Mitochondria are dynamic organelles that frequently divide and fuse together, resulting in the formation of intracellular tubular networks. In yeast and mammals, several factors including Drp1/Dnm1 and Mfn/Fzo1 are known to regulate mitochondrial morphology by controlling membrane fission or fusion. Here, we report the systematic screening of Caenorhabditis elegans mitochondrial proteins required to maintain the morphology of the organelle using an RNA interference feeding library. In C. elegans body wall muscle cells, mitochondria usually formed tubular structures and were severely fragmented by the mutation in fzo-1 gene, indicating that the body wall muscle cells are suitable for monitoring changes in mitochondrial morphology due to gene silencing. Of 719 genes predicted to code for most of mitochondrial proteins, knockdown of >80% of them caused abnormal mitochondrial morphology, including fragmentation and elongation. These findings indicate that most fundamental mitochondrial functions, including metabolism and oxidative phosphorylation, are necessary for maintenance of the tubular networks as well as membrane fission and fusion. This is the first evidence that known mitochondrial activities are prerequisite for regulating the morphology of the organelle. Furthermore, 88 uncharacterized or poorly characterized genes were found in the screening to be implicated in mitochondrial morphology.

Extrahepatic manifestations of hepatitis C virus (HCV) infection occur in 40%- IntroductionMore than 175 million people worldwide are infected with hepatitis C virus (HCV), a positive-strand RNA virus that infects both hepatocytes and peripheral blood mononuclear cells. 1 Chronic HCV infection may lead to hepatitis, liver cirrhosis, hepatocellular carcinomas 2,3 and lymphoproliferative diseases such as B-cell non-Hodgkin lymphoma and mixed-cryoglobulinemia. 1,4-6 B-cell non-Hodgkin lymphoma is a typical extrahepatic manifestation frequently associated with HCV infection 7 with geographic and ethnic variability. 8,9 Based on a meta-analysis, the prevalence of HCV infection in patients with B-cell non-Hodgkin lymphoma is approximately 15%. 8 The HCV envelope protein E2 binds human CD81, 10 a tetraspanin expressed on various cell types including lymphocytes, and activates B-cell proliferation 11 ; however, the precise mechanism of disease onset remains unclear. We previously developed a transgenic mouse model that conditionally expresses HCV cDNA (nucleotides 294-3435), including the viral genes that encode the core, E1, E2, and NS2 proteins, using the Cre/loxP system (in coreϳNS2 [CN2] mice). 12,13 The conditional transgene activation of the HCV cDNA (core, E1, E2, and NS2) protects mice from Fas-mediated lethal acute liver failure by inhibiting cytochrome c release from mitochondria. 13 In HCV-infected mice, persistent HCV protein expression is established by targeted disruption of irf-1, and high incidences of lymphoproliferative disorders are found in CN2 irf-1 ؊/؊ mice. 14 Infection and replication of HCV also occur in B cells, 15,16 although the direct effects, particularly in vivo, of HCV infection on B cells have not been clarified.To define the direct effect of HCV infection on B cells in vivo, we crossed transgenic mice with an integrated full-length HCV genome (Rz) under the conditional Cre/loxP expression system with mice expressing the Cre enzyme under transcriptional control of the B lineage-restricted gene CD19, 17 we addressed the effects of HCV transgene expression in this study. Methods Animal experimentsWild-type (WT), Rz, CD19Cre, RzCD19Cre mice (129/sv, BALB/c, and C57BL/6J mixed background), and MxCre/CN2-29 mice (C57BL/6J background) were maintained in conventional animal housing under specific pathogen-free conditions. All animal experiments were performed according to the guidelines of the Tokyo Metropolitan Institute of Medical Science or the Kumamoto University Subcommittee for Laboratory Animal Care. The protocol was approved by the Institutional Review Boards of both facilities. Measurements of HCV protein and RNAMice were anesthetized and bled, and tissues (spleen, lymph nodes, liver, and tumors) were homogenized in lysis buffer (1% sodium dodecyl sulfate; 0.5% (wt/vol) nonyl phenoxypolyethoxylethanol; 0.15M NaCl; 10 mM For personal use only. on May 9, 2018. by guest www.bloodjournal.org From tris(hydroxymethyl)aminomethane, pH 7.4) using a Dounce homogenizer. The concentration of HCV ...

Mitochondria utilize diverse cytoskeleton-based mechanisms to control their functions and morphology. Here, we report a role for kinesin-like protein KLP6, a newly identified member of the kinesin family, in mitochondrial morphology and dynamics. An RNA interference screen using Caenorhabditis elegans led us to identify a C. elegans KLP-6 involved in maintaining mitochondrial morphology. We cloned a cDNA coding for a rat homolog of C. elegans KLP-6, which is an uncharacterized kinesin in vertebrates. A rat KLP6 mutant protein lacking the motor domain induced changes in mitochondrial morphology and significantly decreased mitochondrial motility in HeLa cells, but did not affect the morphology of other organelles. In addition, the KLP6 mutant inhibited transport of mitochondria during anterograde movement in differentiated neuro 2a cells. To date, two kinesins, KIF1Bα and kinesin heavy chain (KHC; also known as KIF5) have been shown to be involved in the distribution of mitochondria in neurons. Expression of the kinesin heavy chain/KIF5 mutant prevented mitochondria from entering into neurites, whereas both the KLP6 and KIF1Bα mutants decreased mitochondrial transport in axonal neurites. Furthermore, both KLP6 and KIF1Bα bind to KBP, a KIF1-binding protein required for axonal outgrowth and mitochondrial distribution. Thus, KLP6 is a newly identified kinesin family member that regulates mitochondrial morphology and transport.

Mitigation of regulatory T cell-mediated immunosuppression is crucial for optimal in vivo anti-tumor immune responses. In this study, we examined the anti-tumor effect induced by oral ingestion of an immunomodulating diet comprised of Lentinula edodes mycelia (L.E.M.) extract. C57BL ⁄ 6 mice were inoculated subcutaneously in the footpad with B16 melanoma and fed L.E.M. extract. Ingestion of L.E.M. extract significantly inhibited tumor growth, and this in vivo anti-tumor effect was not observed in nude mice, suggesting a T cell-dependent mechanism. In addition, ingestion of L.E.M. extract led to significant restoration of H-2K b -restricted and melanoma-reactive T cells in the spleen and draining lymph nodes of melanoma-bearing mice. Flow cytometry analysis revealed that the percentage of Foxp3 + CD4 + T cells increased in spleen and draining lymph nodes in melanoma-bearing mice, but decreased significantly with ingestion of L.E.M. extract. Importantly, selective depletion of CD8 + T cells abolished the L.E.M.-induced anti-tumor effect, whereas that of CD4 + T cells or CD25 + cells showed no additive influence on the effect. Real-time PCR analysis revealed that ingestion of L.E.M. extract by melanoma-bearing mice decreased the level of Foxp3 mRNA within the tumor tissues, and lowered plasma transforming growth factor (TGF)-b. Furthermore, an in vitro assay revealed that an immunosuppressive activity of CD4 + T cells from melanoma-bearing mice was canceled by ingestion of L.E.M. extract. Our results indicate that oral ingestion of L.E.M. extract restores immune responses of class I-restricted and melanoma-reactive CD8 + T cells in melanoma-bearing mice, presumably by a mitigation of regulatory T cells-mediated immunosuppression. (Cancer Sci 2011; 102: 516-521) A nticancer immunotherapy has received considerable attention recently as a new treatment modality. The immune system is capable of recognizing tumor antigens, and cancerreactive cytotoxic T lymphocytes are the most effective cancer destroying cells.(1-3) Although anticancer immunotherapies, including vaccines and adoptive immunotherapy, have been applied clinically, to date their efficacy has been unsatisfactory.(4) One explanation for the insufficient outcome of anticancer immunotherapy trials is the emergence of immunosuppressive cells, including CD4 + CD25 + regulatory T cells (Tregs) and ⁄ or myeloid-derived suppressor cells, in tumor-bearing states.(5,6) The presence of Tregs correlates strongly with unfavorable prognosis. (7,8) Treatment with antibodies or drugs has been proposed as a means of relieving Treg-mediated immunosuppression.(9-14) Nevertheless, safe and widely applicable treatment modalities to mitigate Treg-mediated immunosuppression in cancer patients must be therapeutically beneficial.A dried powder extracted from Lentinula edodes mycelia (L.E.M.) with hot water before germination and after culturing in a medium composed of bagasse and rice bran, (15) has been reported to exhibit anti-tumor activity and immunomodulatory effects in vit...

Abstract. We previously reported that oral ingestion of Lentinula edodes mycelia (L.E.M.) extract can inhibit the growth of a subcutaneously established melanoma in a T cell-dependent manner via mitigation of regulatory T cell (Treg)-mediated immunosuppression. In this study, we tested the antitumor effect and mechanism of oral ingestion of L.E.M. extract following inoculation of murine colon carcinoma colon-26 (C26) cells into the subserosal space of the cecum (i.c.) of syngeneic mice. In this model, the primary site of the immune response was gut-associated lymphoid tissue (GALT), which is known to be an immunological toleranceinducing site for numerous dietary antigens. Oral ingestion of the L.E.M. extract suppressed the growth of i.c.-inoculated C26 cells in a T cell-dependent manner and restored the T cell response of the mesenteric lymph nodes and the spleen, not only to a tumor antigen-derived peptide, presented on H-2L d molecules, but also to C26 cells. I.c. inoculation of C26 cells increased the potential of CD4 + T cells of the mesenteric lymph nodes to produce transforming growth factor (TGF)-β, but ingestion of the L.E.M. extract decreased the ability of both CD4 + and CD8 + T cells in the mesenteric lymph nodes to produce this immunosuppressive cytokine. Although ingestion of L.E.M. showed only a marginal effect on Tregs in this model, this treatment significantly reduced the plasma levels of TGF-β and IL-6, both of which were increased in the i.c. C26-inoculated mice. In summary, our results indicate that oral ingestion of L.E.M. extract can restore antitumor T cell responses of mice even when the primary antitumor immune response is elicited in GALT, and provide important implications for anticancer immunotherapy of human colon cancer. IntroductionStudies on tumor-reactive cytotoxic T lymphocytes (CTLs) and tumor-related antigens have enabled the design of specific anticancer immunotherapies (1,2). Anticancer vaccines and adoptive immunotherapy have been applied clinically, although their efficacy has been unsatisfactory (3). Although several explanations of their unsatisfactory performance could be proposed, the major reason that these therapies fail is believed to be due to the emergence of immunosuppressive cells, including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) (4,5). In addition, the tumor-bearing state is accompanied by chronic inflammation, and it is thought that the inflammatory state of cancer patients inhibits the efficacy of anticancer immunotherapy (6,7).Lentinula edodes mycelia extract (L.E.M.) is a dried powder of a hot water extract of the mycelia of L. edodes before germination, which were cultured in a medium composed of bagasse and rice bran (8). This L.E.M. extract has been reported to exhibit antitumor activity and immunomodulatory effects both in vitro and in vivo (9,10). L.E.M. can mitigate inflammation in the liver of mice (11), suggesting that it also has an anti-inflammatory effect. In addition, we recently reported that oral ingestion of ...