Automated modular preparative HPLC-MS purification laboratory with enhanced efficiency.

Automated parallel synthesis as tool to increase productivity in chemical synthesis is well-established. However, even more time-consuming than the synthesis process is the following purification of the resulting crude products. To enhance efficiency of the lead optimization process at Bayer CropScience, a high-throughput HPLC/MS-laboratory for the purification of up to 48 crude products per day in the range of 200-400 mg each in one injection per sample has been set up. The use of Covaris technology for HPLC sample preparation, automated aliquotation during fractionation, and a novel evaporation process by combination with freeze-drying are new key technologies applied successfully for the first time in this purification unit facilitating to achieve the targeted efficiency. The whole process is supported by a specially designed IT-landscape covering each step of the workflow. Both the technical instruments used within the laboratory and the workflow and IT platform are described in this article.

Occurrence and non-detectability of maytansinoids in individual plants of the genera Maytenus and Putterlickia.

Individual plants belonging to different species of the family Celastraceae collected from their natural habitats in South Africa (Putterlickia verrucosa (E. Meyer ex Sonder) Szyszyl., Putterlickia pyracantha (L.) Szyszyl., Putterlickia retrospinosa van Wyk and Mostert) and Brazil (Maytenus ilicifolia Mart. ex Reiss., Maytenus evonymoides Reiss., Maytenus aquifolia Mart.) were investigated for the presence of maytansinoids and of maytansine, an ansamycin of high cytotoxic activity. Maytansinoids were not detectable in plants grown in Brazil. Analysis of plants growing in South Africa, however, showed clearly that maytansinoids were present in some individual plants but were not detectable in others. Molecular biological analysis of a Putterlickia verrucosa cell culture gave no evidence for the presence of the aminohydroxybenzoate synthase gene which is unique to the biosynthesis of aminohydroxybenzoate, a precursor of the ansamycins including maytansinoids. Moreover, this gene was not detectable in DNA extracted from the aerial parts of Putterlickia plants. In contrast, observations indicate that this gene may be present in microbes of the rhizosphere of Putterlickia plants. Our observations are discussed with respect to the possibility that the roots of Putterlickia plants may be associated with microorganisms which are responsible for the biosynthesis of maytansine or maytansinoids.

Ultrasound-induced modifications of cytoskeletal components in osteoblast-like SAOS-2 cells.

In clinical and experimental studies an acceleration of fracture healing and increased callus formation induced by low-intensity pulsed ultrasound (LIPUS) has been demonstrated. The exact molecular mechanisms of ultrasound treatment are still unclear. In this study ultrasound transmitted cytoskeletal and growth rate changes of SAOS-2 cells were examined. Osteoblast-like cell lines (SAOS-2) were treated using low-intensity pulsed ultrasound. Cytoskeletal changes were analyzed using rhodamine phalloidine for f-actin staining and indirect immunofluorescence techniques with different monoclonal antibodies against several tubulin modifications. To examine changes of cell number after ultrasound treatment cell counts were done. Significant changes in cytoskeleton structure were detected compared to controls, including an enhancement of stress fiber formation combined with a loss of cell migration after ultrasound application. We further observed that sonication altered the proportion of the more stable microtubules to the more labile microtubule subclass. The labile tyrosinated microtubules appeared highly enhanced, whereas the amount of the more stable acetylated microtubules was remarkably diminished. All these observations were quantified by fluorometric measurements. The centrosomal gamma-tubulin was frequently scattered throughout the cell's cytoplasm, giving rise to additional polyglu-positive microtubular asters, which induced multipolar spindles, leading either to aneuploid mini-or giant cells. Moreover, a significant increase of cell number was noticed in the sonicated group. These experiments demonstrate that ultrasound treatment increases cell number and leads to significant changes of the cytoskeletal structure and composition in vitro.

Ultrasound enhanced endocytotic activity of human fibroblasts.

Although various in vitro studies have shown that low-intensity pulsed ultrasound influences cytoskeletal components and biochemical pathways, the exact biologic mechanisms are still not fully understood. In this study, we analysed the effect of therapeutic ultrasound on the endocytotic activity of human foreskin fibroblasts. Fibroblasts were incubated with two different endocytotic markers (transferrin Alexa 488 and Lucifer yellow; Sigma Bioprobes, Eugene, OR, USA). To evaluate the amount of internalized markers in sonicated and nonsonicated control cells, confocal microscopy and plate reader experiments were performed. Additionally, the structural integrity of the cell membrane was monitored by electron-microscopy. After ultrasound treatment a clear increase (1.6-fold/Lucifer yellow and 1.4-fold/transferrin Alexa 488) of fluorescent marker uptake was detected. Confocal microscopy and plate reader experiments revealed that whole populations of sonicated fibroblasts showed a significant higher fluorescence compared with cells not sonicated (p<0.05; t-test for unpaired samples). The electron microscopic analysis of the cells showed no signs of structural membrane damage or a loosening of the membrane integrity. However, an exceedingly high amount of endocytotic vesicles and clathrin coated pits were observed in the sonicated group.