寺本 英已

The hepatitis C virus (HCV) outbreak that occurred between 1940 and 1999 in a closed leprosy sanatorium located on a small island in Japan was analyzed. The analysis of 318 nucleotides in the NS5B region of HCV allowed us to establish the existence of at least three different HCV strains in this sanatorium.

Oku-Komyo-En is one of the national leprosy sanatoria, located on a small island in Setouchi city, Okayama prefecture of Japan since 1938. Since autopsies were carried out routinely on almost all patients who had died in the sanatorium up to 1980, approximately 1,000 formalin-fixed autopsy tissue samples were available for analysis. When these samples were reviewed, the pathological data indicated a sharp rise in the death rate caused by cirrhosis of the liver and hepatocellular carcinoma (HCC) since 1960 and 1970, respectively. Hepatitis C virus (HCV) infection is a common cause of HCC in Japan. The presence of HCV RNA was demonstrated in paraffin sections prepared from the autopsied liver tissue fixed in formalin for a prolonged period of time, by employing nested RT-PCR using type-specific primers. The data showed that HCV RNA was detectable in samples of the liver archived as early as 1940, representing the liver tissues kept in formalin for up to 67 years. HCV genotypes 1b and 2a were found by RT-PCR at 85.7% and 14.3%, respectively, in patients with leprosy. J. Med. Virol. 82:556-561,2010. (C) 2010 Wiley-Liss, Inc.

This is a case report of granulocytic sarcoma occurring as a nasal lesion prior to the onset of acute myelogenous leukaemia (AML). To understand this case in more detail, we used 40 000 human cDNA microarray to identify the gene expression patterns of nonleukaemic stage bone marrow (BM), AML stage BM and AML stage peripheral blood cells and subsequently define the molecular basis of this disease progression. Of significance, we have tracked the expression profile of BM samples during the course of nonleukaemic to leukaemic progression, and identified a number of genes that may account for the growth potential of leukaemia cells and indicate poor prognosis of this case.

How cyclooxygenase-2 (COX-2) and its proinflammatory metabolite prostaglandin E2 (PGE2) enhance colon cancer progression remains poorly understood. We show that PGE2 stimulates colon cancer cell growth through its heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor, EP2, by a signaling route that involves the activation of phosphoinositide 3-kinase and the protein kinase Akt by free G protein beta gamma subunits and the direct association of the G protein a. subunit with the regulator of G protein signaling (RGS) domain of axin. This leads to the inactivation and release of glycogen synthase kinase 3 beta from its complex with axin, thereby relieving the inhibitory phosphorytation of beta-catenin and activating its signaling pathway. These findings may provide a molecular framework for the future evaluation of chemopreventive strategies for colorectal cancer.

Activated forms of Ras family members are prevalent in many cancers where Ras mutants transduce signals essential for transformation, angiogenesis, invasion and metastasis. As a cancer progression model, we used NIH3T3 cells to explore the mechanism of Ras-induced tumorigenesis. Ras family mutants H-RasV12 and Rit79L strongly induced foci formation, while Rho family mutants RhoA-QL, Rac1-QL and Cdc42-QL were less effective. A comparison of downstream transcriptional targets of Ras and Rho family members using a 26 383 element cDNA microarray revealed that the osteopontin (OPN) gene exhibited the best correlation between magnitude of gene expression change and level of foci formation (r=0.96, P<0.001). In association with H-RasV12- and Rit79L-mediated transformation, foci secreted OPN protein and upregulated the OPN receptor CD44, suggesting the novel initiation of an aberrant OPN-CD44-Rac autocrine pathway. In support of this were the following observations. First, RGD-deficient OPN protein-binding activity was present in H-RasV12- transformed cells but not in control cells, and binding activity was inhibited by the CD44 blocking antibody. Second, foci formation, cell invasion and Rac activity were induced by H-RasV12 and inhibited by the CD44 blocking antibody. Third, foci formation by H-RasV12 was substantially reduced by a short interfering RNA ( siRNA) specifically targeting OPN expression for knockdown. Fourth, H-RasV12- mediated transformation was not blocked by the GRGDS peptide, suggesting that OPN effects were not mediated by the integrins. Lastly, OPN knockdown affected the downstream expression of 160 '2nd tier' genes, and at least a subset of these genes appears to be involved in transformation. Indeed, four genes were selected for knockdown, each resulting in a disruption of foci formation and/or invasion. These results underscore the role of aberrant autocrine signaling and transcriptional networking during tumorigenesis.

The alpha-subunit of G proteins of the G(12/13) family stimulate Rho by their direct binding to the RGS-like (RGL) domain of a family of Rho guanine nucleotide exchange factors (RGL-RhoGEFs) that includes PDZ-RhoGEF (PRG), p115RhoGEF, and LARG, thereby regulating cellular functions as diverse as shape and movement, gene expression, and normal and aberrant cell growth. The structural features determining the ability of Galpha(12/13) to bind RGL domains and the mechanism by which this association results in the activation of RGL-RhoGEFs are still poorly understood. Here, we explored the structural requirements for the functional interaction between Galpha(13) and RGL-RhoGEFs based on the structure of RGL domains and their similarity with the area by which RGS4 binds the switch region of Galpha(i) proteins. Using Galpha(i2), which does not bind RGL domains, as the backbone in which Galpha(13) sequences were swapped or mutated, we observed that the switch region of Galpha(13) is strictly necessary to bind PRG, and specific residues were identified that are critical for this association, likely by contributing to the binding surface. Surprisingly, the switch region of Galpha(13) was not sufficient to bind RGL domains, but instead most of its GTPase domain is required. Furthermore, membrane localization of Galpha(13) and chimeric Galpha(i2) proteins was also necessary for Rho activation. These findings revealed the structural features by which Galpha(13) interacts with RGL domains and suggest that molecular interactions occurring at the level of the plasma membrane are required for the functional activation of the RGL-containing family of RhoGEFs.

We report an autopsy case of Epstein-Barr virus (EBV)-infected malignant lymphoma in a young male who had hypersensitivity to mosquito bites. The autopsy revealed multiple confluent lymphoma lesions in the lungs, and on the right leg irregular-shaped skin ulcers were seen. The left pleural effusion also contained a large number of lymphoma cells. The lymphoma cells were determined as T/NK-cell type cells by immunohistochemistry. EBV DNA was detected most intensively in the lungs and EBV-encoded small RNAs-positive lymphoma cells were also observed in the lungs at a high frequency. EBV latent membrane protein-1 expression and a high Ki-67 labeling indices were noted in the lymphoma cells of the lung lesions. These findings indicate that the development of the malignant lymphoma was associated with the proliferation of EBV-infected lymphoma cells, and the cells that infiltrated the whole the body, especially the lungs, caused the patient's death.

PDZ-RhoGEF, LARG, and p115RhoGEF are members of a newly identified family of Rho-guanine nucleotide exchange factors (GEFs) exhibiting a unique structural feature consisting of the presence of an area of similarity to regulators of G protein signaling (RGS). This RGS-like (RGL) domain provides a functional motif by which Galpha(12) and Galpha(13) can bind and regulate the activity of these RhoGEFs, thus providing a direct link from these heterotrimeric G proteins to Rho. PDZ-RhoGEF and LARG can also be phosphorylated by tyrosine kinases, including FAK, and associate with Plexin B, a semaphorin receptor, which controls axon guidance during development, through their PDZ domain, thereby stimulating Rho. Interestingly, while characterizing a PDZ-RhoGEF antiserum, we found that a transfected PDZ-RhoGEF construct associated with the endogenous PDZ-RhoGEF. Indeed, we observed that PDZ-RhoGEF and LARG can form homo- and hetero-oligomers, whereas p115RhoGEF can only homo-oligomerize, and that this intermolecular interaction was mediated by their unique C-terminal regions. Deletion of the C-terminal tail of PDZ-RhoGEF had no significant effect on the GEF catalytic activity towards Rho in vitro, but resulted in a drastic increase in the ability to stimulate a serum response element reporter and the accumulation of the GTP-bound Rho in vivo. Furthermore, removal of the C-termini of each of the three RGL-containing GEFs unleashed their full transforming potential. Together, these findings suggest the existence of a novel mechanism controlling the activity of PDZ-RhoGEF, LARG, and p115RhoGEF, which involves homo- and hetero-oligomerization through their inhibitory C-terminal region.

The superfamily of small GTP-binding proteins has expanded dramatically in recent years. The Ras family has long been associated with signaling pathways contributing to normal and aberrant cell growth, while Rho-related protein function is to integrate extracellular signals with specific targets regulating cell morphology, cell aggregation, tissue polarity, cell motility and cytokinesis. Recent findings suggest that certain Rho proteins, including RhoA, Rac1 and Cdc42, can also play a role in signal transduction to the nucleus and cell growth control. However, the nature of the genes regulated by Ras and Rho GTPases, as well as their contribution to their numerous biological effects is still largely unknown. To approach these questions, we investigated the global gene expression pattern induced by activated forms of H-Ras, RhoA, Rac1 and Cdc42 using cDNA microarrays comprising 19 117 unique elements. Using this approach, we identified 1184 genes that were up- or downregulated by at least twofold. Hierarchical cluster analysis revealed the existence of patterns of gene regulation both unique and common to H-Ras V12, RhoA QL, Rac1 QL and Cdc42 QL activation. For example, H-Ras V12 upregulated osteopontin and Akt 1, and H-Ras and RhoA stimulated cyclin G1, cyclin-dependent kinase 8, cyclin A2 and HMGI-C, while Rac1 QL and Cdc42 QL upregulated extracellular matrix and cell adhesion proteins such as alpha-actinin 4, procollagen type I and V and neuropilin. Furthermore, H-Ras V12 downregulated by >eightfold 52 genes compared to only three genes by RhoA QL, Rac1 QL and Cdc42 QL. These results provide key information to begin unraveling the complexity of the molecular mechanisms underlying the transforming potential of Ras and Rho proteins, as well as the numerous morphological and cell cycle effects induced by these small GTPases.

A novel protein kinase, polyploidy-associated protein kinase (PAPK), was isolated using a subtraction cDNA library approach from a mouse erythroleukemia cell line that had been induced to polyploidy after serum withdrawal. PAPK shares homology with members of the Ste20/germinal center kinase family of protein kinases and is ubiquitously expressed as two spliced forms, PAPK-A and PAPK-B, that encode for proteins of 418 and 189 amino acids, respectively. The expression of endogenous PAPK-A protein increased after growth factor withdrawal in murine hematopoietic and fibroblast cells. When tested in an in vitro kinase assay, PAPK-A was activated in response to the stress-inducing agent hydrogen peroxide and slightly by fetal calf serum. Biochemical characterization of the PAPK-A-initiated pathway revealed that this novel kinase does not affect MAP kinase activity but can stimulate both c-Jun N-terminal kinase 1 (JNK1) and ERK6/p38gamma. The kinase activity of PAPK appears to be required for the activation of ERK6/p38gamma but not JNK1. When an inducible construct of PAPK-A was expressed in stably transfected NIH3T3 cells, the cells exhibited distinct cytoskeletal changes and became resistant to apoptotic cell death induced by serum withdrawal, effects of PAPK that require its kinase activity. These data suggest that PAPK is a new member of the Ste20/germinal center kinase family that modulates cytoskeletal organization and cell survival.

Objective: Positive-pressure mechanical ventilation can injure the lung, causing oedema and alveolar inflammation, which is termed 'ventilator-induced lung injury' (VILI). We postulated that cyclic stretch upregulates the release of cytokines, which may cause lung damage, and explored which cytokines were released after cyclic stretch in type II alveolar epithelial cells (A549). Methodology: To test this hypothesis, A549 cells were cultured on a silicoelastic membrane and interleukin (IL)-1beta, IL-8, granulocyte-macrophage colony stimulating factor, activin, transforming growth factor (TGF)-beta1, insulin-like growth factor-2 and tumour necrosis factor-alpha mRNA and protein were assessed after stimulation of the cells by cyclic stretch. Results: Cyclic stretch induced activation of protein kinase C and resulted in the release of IL-8 and TGF-beta1 from A549 cells. Conclusions: The release of IL-8 and TGF-beta1 from alveolar epithelial cells may be a contributing factor in alveolitis associated with VILI.

Small GTPases of the Rho family play a central role in cellular processes that involve the reorganization of the actin-based cytoskeleton. Rho-related GTPases, which include Rac and Cdc42, can also regulate gene expression often through the activation of kinase cascades leading to enhanced activity of stress activated protein kinases (SAPKs), including JNK and p38 MAP kinases. As SAPKs are implicated in programmed cell death, these observations suggest that Rho GTPases may promote the initiation of the apoptotic process. However, recent reports suggest that Rho GTPases can have either a protective or a proapoptotic role, depending on the particular cellular context. In an effort to explore the molecular mechanisms underlying these divergent biological activities, we asked whether there was indeed a correlation between the ability to induce SAPKs and apoptosis by Rho family members. We found that although constitutively activated (Q61L) mutants of Rac1, Cdc42, and RhoG, a Rac1 related GTPase of unknown function, potently induce JNK in COS 7 cells, none of these GTPases could induce apoptosis, nor enhance uv-induced cell death. In contrast, Rac1 and RhoG efficiently protected cells from uv-induced apoptosis. Furthermore, we provide evidence that Rac1 and RhoG can activate both apoptotic and anti-apoptotic pathways. Whereas the former is mediated through JNK, the latter is independent on the transcriptional activation of NF-KB, a pro-survival pathway, but results from the direct interaction of these GTPases with phosphatidylinositol 3-kinase (PI3K) and the stimulation of Akt. Together, these findings indicate that members of the Rho family of small GTP-binding proteins can provoke the concomitant stimulation of two counteracting signaling pathways, and that their balance ultimately determines the ability of these GTPases to promote cell survival or death.

A novel branch of the Ras family, Rit, was recently identified. Rit exhibits a distinct C-terminus and effector domain, and does not activate mitogen-activated protein kinase (MAPK) but can cooperate with Raf to transform fibroblasts. Here. we found that when overexpressed, activated mutants of Rit transform NIH 3T3 cells efficiently, and stimulate p38gamma but not MAPK, p38alpha, p38beta, p38delta, or ERK5. Furthermore, we provide evidence that p38gamma activation is required for the ability of Rit to stimulate gene expression and cellular transformation. These findings suggest that this unique GTPase stimulates proliferative pathways distinct from those regulated by other Ras family members. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Positive-pressure mechanical ventilation can injure the lung, causing edema and alveolar inflammation in a complication termed ventilator-induced lung injury (VILI), Cytokines such as interleukin-8 (IL-8) reportedly are important in this inflammatory response. On the other hand. hepatocyte growth factor (HGF) promotes regeneration of the lung, and delays pulmonary fibrosis. We postulated that cyclic stretch upregulates production and release of both of mediators. Human alveolar epithelial cells (A549) cultured on a silicoelastic membrane were tested for mRNA expression and release of IL-8 and HGF after cyclic stretch in vitro. Stretch induced mRNA expression and release of these mediators. The signaling pathway from cyclic stretch to release of IL-8 and HGF appeared to involve protein kinase C in the signal transduction pathway. (C) 2001 Elsevier Science B.V. All rights reserved.

The small GTP-binding Rho proteins control a variety of biological activities, including organization of the actin cytoskeleton, regulation of gene expression and cellular transformation. In contrast, Ras proteins do not induce actin stress fibers, but potently transform cells which exhibit a morphology clearly distinct from that caused by activated forms of Rho. To investigate whether nuclear signaling and oncogenic potential of Rho are a consequence of its profound effect on cytoskeletal organization, we replaced each amino acid in the Rho effector loop with those of Ras, or replaced conserved residues with others known to result in differential signaling capability when introduced into Ras and Rad. These Rho mutants did not gain the ability to induce the MAPK, JNK or p38 pathways but, surprisingly, all Rho effector loop mutants still continued to induce actin stress fiber formation. However, three of these Rho mutants, with substitutions of leucine-39, glutamic acid-39, or cysteine-42, lost the ability to stimulate gene transcription via the serum response factor (SRF) and failed to induce neoplastic transformation. Thus, these results indicate that cytoskeletal changes are not sufficient to induce the transformed phenotype, and that Rho-effector molecules regulating the actin cytostructure are distinct from those signaling to the nucleus and subverting normal growth control.

Stimulation of high affinity IgE Fc receptors (Fc epsilon RI) in basophils and mast cells activates the tyrosine kinases Lyn and Syk and causes the tyrosine phosphorylation of phospholipase C-gamma resulting in the Ca2(+)- and protein kinase C-dependent secretion of inflammatory mediators. Concomitantly, Fc epsilon RI stimulation initiates a number of signaling events resulting in the activation of mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK), which, in turn, regulate nuclear responses, including cytokine gene expression. To dissect the signaling pathway(s) linking Fc epsilon RI to MAPK and JNK, we reconstructed their respective biochemical routes by expression of a chimeric interleukin-2 receptor alpha subunit (Tac)-Fc epsilon RI gamma chain (Tac gamma) in COS-7 cells, Cross-linking of Tac gamma did not affect MAPK in COS-7 cells, but when coexpressed with the tyrosine kinase Syk, Tac gamma stimulation potently induced Syk and Shc tyrosine phosphorylation and MAPK activation. In contrast, Tac gamma did not signal JNK activation, even when coexpressed with Syk. Ectopic expression of a hematopoietic-specific guanine nucleotide exchange factor (GEF), Vav, reconstituted the Tac gamma-induced, Syk- and Rac1-dependent JNR activation; and tyrosine-phosphorylation of Vav by Syk stimulated its GEF activity for Rac1. Thus, these data strongly suggest that Vav plays a critical role linking Fc epsilon RI and Syk to the Rac1-JNK pathway. Furthermore, these findings define a novel signal transduction pathway involving a multimeric cell surface receptor acting on a cytosolic tyrosine kinase, which, in turn, phosphorylates a GEF, thereby regulating its activity toward a small GTP-binding protein and promoting the activation of a kinase cascade.

c-Jun amino-terminal kinases (JNKs) and mitogen-activated protein kinases (MAPKs) are closely related; however, they are independently regulated by a variety of environmental stimuli. Although molecules linking growth factor receptors to MAPKs have been recently identified, little is known about pathways controlling JNK activation. Here, we show that in COS-7 cells, activated Rac1 effectively stimulates MAPK but poorly induces JNK activity. In contrast, mutationally activated Rac1. and Cdc42 GTPases potently activate JNK without affecting MAPK, and oncogenic guanine nucleotide exchange factors for these Rho-like proteins selectively stimulate JNK activity. Furthermore, expression of inhibitory molecules for Rho-related GTPases and dominant negative mutants of Rac1 and Cdc42 block JNK activation by oncogenic exchange factors or after induction by inflammatory cytokines and growth factors. Taken together, these findings strongly support a critical role for Rac1 and Cdc42 in controlling the JNK signaling pathway.