Movement of substrates between blood and brain is known to be influenced by P-glycoprotein (P-gp) at the luminal surface of the endothelium lining brain microvessels and by multidrug resistance associated protein 1 (MRP1) at the basolateral surface of the choroid plexus epithelium. Here, using RT-PCR and Western blotting, we investigate other ABC transporters in both normal and tumour human brain tissue and demonstrate the presence of breast cancer resistance protein (BCRP). Immunofluorescence confocal microscopy demonstrates that BCRP is located at the blood-brain barrier, mainly at the luminal surface of microvessel endothelium. This localization closely resembles that of P-gp. BCRP has several substrates in common with P-gp and may pose an additional barrier to drug access to the brain.

No abstract

Biomedicine has seen tremendous advances in the field of image acquisition. The generation of digital images of high information content has become so straightforward and efficient that the volume of images accumulating in biomedical disciplines is posing significant challenges. Until now, conventional image analysis solutions are generally pixel-based and limited in the amount of information that they extract. However, a software system enabling the complex analysis of biomedical images should not impose restrictions on detection, classification and quantification of structures, but rather allow unlimited freedom to answer exhaustively all conceivable questions about the interactions and relationships between structures. Crucial to this is the precise and robust segmentation of relevant structures in digital micrographs. This challenge involves bringing structure, morphology and context into play. Based on the Definiens Cognition Network Technology, solutions have been deployed for use in biomedicine. The technology is object-oriented, multi-scale, context-driven and knowledge-based. Images are interpreted on the properties of networked image objects, which results in numerous advantages. This approach enables users to bring in detailed expert knowledge and enables complex analyses to be performed with unprecedented accuracy, even on poor quality data or for structures exhibiting heterogeneous properties or variable phenotypes. Extracted structures are the basis for detailed morphometric, structural and relational measurements which can be exported for each individual structure. These data can be used for decision support or correlated against experimental or molecular data, thus bridging classical biomedicine with molecular biology. An overview of the technology is provided with examples from different biomedical applications.

No abstract

The acidic interior of neuroendocrine secretory vesicles provides both an energy gradient for amine‐proton exchangers (VMATs) to concentrate small transmitter molecules, for example catecholamines, and an optimal pH for the prohormone convertases which cleave hormone precursors. There is evidence that VMAT activity modulates prohormone cleavage, but in the absence of measurements of pH in secretory vesicles in intact cells, it has not been possible to establish whether these effects are attributable to raised intravesicular pH due to proton transport through VMATs. Clones were generated of the hamster insulinoma cell line HIT‐T15 expressing a pH‐sensitive form of green fluorescent protein (GFP‐F64L/S65T) targeted to secretory vesicles, with and without co‐expression of VMAT2. In order to study prohormone cleavage, further clones were generated that expressed preprogastrin with and without co‐expression of VMAT2. Confocal microscopy of GFP fluorescence indicated that the pH in the secretory vesicles was 5.6 in control cells, compared with 6.6 in cells expressing VMAT2; the latter was reduced to 5.8 by the VMAT inhibitor reserpine. Using a pulse‐chase labelling protocol, cleavage of 34‐residue gastrin (G34) was found to be inhibited by co‐expression with VMAT2, and this was reversed by reserpine. Similar effects on vesicle pH and G34 cleavage were produced by ammonium chloride. We conclude that VMAT expression confers the linked abilities to store biogenic amines and modulate secretory vesicle pH over a range influencing prohormone cleavage and therefore determining the identity of regulatory peptide secretory products.