Studies on isolated pericytes have been hindered by the lack of specific biochemical markers with which to facilitate identification and purification of this cell type. The pericyte is defined by a strictly anatomical criterion in vivo, namely its anatomical location within the microvascular basement membrane and surrounding the endothelia (1). In dissociated cultures, pericytes have been identified by morphological and growth characteristics (2-5). A recent study (6) has shown that pericytes can be differentiated from endothelial cells and smooth muscle cells by immunofluorescent staining with antibodies directed against actin isoforms. Endothelial cells were shown to express only nonmuscle actin in stress fibers, whereas smooth muscle cells expressed only muscle actin in stress fibers . In contrast, pericytes were shown to express both muscle and nonmuscle actins in stress fibers . The literature contains additional reports of differential distribution of cytoplasmic and cytoskeletal/contractile proteins in vascular cell types (7-9). However, while the differential distribution of cytoplasmic proteins allows identification of vascular cells in vitro, antibodies to these proteins cannot be used as selective agents for purifying cell types as the cells must be fixed and/or permeabilized to make these antigens accessible to antibodies .In this study we report the characterization of an mAb (3G5) that appears to be a specific marker for pericytes within the microvasculature . The 3G5 mAb has previously been shown to react with brain cells, pancreatic islet cells, adrenal medullary cells, thyroid follicular cells, renal glomerular cells, and peripheral blood T lymphocytes (10, 11). Nonetheless, within the microvasculature, the 3G5 mAb is a unique and useful pericyte marker and a selective agent for enriching these cells.

Organic impurities in compound libraries are known to often cause false-positive signals in screening campaigns for new leads, but organic impurities do not fully account for all false-positive results. We discovered inorganic impurities in our screening library that can also cause positive signals for a variety of targets and/or readout systems, including biochemical and biosensor assays. We investigated in depth the example of zinc for a specific project and in retrospect in various HTS screens at Roche and propose a straightforward counter screen using the chelator TPEN to rule out inhibition caused by zinc.

The 13-step synthesis of (+)-isoschizandrin reported herein features a samarium(II) iodide-promoted 8-endo ketyl-olefin coupling to assemble the eight-membered ring present in the target concomitantly with the required functionality and stereochemistry. In constructing (+)-isoschizandrin as a single atropisomer, the synthesis utilizes a kinetic resolution of a seven-membered lactone using a CBS-oxazaborolidine.

Islets of Langerhans consist of four major endocrine cell types assembled in a highly organized manner critical for their function. The molecular forces governing islet cellular architecture are not understood. We determined whether adult rat islet cells carry information necessary for orderly assembly. Dispersed cells from adult rat islets were maintained in static suspension culture for 6 days. During this time the cells reassociated to form numerous aggregates. These aggregates were approximately half the size of native islets with a commensurate reduction in DNA and insulin content. However, both cellular composition and organization were remarkably similar to that of adult rat islets, in which the beta-cells form a central core surrounded by a discontinuous mantle of non-beta-cells. Thus, immunoperoxidase staining showed that in the aggregates, just as in intact islets cultured in parallel, 26% of the cells were non-beta-cells and of these, 94% were clearly peripheral. Non-beta-cells were similarly found to be peripheral, with beta-cells located centrally, even when the ratio of non-beta-cells to beta-cells had been altered. This was achieved by sorting the two cell populations by fluorescence-activated flow cytometry, resulting in aggregates with 79% non-beta-cells and 21% beta-cells. Insulin release from the aggregates was stimulated approximately ninefold by raising glucose from 50 to 300 mg/dl, which was comparable to that found for intact islets. The spontaneous formation of isletlike aggregates displaying appropriate cellular architecture indicates that the signals (molecules) needed for such organization are intrinsic to islet cells and are still expressed by them in adult life.