Cytochrome P450 2B1 mediates complement-dependent sublytic injury in a model of membranous nephropathy

Membranous nephropathy is a disease that affects the filtering units of the kidney, the glomeruli, and results in proteinuria accompanied by loss of kidney function. Passive Heymann nephritis is an experimental model that mimics membranous nephropathy in humans, wherein the glomerular epithelial cell (GEC) injury induced by complement C5b-9 leads to proteinuria. We examined the role of cytochrome P450 2B1 (CYP2B1) in this complement-mediated sublytic injury. Overexpression of CYP2B1 in GECs significantly increased the formation of reactive oxygen species, cytotoxicity, and collapse of the actin cytoskeleton following treatment with anti-tubular brush-border antiserum (anti-Fx1A). In contrast, silencing of CYP2B1 markedly attenuated anti-Fx1A-induced reactive oxygen species generation and cytotoxicity with preservation of the actin cytoskeleton. Gelsolin, which maintains an organized actin cytoskeleton, was significantly decreased by complement C5b-9-mediated injury but was preserved in CYP2B1-silenced cells. In rats injected with anti-Fx1A, the cytochrome P450 inhibitor cimetidine blocked an increase in catalytic iron and ROS generation, reduced the formation of malondialdehyde adducts, maintained a normal distribution of nephrin in the glomeruli, and provided significant protection at the onset of proteinuria. Thus, GEC CYP2B1 contributes to complement C5b-9-mediated injury and plays an important role in the pathogenesis of passive Heymann nephritis.     

Model-based deconvolution of genome-wide DNA binding

Motivation: Chromatin immunoprecipitation followed by hybridizationto a genomic tiling microarray (ChIP-chip) is a routinely usedprotocol for localizing the genomic targets of DNA-binding proteins.The resolution to which binding sites in this assay can be identifiedis commonly considered to be limited by two factors: (1) theresolution at which the genomic targets are tiled in the microarrayand (2) the large and variable lengths of the immunoprecipitatedDNA fragments. Results: We have developed a generative model of binding sitesin ChIP-chip data and an approach, MeDiChI, for efficientlyand robustly learning that model from diverse data sets. Wehave evaluated MeDiChI's performance using simulated data, aswell as on several diverse ChIP-chip data sets collected onwidely different tiling array platforms for two different organisms(Saccharomyces cerevisiae and Halobacterium salinarium NRC-1).We find that MeDiChI accurately predicts binding locations toa resolution greater than that of the probe spacing, even foroverlapping peaks, and can increase the effective resolutionof tiling array data by a factor of 5x or better. Moreover,the method's performance on simulated data provides insightsinto effectively optimizing the experimental design for increasedbinding site localization accuracy and efficacy. Availability: MeDiChI is available as an open-source R package,including all data, from http://baliga.systemsbiology.net/medichi. Contact: dreiss{at}systemsbiology.org Supplementary information: Supplementary data are availableat Bioinformatics online. Associate Editor: Martin Bishop     

Duodenal somatostatinoma of the ampulla of vater diagnosed by endoscopic fine needle aspiration biopsy: a case report

BACKGROUND: Duodenal somatostatinoma is a rare neuroendocrine neoplasm. A better prognosis can be obtained if these tumors are resected at an early clinical stage. Endoscopic punch biopsy has been the method most commonly used for the preoperative diagnosis of neuroendocrine duodenal tumors. To the best of our knowledge, endoscopic fine needle aspiration biopsy (FNAB) of duodenal somatostatinoma has not been reported before. CASE: A 41-year-old, black female presented with upper gastrointestinal bleeding as well as elevated bilirubin, liver enzymes and glucose. Computed tomography, esophagogastroduodenoscopy and endoscopic retrograde cholangiopancreatography (ERCP) detected a mass at the region of the ampulla of Vater partially obstructing the pancreatic duct. The initial punch biopsy yielded only intestinal mucosa. Subsequent endoscopic FNAB suggested the diagnosis of a neuroendocrine neoplasm, as confirmed by additional punch biopsies. Immunohistochemical and electron microscopic studies disclosed somatostatin production by the tumor, which was resected through a modified Whipple procedure. The patient recovered fully. CONCLUSION: This case demonstrates the usefulness of endoscopic FNAB in diagnosing submucosal gastrointestinal neuroendocrine tumors.     

Kinetic consequences of covalent linkage of DNA binding polyamides

Polyamides composed of N-methylpyrrole (Py) and N-methylimidazole (Im) subunits can bind in the minor groove of DNA at predetermined sequences with subnanomolar affinity and high specificity. Covalent linkage of polymer subunits using a gamma-aminobutyric acid linker has been shown to increase both the affinity and specificity of polyamides. Using a fluorescence detected stopped-flow assay, we have studied the differences in association and dissociation kinetics of a series of polyamides representing unlinked, hairpin and cyclic analogues of the four ring polyamide ImPyPyPy-beta-Dp. Whereas the large differences seen in the equilibrium association constants between the unlinked and covalently linked polyamides are primarily due to higher association rate constants, discrimination between matched and mismatched sites by each polyamide can be ascribed in large part to differences in their dissociation rate constants. The consequences of this kinetic behavior for future design are discussed.     

Comprehensive <Emphasis Type="Italic">de novo</Emphasis> structure prediction in a systems-biology context for the archaea <Emphasis Type="Italic">Halobacterium</Emphasis> sp. <Emphasis Type="Italic">NRC-1</Emphasis>

Background  

Large fractions of all fully sequenced genomes code for proteins of unknown function. Annotating these proteins of unknown function remains a critical bottleneck for systems biology and is crucial to understanding the biological relevance of genome-wide changes in mRNA and protein expression, protein-protein and protein-DNA interactions. The work reported here demonstrates that de novo structure prediction is now a viable option for providing general function information for many proteins of unknown function.     

Stat3 beta inhibits gamma-globin gene expression in erythroid cells

We demonstrated previously gamma-globin gene inhibition in K562 cells and primary erythroid progenitors treated with interleukin-6. Although several cis-acting elements have been identified in the globin promoters, the precise mechanism for cytokine-mediated globin gene regulation remains to be elucidated. In this report we demonstrate inhibitors of Stat3 phosphorylation abrogate interleukin-6-mediated gamma gene silencing in erythroid cells. DNA-protein binding studies established Stat3 interaction in the 5'-untranslated gamma-globin promoter region. Furthermore, co-transfection experiments with Stat3 beta demonstrate gamma promoter inhibition in a concentration-dependent manner, which was significantly reversed when the cognate Stat3-binding site in the 5'-untranslated region was mutated. These studies establish a novel mechanism for gamma gene silencing through the STAT signal transduction pathway.     

Role of prodomain in importin-mediated nuclear localization and activation of caspase-2

Caspase-2 is unique among mammalian caspases because it localizes to the nucleus in a prodomain-dependent manner. The caspase-2 prodomain also regulates caspase-2 activity via a caspase recruitment domain that mediates oligomerization of procaspase-2 molecules and their subsequent autoactivation. In this study we sought to map specific functional regions in the caspase-2 prodomain that regulate its nuclear transport and also its activation. Our data indicate that caspase-2 contains a classical nuclear localization signal (NLS) at the C terminus of the prodomain which is recognized by the importin alpha/beta heterodimer. The mutation of a conserved Lys residue in the NLS abolishes nuclear localization of caspase-2 and binding to the importin alpha/beta heterodimer. Although caspase-2 is imported into the nucleus, mutants lacking the NLS were still capable of inducing apoptosis upon overexpression in transfected cells. We define a region in the prodomain that regulates the ability of caspase-2 to form dot- and filament-like structures when ectopically expressed, which in turn promotes cell killing. Our data provides a mechanism for caspase-2 nuclear import and demonstrate that association of procaspase-2 into higher order structures, rather than its nuclear localization, is required for caspase-2 activation and its ability to induce apoptosis.     

Proteomic analysis of an extreme halophilic archaeon,Halobacterium sp. NRC-1

Halobacterium sp. NRC-1 insoluble membrane and soluble cytoplasmic proteins were isolated by ultracentrifugation of whole cell lysate. Using an ion trap mass spectrometer equipped with a C18 trap electrospray ionization emitter/micro-liquid chromatography column, a number of trypsin-generated peptide tags from 426 unique proteins were identified. This represents approximately one-fifth of the theoretical proteome of Halobacterium. Of these, 232 proteins were found only in the soluble fraction, 165 were only in the insoluble membrane fraction, and 29 were in both fractions. There were 72 and 61% previously annotated proteins identified in the soluble and membrane protein fractions, respectively. Interestingly, 57 of previously unannotated proteins found only in Halobacterium NRC-1 were identified. Such proteins could be interesting targets for understanding unique physiology of Halobacterium NRC-1. A group of proteins involved in various metabolic pathways were identified among the expressed proteins, suggesting these pathways were active at the time the cells were collected. This data containing a list of expressed proteins, their cellular locations, and biological functions could be used in future studies to investigate the interaction of the genes and proteins in relation to genetic or environmental perturbations.