Substitution in Amino Acid 70 of Hepatitis C Virus Core Protein Changes the Adipokine Profile via Toll-Like Receptor 2/4 Signaling

Background & Aims

It has been suggested that amino acid (aa) substitution at position 70 from arginine (70R) to glutamine (70Q) in the genotype 1b hepatitis C virus (HCV) core protein is associated with insulin resistance and worse prognosis. However, the precise mechanism is still unclear. The aim of this study was to investigate the impact of the substitution at position 70 in HCV core protein on adipokine production by murine and human adipocytes.

Methods

The influence of treatment with HCV core protein (70R or 70Q) on adipokine production by both 3T3-L1 and human adipocytes were examined with real-time PCR and enzyme-linked immunosorbent assay (ELISA), and triglyceride content was also analyzed. The effects of toll-like receptor (TLR)2/4 inhibition on IL-6 production by 3T3-L1 induced by HCV core protein were examined.

Results

IL-6 production was significantly increased and adiponectin production was reduced without a change in triglyceride content by treatment with 70Q compared to 70R core protein in both murine and human adipocytes. IL-6 induction of 3T3-L1 cells treated by 70Q HCV core protein was significantly inhibited with anti-TLR2 antibody by 42%, and by TLR4 inhibitor by 40%.

Conclusions

Our study suggests that extracellular HCV core protein with substitution at position 70 enhanced IL-6 production and reduced adiponectin production from visceral adipose tissue, which can cause insulin resistance, hepatic steatosis, and ultimately development of HCC.     

Osteopontin Deficiency Accelerates Spontaneous Colitis in Mice with Disrupted Gut Microbiota and Macrophage Phagocytic Activity

Background

Osteopontin (OPN) is a multifunctional protein expressed in a variety of tissues and cells. Recent studies revealed increased OPN expression in the inflamed intestinal tissues of patients with inflammatory bowel disease (IBD). The role of OPN in the pathophysiology of IBD, however, remains unclear.

Aims

To investigate the role of OPN in the development of intestinal inflammation using a murine model of IBD, interleukin-10 knock out (IL-10 KO) mice.

Methods

We compared the development of colitis between IL-10 KO and OPN/IL-10 double KO (DKO) mice. OPN expression in the colonic tissues of IL-10 KO mice was examined by fluorescence in situ hybridization (FISH) analysis. Enteric microbiota were compared between IL-10 KO and OPN/IL-10 DKO mice by terminal restriction fragment length polymorphism analysis. The effect of OPN on macrophage phagocytic function was evaluated by phagocytosis assay.

Results

OPN/IL-10 DKO mice had an accelerated onset of colitis compared to IL-10 KO mice. FISH analysis revealed enhanced OPN synthesis in the colonic epithelial cells of IL-10 KO mice. OPN/IL-10 DKO mice had a distinctly different enteric bacterial profile with a significantly lower abundance of Clostridium subcluster XIVa and a greater abundance of Clostridium cluster XVIII compared to IL-10 KO mice. Intracellular OPN deletion in macrophages impaired phagocytosis of fluorescence particle-conjugated Escherichia coli in vitro. Exogenous OPN enhanced phagocytosis by OPN-deleted macrophages when administered at doses of 1 to 100 ng/ml, but not 1000 ng/ml.

Conclusions

OPN deficiency accelerated the spontaneous development of colitis in mice with disrupted gut microbiota and macrophage phagocytic activity.     

The forespore line of gene expression in Bacillus subtilis

Endospore formation by Bacillus subtilis involves three differentiating cell types, the predivisional cell, the mother cell, and the forespore. Here we report the program of gene expression in the forespore, which is governed by the RNA polymerase sigma factors sigma(F) and sigma(G) and the DNA-binding proteins RsfA and SpoVT. The sigma(F) factor turns on about 48 genes, including the gene for RsfA, which represses a gene in the sigma(F) regulon, and the gene for sigma(G). The sigma(G) factor newly activates 81 genes, including the gene for SpoVT, which turns on (in nine cases) or stimulates (in 11 cases) the expression of 20 genes that had been turned on by sigma(G) and represses the expression of 27 others. The forespore line of gene expression consists of many genes that contribute to morphogenesis and to the resistance and germination properties of the spore but few that have metabolic functions. Comparative genomics reveals a core of genes in the sigma(F) and sigma(G) regulons that are widely conserved among endospore-forming species but are absent from closely related, but non-spore-forming Listeria spp. Two such partially conserved genes (ykoU and ykoV), which are members of the sigma(G) regulon, are shown to confer dry-heat resistance to dormant spores. The ykoV gene product, a homolog of the non-homologous end-joining protein Ku, is shown to associate with the nucleoid during germination. Extending earlier work on gene expression in the predivisional cell and the mother cell, we present an integrated overview of the entire program of sporulation gene expression.     

Bio-photosensor: Cyanobacterial photosystem I coupled with transistor via molecular wire

We report on the first successful output of electrons directly from photosystem I (PSI) of thermophilic cyanobacteria to the gate of a field-effect transistor (FET) by bypassing electron flow via a newly designed molecular wire, i.e., artificial vitamin K₁, and a gold nanoparticle; in short, this newly manufactured photosensor employs a bio-functional unit as the core of the device. Photo-electrons generated by the irradiation of molecular complexes composed of reconstituted PSI on the gate were found to control the FET. This PSI-bio-photosensor can be used to interpret gradation in images. This PSI-FET system is moreover sufficiently stable for use exceeding a period of 1 year.     

Partial requirement of endothelin receptor B in spiral ganglion neurons for postnatal development of hearing

Impairments of endothelin receptor B (Ednrb/EDNRB) cause the development of Waardenburg-Shah syndrome with congenital hearing loss, hypopigmentation, and megacolon disease in mice and humans. Hearing loss in Waardenburg-Shah syndrome has been thought to be caused by an Ednrb-mediated congenital defect of melanocytes in the stria vascularis (SV) of inner ears. Here we show that Ednrb expressed in spiral ganglion neurons (SGNs) in inner ears is required for postnatal development of hearing in mice. Ednrb protein was expressed in SGNs from WT mice on postnatal day 19 (P19), whereas it was undetectable in SGNs from WT mice on P3. Correspondingly, Ednrb homozygously deleted mice (Ednrb(-/-) mice) with congenital hearing loss showed degeneration of SGNs on P19 but not on P3. The congenital hearing loss involving neurodegeneration of SGNs as well as megacolon disease in Ednrb(-/-) mice were markedly improved by introducing an Ednrb transgene under control of the dopamine β-hydroxylase promoter (Ednrb(-/-);DBH-Ednrb mice) on P19. Neither defects of melanocytes nor hypopigmentation in the SV and skin in Ednrb(-/-) mice was rescued in the Ednrb(-/-);DBH-Ednrb mice. Thus, the results of this study indicate a novel role of Ednrb expressed in SGNs distinct from that in melanocytes in the SV contributing partially to postnatal hearing development.     

Phorbol ester-dependent phosphorylation regulates the association of p57/coronin-1 with the actin cytoskeleton

The p57/coronin-1 protein is a member of the coronin family of actin-binding proteins, which are characterized by the presence of WD (tryptophan/aspartic acid) repeats and a coiled-coil motif in the molecule. It is selectively expressed in immune cells and has been suggested to play crucial roles in leukocyte functions, including cell migration and phagocytosis. In this study we examined the effects of p57/coronin-1 phosphorylation on the association of the protein with actin. Treatment of HL60 human leukemic cells or p57/coronin-1-transfected HEK293 cells with phorbol 12-myristate 13-acetate (PMA) reduced the association of p57/coronin-1 with the actin cytoskeleton, as indicated by cell fractionation experiments and by fluorescence microscopic observation. Two-dimensional gel electrophoresis of HL60 cell lysate revealed that p57/coronin-1 was phosphorylated upon PMA stimulation of the cells, giving two major and two minor spots of phosphorylated forms, each with distinct isoelectric points. The p57/coronin-1 molecules associated with the cytoskeleton in PMA-treated HL60 cells were phosphorylated at lower levels than those recovered in the cytosolic fraction. In addition, p57/coronin-1 co-sedimented with F-actin polymerized in vitro had lower phosphorylation levels than the molecules remaining in the supernatant. By affinity chromatographic analysis using anti-p57/coronin-1 antibody-conjugated Sepharose, p57/coronin-1 derived from PMA-treated HL60 cells showed lower affinity for actin than that from untreated cells. Finally, recovery of p57/coronin-1 in the actin cytoskeleton-rich fraction from neutrophil-like differentiated HL60 cells decreased during phagocytosis, concomitant with enhanced phosphorylation of p57/coronin-1. These results strongly suggest that the phosphorylation of p57/coronin-1 down-regulates its association with actin and modulates the reorganization of actin-containing cytoskeleton.     

Ancient ubiquitous protein 1 binds to the conserved membrane-proximal sequence of the cytoplasmic tail of the integrin alpha subunits that plays a crucial role in the inside-out signaling of alpha IIbbeta 3

Modification of the cytoplasmic tails of the integrin alpha(IIb)beta(3) plays an important role in the signal transduction in platelets. We searched for proteins that bind to the alpha(IIb) cytoplasmic tail using the yeast two-hybrid assay with a cDNA library of the megakaryocyte-derived cell line and identified a protein, ancient ubiquitous protein 1 (Aup1), that is ubiquitously expressed in human cells. Observation of UT7/TPO cells expressing a red fluorescent protein-tagged Aup1 indicated its localization in the cytoplasm. Immunoprecipitation of UT7/TPO cells by an antibody for Aup1 revealed that approximately 40% of alpha(IIb) is complexed with Aup1. Binding study with an alpha(IIb) cytoplasmic tail peptide and glutathione S-transferase-Aup1 fusion protein revealed a low affinity (K(d) = 90 microm). Subsequent yeast two-hybrid assay indicated binding of Aup1 to cytoplasmic tails of other integrin alpha subunits. Binding study with the purified Aup1 and various glutathione S-transferase-alpha(IIb) cytoplasmic tail peptides revealed specific binding of Aup1 to the membrane-proximal sequence (KVGFFKR) that is conserved among the integrin alpha subunits and plays a crucial role in the alpha(IIb)beta(3) inside-out signaling. As Aup1 possesses domains related to signal transduction, these results suggest involvement of Aup1 in the integrin signaling.     

Evidence for lateral transfer of the Suilysin gene region of Streptococcus suis

Suilysin is a cholesterol-binding cytolysin encoded by sly in Streptococcus suis. DNA sequence determination of the sly locus in a strain lacking sly revealed the presence of another gene, designated orf102, in the place of sly. No transposable element or long-repeat sequence was found in the close vicinity. Except for six strains whose corresponding loci have been rearranged, all of the remaining 62 strains examined had either sly or orf102 at the same locus and their flanking regions were conserved. The genetic organizations having either sly or orf102 were found in the strains whose 16S rRNA sequences were identical. These results suggest that S. suis acquired sly or orf102 from a foreign source and that these genes subsequently spread among S. suis strains by homologous recombination.