Rat organic anion transporting protein 1A1 (Oatp1a1): purification and phosphopeptide assignment

Rat organic anion transporting protein 1a1 (oatp1a1), a hepatocyte basolateral plasma membrane protein, mediates transport of various amphipathic compounds. Our previous studies indicated that serine phosphorylation of a single tryptic peptide inhibits its transport activity without changing its cell surface content. The site of phosphorylation is unknown and was the subject of the present study. Following immunoaffinity chromatographic purification from rat liver, oatp1a1 was subjected to trypsin digestion and MALDI-TOF. Except for predicted N-glycosylated peptides, 97% of oatp1a1 tryptic peptides were observed. A single tryptic phosphopeptide was found in the C-terminus (aa 626-647), existing in unphosphorylated or singly or doubly phosphorylated forms and sensitive to alkaline phosphatase treatment. The beta-elimination reaction resulted in a mass loss of 98 or 196 Da from this peptide, and subsequent Michael addition with cysteamine increased masses by the predicated 77 and 154 Da, indicating that oatp1a1 can be singly or doubly phosphorylated at serine or threonine residues in the C-terminal sequence SSATDHT (aa 634-640). Subsequent tandem MS/MS analysis revealed that phosphorylation at S634 accounted for all singly phosphorylated peptide, while phosphorylation at S634 and S635 accounted for all doubly phosphorylated peptide. These findings identify the site of oatp1a1 phosphorylation and demonstrate that it is an ordered process, in which phosphorylation at S634 precedes that at S635. The mechanism by which phosphorylation results in loss of transport activity in hepatocytes remains to be established. Whether phosphorylation near the C-terminus inhibits C-terminal oligomerization of oatp1a1, required for normal transport function, can be speculated upon but is as yet unknown.     

Activation of PKR causes amyloid ß-peptide accumulation via de-repression of BACE1 expression

BACE1 is a key enzyme involved in the production of amyloid ß-peptide (Aß) in Alzheimer''s disease (AD) brains. Normally, its expression is constitutively inhibited due to the presence of the 5′untranslated region (5′UTR) in the BACE1 promoter. BACE1 expression is activated by phosphorylation of the eukaryotic initiation factor (eIF)2-alpha, which reverses the inhibitory effect exerted by BACE1 5′UTR. There are four kinases associated with different types of stress that could phosphorylate eIF2-alpha. Here we focus on the double-stranded (ds) RNA-activated protein kinase (PKR). PKR is activated during viral infection, including that of herpes simplex virus type 1 (HSV1), a virus suggested to be implicated in the development of AD, acting when present in brains of carriers of the type 4 allele of the apolipoprotein E gene. HSV1 is a dsDNA virus but it has genes on both strands of the genome, and from these genes complementary RNA molecules are transcribed. These could activate BACE1 expression by the PKR pathway. Here we demonstrate in HSV1-infected neuroblastoma cells, and in peripheral nervous tissue from HSV1-infected mice, that HSV1 activates PKR. Cloning BACE1 5′UTR upstream of a luciferase (luc) gene confirmed its inhibitory effect, which can be prevented by salubrinal, an inhibitor of the eIF2-alpha phosphatase PP1c. Treatment with the dsRNA analog poly (I∶C) mimicked the stimulatory effect exerted by salubrinal over BACE1 translation in the 5′UTR-luc construct and increased Aß production in HEK-APPsw cells. Summarizing, our data suggest that PKR activated in brain by HSV1 could play an important role in the development of AD.     

Characterizing associations and SNP-environment interactions for GWAS-identified prostate cancer risk markers--results from BPC3

Genome-wide association studies (GWAS) have identified multiple single nucleotide polymorphisms (SNPs) associated with prostate cancer risk. However, whether these associations can be consistently replicated, vary with disease aggressiveness (tumor stage and grade) and/or interact with non-genetic potential risk factors or other SNPs is unknown. We therefore genotyped 39 SNPs from regions identified by several prostate cancer GWAS in 10,501 prostate cancer cases and 10,831 controls from the NCI Breast and Prostate Cancer Cohort Consortium (BPC3). We replicated 36 out of 39 SNPs (P-values ranging from 0.01 to 10⁻²⁸). Two SNPs located near KLK3 associated with PSA levels showed differential association with Gleason grade (rs2735839, P = 0.0001 and rs266849, P = 0.0004; case-only test), where the alleles associated with decreasing PSA levels were inversely associated with low-grade (as defined by Gleason grade <8) tumors but positively associated with high-grade tumors. No other SNP showed differential associations according to disease stage or grade. We observed no effect modification by SNP for association with age at diagnosis, family history of prostate cancer, diabetes, BMI, height, smoking or alcohol intake. Moreover, we found no evidence of pair-wise SNP-SNP interactions. While these SNPs represent new independent risk factors for prostate cancer, we saw little evidence for effect modification by other SNPs or by the environmental factors examined.     

The statistics of identifying differentially expressed genes in Expresso and TM4: a comparison

Background  

Analysis of DNA microarray data takes as input spot intensity measurements from scanner software and returns differential expression of genes between two conditions, together with a statistical significance assessment. This process typically consists of two steps: data normalization and identification of differentially expressed genes through statistical analysis. The Expresso microarray experiment management system implements these steps with a two-stage, log-linear ANOVA mixed model technique, tailored to individual experimental designs. The complement of tools in TM4, on the other hand, is based on a number of preset design choices that limit its flexibility. In the TM4 microarray analysis suite, normalization, filter, and analysis methods form an analysis pipeline. TM4 computes integrated intensity values (IIV) from the average intensities and spot pixel counts returned by the scanner software as input to its normalization steps. By contrast, Expresso can use either IIV data or median intensity values (MIV). Here, we compare Expresso and TM4 analysis of two experiments and assess the results against qRT-PCR data.     

Zur Elektronenmikroskopie der Kerneinschlüsse im menschlichen Nebenhodenepithel

Zusammenfassung Es werden verschiedene Typen von Einschlußkörperchen im Karyoplasma der Epithelzellen des menschlichen Nebenhodenepithels beschrieben. Die verschieden strukturierten Kerninklusionen werden als eine Differenzierungsreihe aufgefaßt. Sie beginnt mit kleinen rundlichen Körperchen aus feinfädigem Material und führt über die Ausbildung von Anhäufungen dichter homogener Kugeln zu großen Vakuolen wechselnden Inhaltes.

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Summary Different types of inclusion bodies are described in the karyoplasm of the epithelial cells of the human epididymis. The structural differences between these inclusions are interpreted as being indicative of consecutive stages in the process of their formation. Thus small, spherical bodies consisting of a fine fibrous material are believed to be the initial stages in the formation of the inclusion bodies whereas the dense, homogenous globules are thought to represent a later stage. Large vacuoles containing different materials are regarded as the final stage in this process of differentiation.

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Herrn Professor Dr. W. Bargmann zum 60. Geburtstag gewidmet.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Lipoprotein-associated paf (LA-paf) was found in washed human platelets and monocyte/macrophage-like U937 cells

Beyond cholesterol, inflammatory ether phospholipids such as platelet-activating factor (paf) may play a role in atherogenesis. (1) We detected a paf-like compound (‘LA-paf’) associated with human serum lipoproteins, mainly in LDL but not with the lipoprotein-poor fraction. (2) LA-paf was also found in washed human platelets, from where it was partially released during platelet aggregation in response to paf (50 nM) or thrombin (1 U). In addition, resident monocyte/macrophage-like U937 cells carried huge amounts of LA-paf (41 ng per 10⁷ cells) and metabolized added [³H]paf to a labelled compound co-eluting with the retention time of LA-paf in standard HPLC. (3) Functionally, LA-paf had a comparable potency to synthetic paf, because LA-paf aggregated washed aspirin-treated platelets in a concentration-dependent manner. The specific paf receptor antagonist WEB2086 inhibited the platelet aggregation induced by three distinct LA-paf preparations as compared with synthetic paf with similar inhibitory concentrations (IC₅₀: 35.6 ± 12.8, 24.0 ± 4.0, 38.0 ± 15.8 nM for LA-paf, and 43.6 ± 6.5 nM for synthetic paf), indicating that LA-paf interacted with paf receptors. (4) However, LA-paf had a distinct retention time using high-pressure liquid chromatography (HPLC) as compared with synthetic paf. LA-paf eluted at 9–15 min and synthetic paf at 21–24 min. In addition, total and non-specific [³H]paf binding to intact washed human platelets was affected differently by the two unlabelled agonists: while LA-paf increased total and non-specific (but not specific) binding in a significant manner (P < 0.002 and P < 0.007) as LDL did (P < 0.006 and P < 0.03), synthetic paf decreased total binding (P < 0.03). Similarly, low-density lipoproteins (LDL) increased significantly the total [³H]paf binding. In contrast, paf did not affect specific [¹²⁵I]LDL binding to human fibroblasts. Our results show the presence of LA-paf in lipoproteins,     

Integration of biological networks and gene expression data using Cytoscape

Cytoscape is a free software package for visualizing, modeling and analyzing molecular and genetic interaction networks. This protocol explains how to use Cytoscape to analyze the results of mRNA expression profiling, and other functional genomics and proteomics experiments, in the context of an interaction network obtained for genes of interest. Five major steps are described: (i) obtaining a gene or protein network, (ii) displaying the network using layout algorithms, (iii) integrating with gene expression and other functional attributes, (iv) identifying putative complexes and functional modules and (v) identifying enriched Gene Ontology annotations in the network. These steps provide a broad sample of the types of analyses performed by Cytoscape.     

Immobilization of lymphocytes at surfaces by lectins

Phytohemagglutinin (PHA) and Concanavalin A (ConA) cause normal chicken lymphocytes to adhere to glass and plastic surfaces. Pokeweed mitogen (PWM) does not cause adherence. The effect of ConA was studied in detail. The reaction begins within 15 min at 25 °C and proceeds to completion by 2 h. It is independent of pH and resembles in this respect the spontaneous adherence which can occur in protein-depleted suspensions of chicken lymphocytes. It is distinguished from spontaneous adherence by conducting the reaction in 1% or more serum protein; high concentrations exert a slight restraint, which can be overcome by increasing the concentration of ConA. The reaction is slightly greater at high cell concentrations, is inhibited by 3 mM sodium cyanide, and is effectively blocked by 3 mM iodoacetamide and the α-methyl- -glycosides of glucose and mannose. The reaction is not affected by 2-deoxy- -glucose or N-acetyl glucosamine. Adherent lymphocytes detach when the lectin solution is replaced with lectin-free saline; they readhere when reexposed to ConA or to alloantibody directed against lymphocyte surface antigen. At low concentrations of ConA the large lymphocytes of the bursa of Fabricius adhere more rapidly than the small lymphocytes of the blood and thymus. Mouse lymph node lymphocytes adhere in the same manner as small chicken lymphocytes.     

Gene expression changes in spleens of the wildlife reservoir species, Eurasian wild boar （Sus scrofa）, naturally infected with Brucella suis biovar 2

Brucella suis is responsible for swine brucellosis worldwide.Of the five different 8.suis biovars (bv.), bv.2 appears restricted to Europe where it is frequently isolated from wild boar and hares, can infect pigs and can cause human brucellosis.In this study, the differ-ential gene expression profile was characterized in spleens of Eurasian wild boar naturally infected with B.suis by.2.Of the 20,201 genes analyzed in the microarray, 633 and 1,373 were significantly (fold change＞1.8; P＜0.01) upregulated and downregulated, respectively,in infected wild boar.The analysis was focused on genes that were over represented after conditional test for biological process gene on-tology.Upregulated genes suggested that B.suis bv.2 infection induced cell maturation, migration and/or proliferation in infected animals.The genes downregulated in infected wild boar impaired the activity of several important cellular metabolic pathways such as metabolism,cytoskeleton organization and biogenesis, immune response and lysosomal function and vesicle-mediated transport.In addition, the re-sponse to stress, sperm fertility, muscle development and apoptosis seemed to be also impaired in infected animals.These results sug-gested that B.suis by.2 may use strategies similar to other smooth brucellae to facilitate intraeellular multiplication and the developmentof chronic infections.To our knowledge, this is the first report of the analysis of gene expression profile in hosts infected with B.suis by.2, which is important to understand the molecular mechanisms at the host-pathogen interface in the main reservoir species with possible implications in the zoonotic cycle of the pathogen.