Participation of the extracellular domain in (pro)renin receptor dimerization

The (pro)renin receptor [(P)RR] induces the catalytic activation of prorenin, as well as the activation of the mitogen-activated protein kinase (MAPK) signaling pathway; as such, it plays an important regulatory role in the renin–angiotensin system. (P)RR is known to form a homodimer, but the region participating in its dimerization is unknown. Using glutathione S-transferase (GST) as a carrier protein and a GST pull-down assay, we investigated the interaction of several (P)RR constructs with full-length (FL) (P)RR in mammalian cells. GST fusion proteins with FL (P)RR (GST-FL), the C-terminal M8-9 fragment (GST-M8-9), the extracellular domain (ECD) of (P)RR (GST-ECD), and the (P)RR ECD with a deletion of 32 amino acids encoded by exon 4 (GST-ECDd4) were retained intracellularly, whereas GST alone was efficiently secreted into the culture medium when transiently expressed in COS-7 cells. Immunofluorescence microscopy showed prominent localization of GST-ECD to the endoplasmic reticulum. The GST pull-down analysis revealed that GST-FL, GST-ECD, and GST-ECDd4 bound FLAG-tagged FL (P)RR, whereas GST-M8-9 showed little or no binding when transiently co-expressed in HEK293T cells. Furthermore, pull-down analysis using His-tag affinity resin showed co-precipitation of soluble (P)RR with FL (P)RR from a stable CHO cell line expressing FL h(P)RR with a C-terminal decahistidine tag. These results indicate that the (P)RR ECD participates in dimerization.     

Identification of novel biomarker candidates by proteomic analysis of cerebrospinal fluid from patients with moyamoya disease using SELDI-TOF-MS

Background

Moyamoya disease (MMD) is an uncommon cerebrovascular condition with unknown etiology characterized by slowly progressive stenosis or occlusion of the bilateral internal carotid arteries associated with an abnormal vascular network. MMD is a major cause of stroke, specifically in the younger population. Diagnosis is based on only radiological features as no other clinical data are available. The purpose of this study was to identify novel biomarker candidate proteins differentially expressed in the cerebrospinal fluid (CSF) of patients with MMD using proteomic analysis.

Methods

For detection of biomarkers, CSF samples were obtained from 20 patients with MMD and 12 control patients. Mass spectral data were generated by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) with an anion exchange chip in three different buffer conditions. After expression difference mapping was undertaken using the obtained protein profiles, a comparative analysis was performed.

Results

A statistically significant number of proteins (34) were recognized as single biomarker candidate proteins which were differentially detected in the CSF of patients with MMD, compared to the control patients (p < 0.05). All peak intensity profiles of the biomarker candidates underwent classification and regression tree (CART) analysis to produce prediction models. Two important biomarkers could successfully classify the patients with MMD and control patients.

Conclusions

In this study, several novel biomarker candidate proteins differentially expressed in the CSF of patients with MMD were identified by a recently developed proteomic approach. This is a pilot study of CSF proteomics for MMD using SELDI technology. These biomarker candidates have the potential to shed light on the underlying pathogenesis of MMD.

     

Positively charged acyl derivatives of carbohydrates as promising transfection agents

A convenient approach to the synthesis of mono- and polycationic glycolipid amphiphiles is suggested. The compounds obtained can be used for study of the structure-activity relationship and determination of the effect of hydrophobic and cationic domains on transfection efficiency.     

Differential role of TLR- and RLR-signaling in the immune responses to influenza A virus infection and vaccination

The innate immune system recognizes influenza A virus via TLR 7 or retinoic acid-inducible gene I in a cell-type specific manner in vitro, however, physiological function(s) of the MyD88- or interferon-beta promoter stimulator 1 (IPS-1)-dependent signaling pathways in antiviral responses in vivo remain unclear. In this study, we show that although either MyD88- or IPS-1-signaling pathway was sufficient to control initial antiviral responses to intranasal influenza A virus infection, mice lacking both pathways failed to show antiviral responses, resulting in increased viral load in the lung. By contrast, induction of B cells or CD4 T cells specific to the dominant hemagglutinin or nuclear protein Ags respectively, was strictly dependent on MyD88 signaling, but not IPS-1 signaling, whereas induction of nuclear protein Ag-specific CD8 T cells was not impaired in the absence of either MyD88 or IPS-1. Moreover, vaccination of TLR7- and MyD88-deficient mice with inactivated virus failed to confer protection against a lethal live virus challenge. These results strongly suggest that either the MyD88 or IPS-1 signaling pathway is sufficient for initial antiviral responses, whereas the protective adaptive immune responses to influenza A virus are governed by the TLR7-MyD88 pathway.     

Critical role of the Fc receptor gamma-chain on APCs in the development of allergen-induced airway hyperresponsiveness and inflammation

The FcR common gamma-chain (FcRgamma) is an essential component of the receptors FcepsilonRI, FcgammaRI, and FcgammaRIII, which are expressed on many inflammatory cell types. The role of these receptors in the initiation or maintenance of allergic inflammation has not been well defined. FcRgamma-deficient (FcRgamma(-/-)) and control (wild-type (WT)) mice were sensitized and subsequently challenged with OVA. Following sensitization and challenge to OVA, FcRgamma-deficient (FcRgamma(-/-)) mice developed comparable levels of IgE and IgG1 as WT mice. However, numbers of eosinophils, levels of IL-5, IL-13, and eotaxin in bronchoalveolar lavage fluid, and mononuclear cell (MNC) proliferative responses to OVA were significantly reduced, as was airway hyperresponsiveness (AHR) to inhaled methacholine. Reconstitution of FcRgamma(-/-) mice with whole spleen MNC from WT mice before sensitization restored development of AHR and the numbers of eosinophils in bronchoalveolar lavage fluid; reconstitution after sensitization but before OVA challenge only partially restored these responses. These responses were also restored when FcRgamma(-/-) mice received T cell-depleted MNC, T and B cell-depleted MNC, or bone marrow-derived dendritic cells before sensitization from FcR(+/+) or FcgammaRIII-deficient but not FcRgamma(-/-) mice. The expression levels of FcgammaRIV on bone marrow-derived dendritic cells from FcR(+/+) mice were found to be low. These results demonstrate that expression of FcRgamma, most likely FcgammaRI, on APCs is important during the sensitization phase for the development of allergic airway inflammation and AHR.     

LAT and NTAL mediate immunoglobulin E-induced sustained extracellular signal-regulated kinase activation critical for mast cell survival

Immunoglobulin E (IgE) induces mast cell survival in the absence of antigen (Ag) through the high-affinity IgE receptor, Fcepsilon receptor I (FcepsilonRI). Although we have shown that protein tyrosine kinase Syk and sustained extracellular signal-regulated kinase (Erk) activation are required for IgE-induced mast cell survival, how Syk couples with sustained Erk activation is still unclear. Here, we report that the transmembrane adaptors LAT and NTAL are phosphorylated slowly upon IgE stimulation and that sustained but not transient Erk activation induced by IgE was inhibited in LAT(-/-) NTAL(-/-) bone marrow-derived mast cells (BMMCs). IgE-induced survival requires Ras activation, and both were impaired in LAT(-/-) NTAL(-/-) BMMCs. Sos was preferentially required for FcepsilonRI signals by IgE rather than IgE plus Ag. Survival impaired in LAT(-/-) NTAL(-/-) BMMCs was restored to levels comparable to those of the wild type by membrane-targeted Sos, which bypasses the Grb2-mediated membrane recruitment of Sos. The IgE-induced survival of BMMCs lacking Gads, an adaptor critical for the formation of the LAT-SLP-76-phospholipase Cgamma (PLCgamma) complex, was observed to be normal. IgE stimulation induced the membrane retention of Grb2-green fluorescent protein fusion proteins in wild-type but not LAT(-/-) NTAL(-/-) BMMCs. These results suggest that LAT and NTAL contribute to the maintenance of Erk activation and survival through the membrane retention of the Ras-activating complex Grb2-Sos and, further, that the LAT-Gads-SLP-76-PLCgamma and LAT/NTAL-Grb2-Sos pathways are differentially required for degranulation and survival, respectively.     

Light activation of an innate olfactory avoidance response in Drosophila

How specific sensory stimuli evoke specific behaviors is a fundamental problem in neurobiology. In Drosophila, most odorants elicit attraction or avoidance depending on their concentration, as well as their identity [1]. Such odorants, moreover, typically activate combinations of glomeruli in the antennal lobe of the brain [2-4], complicating the dissection of the circuits translating odor recognition into behavior. Carbon dioxide (CO2), in contrast, elicits avoidance over a wide range of concentrations [5, 6] and activates only a single glomerulus, V [5]. The V glomerulus receives projections from olfactory receptor neurons (ORNs) that coexpress two GPCRs, Gr21a and Gr63a, that together comprise a CO2 receptor [7-9]. These CO2-sensitive ORNs, located in the ab1 sensilla of the antenna, are called ab1c neurons [10]. Genetic silencing of ab1c neurons indicates that they are necessary for CO2-avoidance behavior [5]. Whether activation of these neurons alone is sufficient to elicit this behavior, or whether CO2 avoidance requires additional inputs (e.g., from the respiratory system), remains unclear. Here, we show that artificial stimulation of ab1c neurons with light (normally attractive to flies) elicits the avoidance behavior typical of CO2. Thus, avoidance behavior appears hardwired into the olfactory circuitry that detects CO2 in Drosophila.     

4-Pyridone derivatives as new inhibitors of bacterial enoyl-ACP reductase FabI

Bacterial FAS provides essential fatty acids for use in the assembly of key cellular components. Among them, FabI is an enoyl-ACP reductase which catalyzes the final and rate-limiting step of bacterial FAS. It is a potential target for selective antibacterial action, because it shows low overall sequence homology with mammalian enzymes. Until today, various compounds have been reported as inhibitors of bacterial FabI-inhibitory compounds. To discover novel small-molecular FabI inhibitors, we initially screened our compound library for inhibitory activity toward FabI of Escherichia coli. And discovered 4-pyridone derivatives as a lead compound. Structure optimization studies yielded 4-pyridone derivatives 7n having strong FabI-inhibitory and antibacterial activities against Staphylococcus aureus. There have been no reports concerning 4-pyridone derivatives as FabI inhibitor.     

Phosphatidylinositol 3-Kinase Class II α-Isoform PI3K-C2α Is Required for Transforming Growth Factor β-induced Smad Signaling in Endothelial Cells

We have recently demonstrated that the PI3K class II-α isoform (PI3K-C2α), which generates phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphates, plays crucial roles in angiogenesis, by analyzing PI3K-C2α knock-out mice. The PI3K-C2α actions are mediated at least in part through its participation in the internalization of VEGF receptor-2 and sphingosine-1-phosphate receptor S1P₁ and thereby their signaling on endosomes. TGFβ, which is also an essential angiogenic factor, signals via the serine/threonine kinase receptor complex to induce phosphorylation of Smad2 and Smad3 (Smad2/3). SARA (Smad anchor for receptor activation) protein, which is localized in early endosomes through its FYVE domain, is required for Smad2/3 signaling. In the present study, we showed that PI3K-C2α knockdown nearly completely abolished TGFβ1-induced phosphorylation and nuclear translocation of Smad2/3 in vascular endothelial cells (ECs). PI3K-C2α was necessary for TGFβ-induced increase in phosphatidylinositol 3,4-bisphosphates in the plasma membrane and TGFβ receptor internalization into the SARA-containing early endosomes, but not for phosphatidylinositol 3-phosphate enrichment or localization of SARA in the early endosomes. PI3K-C2α was also required for TGFβ receptor-mediated formation of SARA-Smad2/3 complex. Inhibition of dynamin, which is required for the clathrin-dependent receptor endocytosis, suppressed both TGFβ receptor internalization and Smad2/3 phosphorylation. TGFβ1 stimulated Smad-dependent VEGF-A expression, VEGF receptor-mediated EC migration, and capillary-like tube formation, which were all abolished by either PI3K-C2α knockdown or a dynamin inhibitor. Finally, TGFβ1-induced microvessel formation in Matrigel plugs was greatly attenuated in EC-specific PI3K-C2α-deleted mice. These observations indicate that PI3K-C2α plays the pivotal role in TGFβ receptor endocytosis and thereby Smad2/3 signaling, participating in angiogenic actions of TGFβ.