Mrp1 Multidrug Resistance-Associated Protein and P-Glycoprotein Expression in Rat Brain Microvessel Endothelial Cells

Abstract: Two membrane glycoproteins acting as energy-dependent efflux pumps, mdr -encoded P-glycoprotein (P-gp) and the more recently described multidrug resistance-associated protein (MRP), are known to confer cellular resistance to many cytotoxic hydrophobic drugs. In the brain, P-gp has been shown to be expressed specifically in the capillary endothelial cells forming the blood-brain barrier, but localization of MRP has not been well characterized yet. Using RT-PCR and immunoblot analysis, we have compared the expression of P-gp and Mrp1 in homogenates, isolated capillaries, primary cultured endothelial cells, and RBE4 immortalized endothelial cells from rat brain. Whereas the mdr1a P-gp-encoding mRNA was specifically detected in brain microvessels and mdr1b mRNA in brain parenchyma, mrp1 mRNA was present both in microvessels and in parenchyma. However, Mrp1 was weakly expressed in microvessels. Mrp1 expression was higher in brain parenchyma, as well as in primary cultured brain endothelial cells and in immortalized RBE4 cells. This Mrp1 overexpression in cultured brain endothelial cells was less pronounced when the cells were cocultured with astrocytes. A low Mrp activity could be demonstrated in the endothelial cell primary monocultures, because the intracellular [³H]vincristine accumulation was increased by several MRP modulators. No Mrp activity was found in the cocultures or in the RBE4 cells. We suggest that in rat brain, Mrp1, unlike P-gp, is not predominantly expressed in the blood-brain barrier endothelial cells and that Mrp1 and the mdr1b P-gp isoform may be present in other cerebral cells.     

Characterization of Arsenate Uptake by Cultured Primary Rat Astrocytes

Arsenate is known to be accumulated by cultured astrocytes and to stimulate astrocytic glutathione export, but the arsenate uptake into astrocytes has not been characterized so far. To address this topic, we have exposed primary rat astrocyte cultures to arsenate and determined the cellular arsenic content by atomic absorption spectroscopy. Viable astrocytes accumulated arsenate in a time- and concentration-dependent manner. Their cellular arsenic content increased almost proportional with time for up to 60 min after application of arsenate. Analysis of the concentration-dependent increase in the specific arsenic content of the cells after 30 min of arsenate exposure revealed that cultured astrocytes take up arsenate with saturable kinetics by a transport process that has apparent K_M- and V_max-values of 1.7 ± 0.2 mM and 28 ± 4 nmol/(mg protein × 30 min), respectively. Arsenate uptake in viable astrocytes was strongly inhibited by the presence of phosphate or by lowering the incubation temperature to 4 °C and was completely abolished in a sodium ion-free medium. These results strongly suggest that the saturable temperature-dependent arsenate uptake into astrocytes is mediated by a sodium-dependent phosphate cotransporter.     

Mapping of the Physcomitrella patens proteome

The moss Physcomitrella patens is unique among land plants due to the high rate of homologous recombination in its nuclear DNA. The feasibility of gene targeting makes Physcomitrella an unrivalled model organism in the field of plant functional genomics. To further extend the potentialities of this seed-less plant we aimed at exploring the P. patens proteome. Experimental conditions had to be adopted to meet the special requirements connected to the investigations of this moss. Here we describe the identification of 306 proteins from the protonema of Physcomitrella. Proteins were separated by two dimensional electrophoresis, excised form the gel and analysed by means of mass spectrometry. This reference map will lay the basis for further profound studies in the field of Physcomitrella proteomics.     

Loss of GH3 function does not affect phytochrome-mediated development in a moss, Physcomitrella patens

Auxin-induced gene expression is described for a variety of different genes including the SAUR-, Aux/IAA- and GH3-families, members of which have been found in seed plants. The precise function of GH3-like proteins in plant development is not well characterised yet. Mutant analysis in Arabidopsis thaliana indicates a possible role for GH3-like proteins in connecting auxin and light signal transduction. Here, we report the isolation of three different GH3-like homologues from a lower land plant, the moss Physcomitrella patens. Two of the GH3-like homologues were chosen for further characterisation. Both genes are expressed in gametophytic tissues, with expression starting very early in moss development. Knockout plants were generated and analysed. In comparison to white-light growth, cultivation of the wild type and knockout plants under red-light conditions resulted in a delay in gametophytic tissue development. The leafy moss plants displayed an elongated phenotype. Growth delay and elongation were even stronger under far-red light conditions. No obvious differences between wild type and knockout plants could be detected under the examined conditions, indicating functional redundancy of the two genes.     

Landscape diversity patterns and endemism of Araceae in Ecuador

Araceae is one of the largest herb families in tropical America, but its patterns of diversity and endemism are poorly known. We used predictive distribution modelling in GIS to study Araceae richness on a landscape scale in Ecuador. Modelling was based on georeferenced herbarium collections with humidity and mean annual temperature as climatic variables. Variation partitioning using multiple regression showed that humidity and altitude were main factors in explaining Araceae diversity patterns. Endemism was less well explained by present climatic factors. Unlike common diversity patterns of herbs or epiphytes, Araceae richness was highest in the eastern (Amazon) lowland rain forest with a secondary centre on the Andean foothills of northwestern Ecuador. The peak in endemism was on the western slopes of the Andes, corresponding to areas that have been severely affected by human activities and deforestation. Eastern lowland (Amazonian) forests were poor in endemic Araceae.     

PIGMENT TURNOVER IN THE MARINE DIATOM THALASSIOSIRA WEISSFLOGII. I. THE 14CO2-LABELING KINETICS OF CHLOROPHYLL a1

The turnover of chlorophyll a (chl a) was investigated in the diatom Thalassiosira weissflogii (Grunow) Fryxell and Hasle using a new method based on the incorporation of ¹⁴C into chl a. The alga was maintained in its exponential growth phase under continuous light; ¹⁴C was supplied as bicarbonate. The time course of label accumulation into the tetrapyrrole ring and the phytol side chain was determined for time periods equivalent to 1–2 cell doublings. The labeling kinetics of the tetrapyrrole ring and the phytol side chain were described satisfactorily by a simple precursor-pigment model with two free parameters, the precursor turnover rate and the pigment turnover rate, both having dimensions of time^?1. The model was fit to the experimental data to determine the values of these two free parameters. The turnover rates of the tetrapyrrole ring and the phytol side chain were not significantly different, ranging from 0.01 to 0.1 per day. These rates are equivalent to turnover times ranging from days to weeks. Growth rate-normalized turnover rates did not vary with irradiance (7.5–825 μE · m^?2· s^?1). The precursor turnover rates of the tetrapyrrole ring and the phytol side chain differed by an order of magnitude. These results indicate that chl a is not degraded significantly in cultures of T. weissflogii grown under continuous light. Neither irradiance nor growth rate affected growth rate-normalized chlorophyll turnover rates. Our results are inconsistent with the hypothesis that steady-state cellular concentrations of chl a are maintained by a dynamic equilibrium between rates of synthesis and degradation.     

Rhodopsin 5- and Rhodopsin 6-mediated clock synchronization in Drosophila melanogaster is independent of retinal phospholipase C-β signaling

Circadian clocks of most organisms are synchronized with the 24-hour solar day by the changes of light and dark. In Drosophila, both the visual photoreceptors in the compound eyes as well as the blue-light photoreceptor Cryptochrome expressed within the brain clock neurons contribute to this clock synchronization. A specialized photoreceptive structure located between the retina and the optic lobes, the Hofbauer-Buchner (H-B) eyelet, projects to the clock neurons in the brain and also participates in light synchronization. The compound eye photoreceptors and the H-B eyelet contain Rhodopsin photopigments, which activate the canonical invertebrate phototransduction cascade after being excited by light. We show here that 2 of the photopigments present in these photoreceptors, Rhodopsin 5 (Rh5) and Rhodopsin 6 (Rh6), contribute to light synchronization in a mutant (norpA(P41) ) that disrupts canonical phototransduction due to the absence of Phospholipase C-β (PLC-β). We reveal that norpA(P41) is a true loss-of-function allele, resulting in a truncated PLC-β protein that lacks the catalytic domain. Light reception mediated by Rh5 and Rh6 must therefore utilize either a different (nonretinal) PLC-β enzyme or alternative signaling mechanisms, at least in terms of clock-relevant photoreception. This novel signaling mode may distinguish Rhodopsin-mediated irradiance detection from image-forming vision in Drosophila.     

Sensitive detection of idiotypic platelet-reactive alloantibodies by an electrical protein chip

To prevent and treat immune-mediated platelet disorders (e.g. neonatal allo-immune thrombocytopenia and platelet transfusion refractoriness) the causative idiotypic platelet-reactive antibodies have to be detected with high sensitivity and specificity. The "Monoclonal Antibody Immobilization Platelet Assay" (MAIPA) is the diagnostic gold standard for immunotyping sera with respect to alloantibodies against human platelet antigens (HPA). However, it is labor-intensive and time-consuming. In this work, an automated protein chip assay (enzyme-linked sandwich immunoassay) based on interdigitated gold microelectrodes in combination with an electrical read-out system was developed and optimized. For this purpose, specific capture antibodies were immobilized on the gold electrodes. The binding of the target is detected via an enzyme-labeled detection antibody by a redox-recycling process that corresponds to the amount of bound target molecule. With this electrical chip assay it is possible to detect antibodies against HPA-1a, HPA-5b and HLA with high sensitivity and specificity in less than half the duration of the MAIPA protocol with similar intra- and interassay variance.     

GIT1 Phosphorylation on Serine 46 by PKD3 Regulates Paxillin Trafficking and Cellular Protrusive Activity

The continuous assembly and disassembly of focal adhesions is required for efficient cell spreading and migration. The G-protein-coupled receptor kinase-interacting protein 1 (GIT1) is a multidomain protein whose dynamic localization to sites of cytoskeletal remodeling is critically involved in the regulation of these processes. Here we provide evidence that the subcellular localization of GIT1 is regulated by protein kinase D3 (PKD3) through direct phosphorylation on serine 46. GIT1 phosphorylation on serine 46 was abrograted by PKD3 depletion, thereby identifying GIT1 as the first specific substrate for this kinase. A GIT1 S46D phosphomimetic mutant localized to motile, paxillin-positive cytoplasmic complexes, whereas the phosphorylation-deficient GIT1 S46A was enriched in focal adhesions. We propose that phosphorylation of GIT1 on serine 46 by PKD3 represents a molecular switch by which GIT1 localization, paxillin trafficking, and cellular protrusive activity are regulated.