Hepatitis C virus (HCV) is a causative agent of chronic hepatitis and hepatocellular carcinoma. The core protein of HCV packages the viral RNA genome to form a nucleocapsid. In addition to its function as a structural protein, core protein is involved in regulation of cellular transcription, virus-induced transformation, and pathogenesis. To gain insights into cellular functions of the core protein by identification of cellular proteins interacting with the core protein, we employed a proteomic approach. Hepatocytes soluble cytoplasmic proteins were applied to the core proteins immobilized on Ni-nitrilotriacetic resin and total bound cellular proteins were resolved by 2-DE. Analyses of interacting proteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry allowed identification of 14 cellular proteins binding to the core protein. These proteins include DEAD-box polypeptide 5, similar in function to a known protein identified previously by yeast two-hybrid screening and 13 newly identified cellular proteins. Interestingly, nine protein spots were identified as intermediate microfilament proteins, including cytokeratins (five spots for cytokeratin 8, two for cytokeratin 19, and one for cytokeratin 18) and vimentin. Cytokeratin 8 and vimentin, which were previously shown to be involved in the infection processes of other viruses, were further analyzed to confirm their in vivo interactions with the core protein by immunoblotting and immunofluorescence microscopy. We discuss the functional implications of the interactions of the core protein with newly identified cellular proteins in HCV infection and pathogenesis.

The prostaglandin-evoked cAMP production was studied in human neuroblastoma SK-N-BE(2)C cells during neuronal differentiation induced by all-trans retinoic acid. The incubation with 5 microM all-trans retinoic acid for 4-6 days promoted neurite outgrowth of cells. After differentiation, prostaglandin E(2) (PGE(2))-induced cAMP production was dramatically increased, whereas forskolin- and AlF-induced cAMP productions were not changed. The increase reached maximum after 4-days of incubation with all-trans retinoic acid. The differentiation caused an increase in the maximal response and a decrease in the half-maximal effective concentration of the PGE(2)-induced cAMP production. In addition, the binding of [(3)H]PGE(2) to membrane receptors was enhanced in differentiated cells. However, the order of potency of the various prostaglandins (PGE(1) = PGE(2) > PGD(2) = PGF(2alpha) = PGI(2)) in cAMP production did not change during the differentiation, suggesting that mainly E-prostanoid (EP) receptors were involved. Butaprost, an EP(2) receptor specific agonist, increased the cAMP level in a concentration dependent manner and had a similar potentiating effect on cAMP production as PGE(2) upon differentiation. Northern blot analysis using the human cDNA probes shows that the EP(2) mRNA level was about seven times higher in differentiated cells, while the dopamine beta-hydroxylase (DBH) mRNA completely disappeared. Our results, thus, suggest that elevated gene expression of the prostanoid EP(2) receptor results in an increase in the PGE(2)-evoked cAMP production in SK-N-BE(2)C cells during neuronal differentiation.

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Objective-The serum level of tumor necrosis factor-␣ (TNF-␣) is in the picomolar range under inflammatory conditions.We investigated whether these picomolar levels of TNF-␣ directly modulate the functional activities of circulating monocytes. Methods and Results-In THP-1 monocytes treated with TNF-␣ (1 to 100 pmol/L/30 minutes), cytosolic RhoA small GTPase rapidly translocated to the plasma membrane via functionally active ezrin/radixin/moesin (ERM) complex, a cytoskeletal linker, and subsequent actin polymerization through NF-B activation. The threonine phosphorylation of ERM was accomplished by the activation of TNF receptor type I (TNFRI) and signaling pathways involving PI3K and an atypical PKC; ie, PKC. The TNF-␣-treated monocytes (10 pmol/L) displayed more potent and prolonged generation of GTP-bound RhoA in response to secondary stimulation with RhoA-activating monocyte chemoattractant protein-1 (MCP-1). Clearly, human circulating monocytes preconditioned by 10 pmol/L TNF-␣ augmented MCP-1-mediated chemotaxis and firm adhesion on VCAM-1 and ICAM-1 in vitro and ex vivo. The elevation of serum TNF-␣ (Ͼ5 pmol/L within 16 hours), which was introduced by intraperitoneal injection of mouse-specific TNF-␣ to C57/BL6 mice, enhanced the number of CD80ϩ monocytes transmigrating to the JE/MCP-1-injected intraperitoneal space. T umor necrosis factor (TNF)-␣ is one of the representative cytokines modulating immune and inflammatory responses at the site of local inflammation. TNF-␣, mainly produced by macrophages, 1 regulates genes responsible for the recruitment of monocytes, such as VCAM-1 and E-selectin 2 and monocyte chemoattractant protein-1 (MCP-1), by endothelial cells. 3 The crucial role of TNF-␣ in monocyte recruitment was confirmed previously in vivo, whereby genetic disruption of a specific TNF receptor I (TNFRI or p55) resulted in less severe infiltration of inflammatory leukocytes to the wound area. 4 TNF-␣ additionally triggers exacerbated inflammatory responses in recruited monocytes/macrophages, such as production of inflammatory cytokines, oxygen radicals, 5 and matrix metalloproteinase-9. 6 We previously showed that activities of monocytes determining the efficiency of recruitment are affected by a number of factors in blood (ie, lipoproteins, C-reactive protein, and pharmacological agents, such as a HMG-CoA reductase inhibitor and a PPAR␥ agonist [see review 7 ]). The serum level of TNF-␣, which is nearly undetectable at steady-state, is profoundly elevated (to the picomolar range) under inflammatory conditions. 8 Interestingly, TNF-␣ shows high-affinity binding to specific receptors, such as TNFRI (p55 or CD120a) and TNFRII (p75 or CD120b) 9 in myeloid cell lines, including monocytes, with dissociation constants (K d ) of 6.5 to 7.1ϫ10 Ϫ11 mol/L. 10,11 Therefore, we hypothesized that the functional activities of circulating monocytes are affected by picomolar levels of TNF-␣ in the blood circulation. Conclusions-PicomolarIn the present study, we elucidate the mechanism by which monocyte recruitme...