspects of behaviour clearly indicated stress symptoms in both fish species, therefore it proved to be a relevant method to characterise the impact of toxic substances in the environment and for biomonitoring.