Targeted disruption of the Lasp-1 gene is linked to increases in histamine-stimulated gastric HCl secretion

Lasp-1 (LIM and SH3 domain protein 1) is a multidomain actin-binding protein that is differentially expressed within epithelial tissues and brain. In the gastric mucosa, Lasp-1 is highly expressed in the HCl-secreting parietal cell, where it is prominently localized within the F-actin-rich subcellular regions. Histamine-induced elevation of parietal cell [cAMP]i increases Lasp-1 phosphorylation, which is correlated with activation of HCl secretion. To determine whether Lasp-1 is involved in the regulation of HCl secretion in vivo, we generated a murine model with a targeted disruption of the Lasp-1 gene. Lasp-1-null mice had slightly lower body weights but developed normally and had no overt phenotypic abnormalities. Basal HCl secretion was unaffected by loss of Lasp-1, but histamine stimulation induced a more robust acid secretory response in Lasp-1-null mice compared with wild-type littermates. A similar effect of histamine was observed in isolated gastric glands on the basis of measurements of accumulation of the weak base [14C]aminopyrine. In addition, inhibition of the acid secretory response to histamine by H2 receptor blockade with ranitidine proceeded more slowly in glands from Lasp-1-null mice. These findings support the conclusion that Lasp-1 is involved in the regulation of parietal HCl secretion. We speculate that cAMP-dependent phosphorylation of Lasp-1 alters interactions with F-actin and/or endocytic proteins that interact with Lasp-1, thereby regulating the trafficking/activation of the H+, K+-ATPase (proton pump).     

The Impact and Desirability of News of Risk for Schizophrenia

In studies of schizotypy, investigators seldom inform participants that they are engaged in research designed to shed light on risk for schizophrenia. Such nondisclosure is justified in part by the argument that disclosure of risk status may be harmful. However, there is little evidence that this is the case. Harm arising from disclosure of risk status was examined in two experiments. In the first, participants (n = 114 psychology undergraduates) were asked to anticipate their reactions to news of risk for schizophrenia, depression, cancer, and diabetes, and also to indicate whether they would want to know their schizophrenia risk status. Participants anticipated schizophrenia risk would have a negative impact that was significantly greater than depression or diabetes risk but similar to cancer risk. The anticipated impact of schizophrenia risk was predicted by expectations of stigmatization as well as confidence in the accuracy of biological screening. Although 81% indicated a preference for knowing their risk status, just 11% were prepared to undergo an assessment to find out. In the second, a between-subjects deception paradigm was used to inform participants (n = 144 psychology undergraduates) they had an enzyme deficiency that placed them at increased risk for schizophrenia, cancer, or depression. Impact was assessed using prospective self-report and salivary cortisol and retrospective self-report. Impact was modeled using measures of stigmatization and health locus of control. Retrospectively, schizophrenia, cancer, and depression risk had strong negative impacts relative to a control group, but there was no effect on prospective measures. Together, the findings suggest that news of risk for schizophrenia has the potential to engender distress, although participants’ anticipations and reflections of responses are not corroborated in prospectively measured outcomes.     

Bark structure and mode of canopy regeneration in trees of Melia azedarach L.

Summary The bark texture of Melia azedarach L. changes from smooth to furrowed as trees age. In trees that were cut down, those with smooth bark sprouted below the cut from suppressed buds; trees with thick, furrowed bark sprouted at the edge of the cut surface from adventitious buds. The trees that had thin, furrowed bark sprouted mainly at the edge of the cut from adventitious buds, but sometimes also from suppressed buds in cracks. The relationship between sprouting pattern and tree architecture are discussed.     

The catalytic histidine dyad of high density lipoprotein-associated serum paraoxonase-1 (PON1) is essential for PON1-mediated inhibition of low density lipoprotein oxidation and stimulation of macrophage cholesterol efflux

High density lipoprotein (HDL)-associated paraoxonase-1 (PON1) anti-atherogenic properties in macrophages, i.e. inhibition of cell-mediated oxidation of low density lipoprotein (LDL) and stimulation of cholesterol efflux, were studied using recombinant variants of PON1 and apoA-I expressed in Escherichia coli and reconstituted HDL (rHDL) particles composed of phosphatidylcholine/free cholesterol (PC/FC) and apoA-I. PON1 lactonase activity is stimulated by apoA-I by approximately 7-fold relative to PC/FC particles. Wild-type (WT) PON1 bound to rHDL inhibited macrophage-mediated LDL oxidation and stimulated cholesterol efflux from the cells to 2.3- and 3.2-fold greater extents, respectively, compared with WT PON1 bound to PC/FC particles without apoA-I. We also tested PON1 catalytic histidine dyad mutants (H115Q and H134Q) that are properly folded and that bind HDL in a similar mode compared with WT PON1, but that exhibit almost no lactonase activity. These could not inhibit macrophage-mediated LDL oxidation or stimulate rHDL-mediated cholesterol efflux from the cells. Furthermore, whereas HDL-bound WT PON1 induced the formation of lysophosphatidylcholine (LPC) in macrophages, the His dyad mutants did not, suggesting that the above anti-atherogenic properties of HDL-associated PON1 involve LPC release. Indeed, enrichment of macrophages with increasing concentrations of LPC resulted in inhibition of the cells' capability to oxidize LDL and in stimulation of HDL-mediated cholesterol efflux from the macrophages in an LPC dose-dependent manner. Thus, we provide the first direct indication that the anti-atherogenic properties of PON1 are related to its lipolactonase activity and propose a model in which PON1 acts as a lipolactonase to break down oxidized lipids and to generate LPC.     

Lignocellulose affects Mn2+ regulation of peroxidase transcript levels in solid-state cultures of Pleurotus ostreatus

The effect of Mn2+ amendment on peroxidase gene expression was studied during Pleurotus ostreatus growth on cotton stalks. Four peroxidase-encoding genes were expressed differentially and in a manner different from that observed in defined media. Mn2+ affects mnp3 expression even 2 h after its addition to the cultures, suggesting a direct effect of the metal ion on expression.     

Binding of a photoaffinity analogue of glutathione to MRP1 (ABCC1) within two cytoplasmic regions (L0 and L1) as well as transmembrane domains 10-11 and 16-17

MRP1 (or ABCC1) is an ABC membrane protein that transports a wide range of natural products as well as glutathione (GSH)-, glucuronate-, and sulfate-conjugated metabolites. In addition, free GSH is required for MRP1 to transport several chemotherapeutic drugs. However, the mechanisms regulating the influence of GSH on MRP1 is poorly understood, and the location of GSH binding site(s) within MRP1 have yet to be determined. To address these issues, we have synthesized a [(125)I] labeled azido-derivative of GSH (IAAGSH) to photoaffinity label MRP1. Our results revealed that IAAGSH labeled MRP1 with high specificity, and binding was inhibited by MRP1 substrates leukotriene C(4) and MK571. Interestingly, verapamil and vincristine enhanced IAAGSH photolabeling of MRP1, in agreement with observations that both drugs enhance GSH transport. We observed GSH to be the best inhibitor of photoaffinity labeling, as compared to oxidized glutathione (GSSG) and four different GSH alkyl derivatives. These observations indicate that IAAGSH interacted with MRP1 in a similar manner as unmodified GSH. Moreover, using eight MRP1-HA variants, each containing hemagglutinin A (HA) epitopes inserted at different sites in MRP1, we mapped the GSH binding sites in MRP1. Our GSH analogue photoaffinity labeled four MRP1 polypeptides that were located within two cytoplasmic domains in linker sequences (L0 and L1) as well as transmembrane domains 10-11 and 16-17. The photoaffinity labeling of polypeptides within L0 and L1 domains is further confirmed using two MRP1-specific monoclonal antibodies (MRPr1 and QCRL1) with epitopes within the linker domains. Taken together, this study provides the most precise information to date on the location of GSH binding sites in MRP1.     

The myelin-associated oligodendrocytic basic protein region MOBP15-36 encompasses the immunodominant major encephalitogenic epitope(s) for SJL/J mice and predicted epitope(s) for multiple sclerosis-associated HLA-DRB1*1501

Autoimmune response to the myelin-associated oligodendrocytic basic protein (MOBP), a CNS-specific myelin constituent, was recently suggested to play a role in the pathogenesis of multiple sclerosis (MS). The pathogenic autoimmune response to MOBP and the associated pathology in the CNS have not yet been fully investigated. In this study, we have characterized the clinical manifestations, pathology, T cell epitope-specificity, and TCRs associated with experimental autoimmune encephalomyelitis (EAE) induced in SJL/J mice with recombinant mouse MOBP (long isoform, 170 aa). Analysis of encephalitogenic MOBP-reactive T cells for reactivity to overlapping MOBP peptides defined MOBP15-36 as their major immunodominant epitope. Accordingly, MOBP15-36 was demonstrated to be the major encephalitogenic MOBP epitope for SJL/J mice, inducing severe/chronic clinical EAE associated with intense perivascular and parenchymal infiltrations, widespread demyelination, axonal loss, and remarkable optic neuritis. Molecular modeling of the interaction of I-A(s) with MOBP15-36, together with analysis of the MOBP15-36-specific T cell response to truncated peptides, suggests MOBP20-28 as the core sequence for I-A(s)-restricted recognition of the encephalitogenic region MOBP15-36. Although highly focused in their epitope specificity, the encephalitogenic MOBP-reactive T cells displayed a widespread usage of TCR Vbeta genes. These results would therefore favor epitope-directed, rather than TCR-targeted, approaches to therapy of MOBP-associated pathogenic autoimmunity. Localization by molecular modeling of a potential HLA-DRB1*1501-associated MOBP epitope within the encephalitogenic MOBP15-36 sequence suggests the potential relevance of T cell reactivity against MOBP15-36 to MS. The reactivity to MOBP15-36 detected in MS shown here and in another study further emphasizes the potential significance of this epitope for MS.     

YND1 interacts with CDC55 and is a novel mediator of E4orf4-induced toxicity

Adenovirus E4orf4 (early region 4 open reading frame 4) protein induces protein phosphatase 2A-dependent non-classical apoptosis in mammalian cells and irreversible growth arrest in Saccharomyces cerevisiae. Oncogenic transformation sensitizes cells to E4orf4-induced cell death. To uncover additional components of the E4orf4 network required for induction of its unique mode of apoptosis, we used yeast genetics to select gene deletions conferring resistance to E4orf4. Deletion of YND1, encoding a yeast Golgi apyrase, conferred partial resistance to E4orf4. However, Ynd1p apyrase activity was not required for E4orf4-induced toxicity. Ynd1p and Cdc55p, the yeast protein phosphatase 2A-B subunit, contributed additively to E4orf4-induced toxicity. Furthermore, concomitant overexpression of one and deletion of the other was detrimental to yeast growth, demonstrating a functional interaction between the two proteins. YND1 and CDC55 also interacted genetically with CDC20 and CDH1/HCT1, encoding activating subunits of the anaphase-promoting complex/cyclosome. In addition to their functional interaction, Ynd1p and Cdc55p interacted physically, and this interaction was disrupted by E4orf4, which remained associated with both proteins. The results suggested that Ynd1p and Cdc55p share a common downstream target whose balanced modulation by the two E4orf4 partners is crucial to viability. Disruption of this balance by E4orf4 may lead to cell death. NTPDase-4/Lalp70/UDPase, the closest mammalian homologue of Ynd1p, associated with E4orf4 in mammalian cells, suggesting that the results in yeast are relevant to the mammalian system.