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Bladder cancer is an often widely disseminated and deadly cancer. To block the malignant outgrowth of bladder cancer, we must elucidate the molecular-level characteristics of not only bladder cancer cells but also their surrounding milieu. As part of this effort, we have long been studying extracellular S100A8/A9, which is elevated by the inflammation associated with certain cancers. Extracellularly enriched S100A8/A9 can hasten a shift to metastatic transition in multiple types of cancer cells. Intriguingly, high-level S100A8/A9 has been detected in the urine of bladder-cancer patients, and the level increases with the stage of malignancy. Nonetheless, S100A8/A9 has been investigated mainly as a potential biomarker of bladder cancers, and there have been no investigations of its role in bladder-cancer growth and metastasis. We herein report that extracellular S100A8/A9 induces upregulation of growth, migration and invasion in bladder cancer cells through its binding with cell-surface Toll-like receptor 4 (TLR4). Our molecular analysis revealed the TLR4 downstream signal that accelerates such cancer cell events. Tumor progression locus 2 (TPL2) was a key factor facilitating the aggressiveness of cancer cells. Upon binding of S100A8/A9 with TLR4, TPL2 activation was enhanced by an action with a TLR4 adaptor molecule, TIR domain-containing adaptor protein (TIRAP), which in turn led to activation of the mitogen-activated protein kinase (MAPK) cascade of TPL2. Finally, we showed that sustained inhibition of TLR4 in cancer cells effectively dampened cancer survival in vivo. Collectively, our results indicate that the S100A8/A9-TLR4-TPL2 axis influences the growth, survival, and invasive motility of bladder cancer cells.

DOI： 10.1016/j.bbrc.2022.09.116

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Secondary lymphoid tissues, such as the spleen and lymph nodes (LNs), contribute to breast cancer development and metastasis in both anti- and pro-tumoral directions. Although secondary lymphoid tissues have been extensively studied, very little is known about the immune conversion in mesenteric LNs (mLNs) during breast cancer development. Here, we demonstrate inflammatory immune conversion of mLNs in a metastatic 4T1 breast cancer model. Splenic T cells were significantly decreased and continuously suppressed IFN-γ production during tumor development, while myeloid-derived suppressor cells (MDSCs) were dramatically enriched. However, T cell numbers in the mLN did not decrease, and the MDSCs only moderately increased. T cells in the mLN exhibited conversion from a pro-inflammatory state with high IFN-γ expression to an anti-inflammatory state with high expression of IL-4 and IL-10 in early- to late-stages of breast cancer development. Interestingly, increased migration of CD103+CD11b+ dendritic cells (DCs) into the mLN, along with increased (1→3)-β-D-glucan levels in serum, was observed even in late-stage breast cancer. This suggests that CD103+CD11b+ DCs could prime cancer-reactive T cells. Together, the data indicate that the mLN is an important lymphoid tissue contributing to breast cancer development.

DOI： 10.3390/ijms231911035

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

The dissection of the complex multistep process of metastasis exposes vulnerabilities that could be exploited to prevent metastasis. To search for possible factors that favor metastatic outgrowth, we have been focusing on secretory S100A8/A9. A heterodimer complex of the S100A8 and S100A9 proteins, S100A8/A9 functions as a strong chemoattractant, growth factor, and immune suppressor, both promoting the cancer milieu at the cancer-onset site and cultivating remote, premetastatic cancer sites. We previously reported that melanoma cells show lung-tropic metastasis owing to the abundant expression of S100A8/A9 in the lung. In the present study, we addressed the question of why melanoma cells are not metastasized into the brain at significant levels in mice despite the marked induction of S100A8/A9 in the brain. We discovered the presence of plasma histidine-rich glycoprotein (HRG), a brain-metastasis suppression factor against S100A8/A9. Using S100A8/A9 as an affinity ligand, we searched for and purified the binding plasma proteins of S100A8/A9 and identified HRG as the major protein on mass spectrometric analysis. HRG prevents the binding of S100A8/A9 to the B16-BL6 melanoma cell surface via the formation of the S100A8/A9 complex. HRG also inhibited the S100A8/A9-induced migration and invasion of A375 melanoma cells. When we knocked down HRG in mice bearing skin melanoma, metastasis to both the brain and lungs was significantly enhanced. The clinical examination of plasma S100A8/A9 and HRG levels showed that lung cancer patients with brain metastasis had higher S100A8/A9 and lower HRG levels than nonmetastatic patients. These results suggest that the plasma protein HRG strongly protects the brain and lungs from the threat of melanoma metastasis.

DOI： 10.3390/ijms231810300

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Follicular dendritic cells (FDCs) play a crucial role in generating high-affinity antibody-producing B cells during the germinal center (GC) reaction. Herein, we analysed the altered gene expression profile of a mouse FDC line, FL-Y, following lymphotoxin β receptor stimulation, and observed increased Slam-family member 8 (Slamf8) mRNA expression. Forced Slamf8 expression and SLAMF8-Fc addition enhanced the ability of FL-Y cells to induce FDC-induced monocytic cell (FDMC) differentiation. FDMCs accelerated GC-phenotype proliferation in cultured B cells, suggesting that they are capable of promoting GC responses. Furthermore, a pulldown assay showed that SLAMF8-Fc could bind to SLAMF8-His. Overall, the homophilic interaction of SLAMF8 promotes FDMC differentiation and SLAMF8 might act as a novel regulator of GC responses by regulating FDMC differentiation.

DOI： 10.1002/1873-3468.14468

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The role of Dickkopf-3 (Dkk3)/REIC (The Reduced Expression in Immortalized Cells), a Wnt-signaling inhibitor, in male reproductive physiology remains unknown thus far. To explore the functional details of Dkk3/REIC in the male reproductive process, we studied the Dkk3/REIC knock-out (KO) mouse model. By examining testicular sections and investigating the sperm characteristics (count, vitality and motility) and ultrastructure, we compared the reproductive features between Dkk3/REIC-KO and wild-type (WT) male mice. To further explore the underlying molecular mechanism, we performed RNA sequencing (RNA-seq) analysis of testicular tissues. Our results showed that spermiation failure existed in seminiferous tubules of Dkk3/REIC-KO mice, and sperm from Dkk3/REIC-KO mice exhibited inferior motility (44.09 ± 8.12% vs. 23.26 ± 10.02%, p < 0.01). The Ultrastructure examination revealed defects in the sperm fibrous sheath of KO mice. Although the average count of Dkk3/REIC-KO epididymal sperm was less than that of the wild-types (9.30 ± 0.69 vs. 8.27 ± 0.87, ×106), neither the gap (p > 0.05) nor the difference in the sperm vitality rate (72.83 ± 1.55% vs. 72.50 ± 0.71%, p > 0.05) were statistically significant. The RNA-seq and GO (Gene Oncology) enrichment results indicated that the differential genes were significantly enriched in the GO terms of cytoskeleton function, cAMP signaling and calcium ion binding. Collectively, our research demonstrates that Dkk3/REIC is involved in the process of spermiation, fibrous sheath integrity maintenance and sperm motility of mice.

DOI： 10.3390/genes13020285

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Serum autoantibody to cancer/testis antigens (CTAs) is a critical biomarker that reflects the antitumor immune response. Quantitative and multiplexed anti-CTA detection arrays can assess the immune status in tumors and monitor therapy-induced antitumor immune reactions. Most full-length recombinant CTA proteins tend to aggregate. Cysteine residue-specific S-cationization techniques facilitate the preparation of water-soluble and full-length CTAs. Combined with Luminex technology, we designed a multiple S-cationized antigen-immobilized bead array (MUSCAT) assay system to evaluate multiple serum antibodies to CTAs. Reducible S-alkyl-disulfide-cationized antigens in cytosolic conditions were employed to develop rabbit polyclonal antibodies as positive controls. These control antibodies sensitively detected immobilized antigens on beads and endogenous antigens in human lung cancer-derived cell lines. Rabbit polyclonal antibodies successfully confirmed the dynamic ranges and quantitative MUSCAT assay results. An immune monitoring study was conducted using the serum samples on an adenovirus-mediated REIC/Dkk-3 gene therapy clinical trial that showed a successful clinical response in metastatic castration-resistant prostate cancer. Autoantibody responses were closely related to clinical outcomes. Notably, upregulation of anti-CTA responses was monitored before tumor regression. Thus, quantitative monitoring of anti-CTA antibody biomarkers can be used to evaluate the cancer-immunity cycle. A quality-certified serum autoantibody monitoring system is a powerful tool for developing and evaluating cancer immunotherapy.

DOI： 10.3389/fonc.2022.869393

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To develop combination immunotherapies for gastric cancers, immunologically well-characterized preclinical models are crucial. Here, we leveraged two transplantable murine gastric cancer cell lines, YTN2 and YTN16, derived from the same parental line but differing in their susceptibility to immune rejection. We established their differential sensitivity to immune checkpoint inhibitors (ICI) and identified neoantigens. Although anti-CTLA-4 mAbs eradicated YTN16 tumors in 4 of 5 mice, anti-PD-1 and anti-PD-L1 mAbs failed to eradicate YTN16 tumors. Using whole-exome and RNA sequencing, we identified two and three neoantigens in YTN2 and YTN16, respectively. MHC class I ligandome analysis detected the expression of only one of these neoantigens, mutated Cdt1, but the exact length of MHC binding peptide was determined. Dendritic cell vaccine loaded with neoepitope peptides and adoptive transfer of neoantigen-specific CD8+ T cells successfully inhibited the YTN16 tumor growth. Targeting mutated Cdt1 had better efficacy for controlling the tumor. Therefore, mutated Cdt1 was the dominant neoantigen in these tumor cells. More mCdt1 peptides were bound to MHC class I and presented on YTN2 surface than YTN16. This might be one of the reasons why YTN2 was rejected while YTN16 grew in immune-competent mice.

DOI： 10.3390/cancers14010106

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Transient expression of human intracellular proteins in human embryonic kidney (HEK) 293 cells is a reliable system for obtaining soluble proteins with biologically active conformations. Contrary to conventional concepts, we found that recombinantly expressed intracellular cancer-testis antigens (CTAs) showed frequent aggregation in HEK293 cells. Although experimental subcellular localization of recombinant CTAs displayed proper cytosolic or nuclear localization, some proteins showed aggregated particles in the cell. This aggregative property was not observed in recombinant housekeeping proteins. No significant correlation was found between the aggregative and biophysical properties, such as hydrophobicity, contents of intrinsically disordered regions and expression levels, of CTAs. These results can be explained in terms of structural instability of CTAs, which are specifically expressed in the testis and aberrantly expressed in cancer cells and function as a hub in the protein-protein network using intrinsically disordered regions. Hence, we speculate that recombinantly expressed CTAs failed to form this protein complex. Thus, unfolded CTAs formed aggregated particles in the cell.

DOI： 10.1093/jb/mvab081

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DOI： 10.34565/seibutsukogaku.99.4_162

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DOI： 10.34565/seibutsukogaku.99.4_180

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Optimized conditions are needed to refold recombinant proteins from bacterial inclusion bodies into their biologically active conformations. In this study, we found two crucial requirements for efficient refolding of cationic tetrameric chicken avidin. The first step is to eliminate nucleic acid contaminants from the bacterial inclusion body. The electrostatic interactions between the remaining nucleic acids and proteins strongly enhanced protein aggregation during the refolding process. The cysteine specific reversible S-cationization procedure was successfully employed for large-scale preparation of nucleic acid free denatured protein without purification tag system. The second step is the intramolecular disulfide formation prior to refolding in dialysis removing denaturant. Disulfide intact monomeric avidin showed efficient formation of biologically active tetrameric conformation during the refolding process. Using this optimized refolding procedure, highly cationic avidin derivative designed as an intracellular delivery carrier of biotinylated protein was successfully prepared.

DOI： 10.1002/btpr.3031

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/btpr.3031

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INTRODUCTION: Programmed cell death-1 (PD-1) inhibitors effectively treat NSCLC and prolong survival. Robust biomarkers for predicting clinical benefits of good response and long survival with anti-PD-1 therapy have yet to be identified; therefore, predictive biomarkers are needed to select patients with benefits. METHODS: We conducted a prospective study to explore whether serum antibody against NY-ESO-1 and/or XAGE1 cancer-testis antigens predicted primarily good clinical response and secondarily long survival with anti-PD-1 therapy for NSCLC. The serum antibody was detected by enzyme-linked immunosorbent assay, and tumor immune microenvironment and mutation burden were analyzed by immunohistochemistry and next-generation sequencing. RESULTS: In the discovery cohort (n = 13), six antibody-positive NSCLC cases responded to anti-PD-1 therapy (two complete and four partial responses), whereas seven antibody-negative NSCLC cases did not. Antibody positivity was associated with good response and survival, regardless of tumor programmed death ligand 1 (PD-L1) expression, mutation burden, and CD8+ T-cell infiltration. In the validation cohort (n = 75), 17 antibody-positive NSCLC cases responded well to anti-PD-1 therapy as compared with 58 negative NSCLC cases (objective response rate 65% versus 19%, p = 0.0006) and showed significantly prolonged progression-free survival and overall survival. Antibody titers highly correlated with tumor reduction rates. In the multivariate analysis, response biomarkers were tumor programmed death ligand 1 expression and antibody positivity, and only antibody positivity was a significantly better predictive biomarker of progression-free survival (hazard ratio = 0.4, p = 0.01) and overall survival (hazard ratio = 0.2, p = 0.004). CONCLUSIONS: Our results suggest that NY-ESO-1 and/or XAGE1 serum antibodies are useful biomarkers for predicting clinical benefits in anti-PD-1 therapy for NSCLC and probably for other cancers.

DOI： 10.1016/j.jtho.2019.08.008

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S100A11, a member of the S100 family of proteins, is actively secreted from pancreatic ductal adenocarcinoma (PDAC) cells. However, the role of the extracellular S100A11 in PDAC progression remains unclear. In the present study, we investigated the extracellular role of S100A11 in crosstalking between PDAC cells and surrounding fibroblasts in PDAC progression. An abundant S100A11 secreted from pancreatic cancer cells stimulated neighboring fibroblasts through receptor for advanced glycation end products (RAGE) upon S100A11 binding and was followed by not only an enhanced cancer cell motility in vitro but also an increased number of the PDAC-derived circulating tumor cells (CTCs) in vivo. Mechanistic investigation of RAGE downstream in fibroblasts revealed a novel contribution of a mitogen-activated protein kinase kinase kinase (MAPKKK), tumor progression locus 2 (TPL2), which is required for positive regulation of PDAC cell motility through induction of cyclooxygenase 2 (COX2) and its catalyzed production of prostaglandin E2 (PGE2), a strong chemoattractive fatty acid. The extracellularly released PGE2 from fibroblasts was required for the rise in cellular migration as well as infiltration of their adjacent PDAC cells in a coculture setting. Taken together, our data reveal a novel role of the secretory S100A11 in PDAC disseminative progression through activation of surrounding fibroblasts triggered by the S100A11-RAGE-TPL2-COX2 pathway. The findings of this study will contribute to the establishment of a novel therapeutic antidote to PDACs that are difficult to treat by regulating cancer-associated fibroblasts (CAFs) through targeting the identified pathway.

DOI： 10.3727/096504019X15555408784978

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The metastatic dissemination of cancer cells to remote areas of the body is the most problematic aspect in cancer patients. Among cancers, melanomas are notoriously difficult to treat due to their significantly high metastatic potential even during early stages. Hence, the establishment of advanced therapeutic approaches to regulate metastasis is required to overcome the melanoma disease. An accumulating mass of evidence has indicated a critical role of extracellular S100A8/A9 in melanoma distant metastasis. Lung S100A8/A9 is induced by melanoma cells from distant organs and it attracts these cells to its enriched lung environment since melanoma cells possess several receptors that sense the S100A8/A9 ligand. We hence aimed to develop a neutralizing antibody against S100A8/A9 that would efficiently block melanoma lung metastasis. Our protocol provided us with one prominent antibody, Ab45 that efficiently suppressed not only S100A8/A9-mediated melanoma mobility but also lung tropic melanoma metastasis in a mouse model. This prompted us to make chimeric Ab45, a chimera antibody consisting of mouse Ab45-Fab and human IgG2-Fc. Chimeric Ab45 also showed significant inhibition of the lung metastasis of melanoma. From these results, we have high hopes that the newly produced antibody will become a potential biological tool to block melanoma metastasis in future clinical settings.

DOI： 10.1002/ijc.31982

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Metastatic breast cancer is the leading cause of cancer-associated death in women. The progression of this fatal disease is associated with inflammatory responses that promote cancer cell growth and dissemination, eventually leading to a reduction of overall survival. However, the mechanism(s) of the inflammation-boosted cancer progression remains unclear. In this study, we found for the first time that an extracellular cytokine, S100A8/A9, accelerates breast cancer growth and metastasis upon binding to a cell surface receptor, melanoma cell adhesion molecule (MCAM). Our molecular analyses revealed an important role of ETS translocation variant 4 (ETV4), which is significantly activated in the region downstream of MCAM upon S100A8/A9 stimulation, in breast cancer progression in vitro as well as in vivo. The MCAM-mediated activation of ETV4 induced a mobile phenotype called epithelial-mesenchymal transition (EMT) in cells, since we found that ETV4 transcriptionally upregulates ZEB1, a strong EMT inducer, at a very high level. In contrast, downregulation of either MCAM or ETV4 repressed EMT, resulting in greatly weakened tumor growth and lung metastasis. Overall, our results revealed that ETV4 is a novel transcription factor regulated by the S100A8/A9-MCAM axis, which leads to EMT through ZEB1 and thereby to metastasis in breast cancer cells. Thus, therapeutic strategies based on our findings might improve patient outcomes.

DOI： 10.1016/j.neo.2019.04.006

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Since metastasis accounts for the majority of cancer-associated deaths, studies on the mechanisms of metastasis are needed to establish innovative strategies for cancer treatment. We previously reported that melanoma cell adhesion molecule (MCAM) functions as a critical receptor for S100A8/A9, and binding of S100A8/A9 to MCAM results in the migration of melanoma cells to lung tissue. However, the critical role of MCAM in the original melanoma skin lesion is still not clear. In this study, we aimed to determine the importance of the S100A8/A9-MCAM axis in melanoma dissemination in a skin lesion as a critical early step for metastasis. Mechanistic studies revealed the downstream signaling of MCAM that signaled the induction of metastasis. S100A8/A9-MCAM binding activates mitogen-activated protein kinase kinase kinase 8 (MAP3K8), also termed TPL2, leading to strong activation of the transcription factor ETV4 and subsequent induction of matrix metalloproteinase-25 (MMP25), and finally to induction of melanoma lung tropic metastasis. Collectively, our results demonstrate a crucial role of the S100A8/A9-MCAM signaling axis in metastatic onset of melanoma cells and indicate that strategies targeting the identified pathway may be useful for the establishment of innovative anti-cancer therapies.

DOI： 10.1016/j.canlet.2019.03.023

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The fertile stroma in pancreatic ductal adenocarcinomas (PDACs) has been suspected to greatly contribute to PDAC progression. Since the main cell constituents of the stroma are fibroblasts, there is crosstalking(s) between PDAC cells and surrounding fibroblasts in the stroma, which induces a fibroblast proliferation burst. We have reported that several malignant cancer cells including PDAC cells secrete a pronounced level of S100A11, which in turn stimulates proliferation of cancer cells via the receptor for advanced glycation end products (RAGE) in an autocrine manner. Owing to the RAGE+ expression in fibroblasts, the extracellular abundant S100A11 will affect adjacent fibroblasts. In this study, we investigated the significance of the paracrine axis of S100A11-RAGE in fibroblasts for their proliferation activity. In in vitro settings, extracellular S100A11 induced upregulation of fibroblast proliferation. Our mechanistic studies revealed that the induction is through RAGE-MyD88-mTOR-p70 S6 kinase upon S100A11 stimulation. The paracrine effect on fibroblasts is linked mainly to triggering growth but not cellular motility. Thus, the identified pathway might become a potential therapeutic target to suppress PDAC progression through preventing PDAC-associated fibroblast proliferation.

DOI： 10.3727/096504018X15433161908259

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Within the "seed and soil" theory of organ tropic cancer metastasis is a growing compilation of evidence that S100A8/A9 functions as a soil signal that attracts cancer cells to certain organs, which prove beneficial to their growth. S100A8/A9-sensing receptors including Toll-like receptor 4 (TLR4), advanced glycation end products (RAGE), and also important receptors we recently succeeded in identifying (EMMPRIN, NPTNβ, MCAM, and ALCAM) have the potential to become promising therapeutic targets. In our study, we prepared extracellular regions of these novel molecules and fused them to human IgG2-Fc to extend half-life expectancy, and we evaluated the anti-metastatic effects of the purified decoy proteins on metastatic cancer cells. The purified proteins markedly suppressed S100A8/A9-mediated lung tropic cancer metastasis. We hence expect that our novel biologics may become a prominent medicine to prevent cancer metastasis in clinical settings through cutting the linkage between "seed and soil".

DOI： 10.1002/ijc.31945

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Compiling evidence indicates an unusual role of extracellular S100A8/A9 in cancer metastasis. S100A8/A9 secreted from either cancer cells or normal cells including epithelial and inflammatory cells stimulates cancer cells through S100A8/A9 sensor receptors in an autocrine or paracrine manner, leading to cancer cell metastatic progression. We previously reported a novel S100A8/A9 receptor, neuroplastin-β (NPTNβ), which plays a critical role in atopic dermatitis when it is highly activated in keratinocytes by an excess amount of extracellular S100A8/A9 in the inflammatory skin lesion. Interestingly, our expression profiling of NPTNβ showed significantly high expression levels in lung cancer cell lines in a consistent manner. We hence aimed to determine the significance of NPTNβ as an S100A8/A9 receptor in lung cancer. Our results showed that NPTNβ has strong ability to induce cancer-related cellular events, including anchorage-independent growth, motility and invasiveness, in lung cancer cells in response to extracellular S100A8/A9, eventually leading to the expression of a cancer disseminative phenotype in lung tissue in vivo. Mechanistic investigation revealed that binding of S100A8/A9 to NPTNβ mediates activation of NFIA and NFIB and following SPDEF transcription factors through orchestrated upstream signals from TRAF2 and RAS, which is linked to anchorage-independent growth, motility and invasiveness. Overall, our results indicate the importance of the S100A8/A9-NPTNβ axis in lung cancer disseminative progression and reveal a pivotal role of its newly identified downstream signaling, TRAF2/RAS-NFIA/NFIB-SPDEF, in linking to the aggressive development of lung cancers.

DOI： 10.1002/mc.22987

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We previously identified novel S100A8/A9 receptors, extracellular matrix metalloproteinase inducer (EMMPRIN), melanoma cell adhesion molecule (MCAM), activated leukocyte cell adhesion molecule (ALCAM), and neuroplastin (NPTN) β, that are critically involved in S100A8/A9-mediated cancer metastasis and inflammation when expressed at high levels. However, little is known about the presence of any cancer-specific mechanism(s) that modifies these receptors, further inducing upregulation at protein levels without any transcriptional regulation. Expression levels of glycosyltransferase-encoding genes were examined by a PCR-based profiling array followed by confirmation with quantitative real-time PCR. Cell migration and invasion were assessed using a Boyden chamber. Western blotting was used to examine the protein level, and the RNA level was examined by Northern blotting. Immunohistochemistry was used to examine the expression pattern of β-1,3-galactosyl-O-glycosyl-glycoprotein β-1,6-N-acetylglucosaminyltransferase 3 (GCNT3) and MCAM in melanoma tissue. We found that GCNT3 is overexpressed in highly metastatic melanomas. Silencing and functional inhibition of GCNT3 greatly suppressed migration and invasion of melanoma cells, resulting in the loss of S100A8/A9 responsiveness. Among the novel S100A8/A9 receptors, GCNT3 favorably glycosylates the MCAM receptor, extending its half-life and leading to further elevation of S100A8/A9-mediated cellular motility in melanoma cells. GCNT3 expression is positively correlated to MCAM expression in patients with high-grade melanomas. Collectively, our results showed that GCNT3 is an upstream regulator of MCAM protein and indicate the possibility of a potential molecular target in melanoma therapeutics through abrogation of the S100A8/A9-MCAM axis.

DOI： 10.3727/096504017X15031557924123

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC  

Current ageing theories are far from satisfactory because of the many determinants involved in ageing. The well-known rate-of-living theory assumes that the product (lifetime energy expenditure, LEE) of maximum lifespan (MLS) and mass-specific basal metabolic rate (msBMR) is approximately constant. Although this theory provides a significant inverse correlation between msBMR and MLS as a whole for mammals, it remains problematic for two reasons. First, several interspecies studies within respective orders (typically within rodents) have shown no inverse relationships between msBMR and MLS. Second, LEE values widely vary in mammals and birds. Here, to solve these two problems, we introduced a new quantity designated as mitochondrial (mt) lifetime energy output, mtLEO = MLS x mtMR, in place of LEE, by using the mt metabolic rate (mtMR) per mitochondrion. Thereby, we found that mtLEO values were distributed more narrowly than LEE ones, and strongly correlated with the four amino-acid variables (AAVs) of Ser, Thr and Cys contents and hydrophobicity of mtDNA-encoded membrane proteins (these variables were related to the stability of these proteins). Consequently, only these two mt items, mtMR and the AAVs, solved the above-mentioned problems in the rate-of-living theory, and thus extensively improved the correlation with MLS compared with that given by LEE.

DOI： 10.1098/rsob.170083

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Many extracellular globular proteins have evolved to possess disulphide bonds in their native conformations, which aids in thermodynamic stabilisation. However, disulphide bond breakage by heating leads to irreversible protein denaturation through disulphide-thiol exchange reactions. In this study, we demonstrate that methanethiosulphonate (MTS) specifically suppresses the heat-induced disulphide-thiol exchange reaction, thus improving the heat-resistance of proteins. In the presence of MTS, small globular proteins that contain disulphides can spontaneously refold from heat-denatured states, maintaining wild-type disulphide pairing. Because the disulphide-thiol exchange reaction is triggered by the generation of catalytic amounts of perthiol or thiol, rapid and specific perthiol/thiol protection by MTS reagents prevents irreversible denaturation. Combining MTS reagents with another additive that suppresses chemical modifications, glycinamide, further enhanced protein stabilisation. In the presence of these additives, reliable remnant activities were observed even after autoclaving. However, immunoglobulin G and biotin-binding protein, which are both composed of tetrameric quaternary structures, failed to refold from heat-denatured states, presumably due to chaperon requirements. Elucidation of the chemical modifications involved in irreversible thermoinactivation is useful for the development of preservation buffers with optimum constitutions for specific proteins. In addition, the impact of disulphide bond breakage on the thermoinactivation of proteins can be evaluated using MTS reagents.

DOI： 10.1038/s41598-017-12748-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

We developed and validated a novel hTERT/CMV promoter element-driven gene expression cassette that can robustly enhance cancer-specific gene expression. The following gene expressional elements were located in tandem within the plasmid construct: [hTERT core promoter, cytomegalovirus (CMV) minimized promoter, RU5' sequence, an inserted gene, BGH polyA, hTERT enhancer]; this is hereafter referred to as the hT/Cm-R-hT construct. Using various human cancer cell lines and normal cells, the cancer-specific transcription of the green fluorescent protein (GFP) gene was examined by western blotting and fluorescence microscopy. Cancer-specific gene expression was robustly achieved in the hT/Cm-R-hT plasmid in comparison to the other control hT/Cm-driven construct. Notably, the expression level of GFP observed in the hT/Cm-RhT-driven construct was superior to that of the control plasmid with the conventional CMV promoter in HEK293 cells, which are known to possess higher hTERT activity than normal cells. We next examined the availability of hT/Cm-R-hT in detecting the target GFP expressing cancer cells from human peripheral blood mononuclear cells (PBMCs). The hT/Cm-R-hT plasmid successfully induced cancer-specific gene expression; the robust expression of GFP was observed in target HeLa cancer cells, whereas GFP was not visibly expressed in normal PBMCs. The plasmid allowed for the selective visualization of viable HeLa cancer cells in mixed cell cultures containing up to 10000-fold more PBMCs. These findings indicate that the hT/Cm-R-hT expressional system is a valuable tool for detecting viable cancer cells mixed with normal cells. The current system can therefore be applied to the in vitro detection of cancer cells that are disseminated in the blood and other types of body fluid in vivo. Since the current system can also be applied to other types of vectors, including virus vectors, this approach using the hTERT promoter-based construct is expected to become a valuable tool for enhancing cancer specific gene expression.

DOI： 10.3892/or.2017.5710

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Reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3) overexpression, induced using an adenovirus (Ad)-REIC, has been revealed to have a dramatic therapeutic effect on multiple types of cancer. To achieve an improved therapeutic effect from Ad-REIC on cancer, our group previously developed an enhanced gene expression system, the C-TSC cassette [cytomegalovirus (CMV)-RU5' located upstream (C); another promoter unit composed of triple tandem promoters, human telomerase reverse transcriptase (hTERT), simian virus 40 and CMV, located downstream of the cDNA (TSC); plus a polyadenylation (polyA) signal]. When applied to the conventional Ad-REIC, this novel system induced the development of an enhanced product, Ad-C-TSC-REIC, which exhibited a noticeable anticancer effect. However, there were difficulties in terms of Ad-C-TSC-REIC productivity in HEK293 cells, which are a widely used donor cell line for viral production. Productivity of Ad-C-TSC-REIC was significantly reduced compared with the conventional Ad-REIC, as the Ad-C-TSC-REIC had a significantly higher ability to induce apoptotic cell death of not only various types of cancer cell, but also HEK293 cells. The present study aimed to overcome this problem by modifying the C-TSC structure, resulting in an improved candidate: A C-T cassette (C: CMV-RU5' located upstream; T: another promoter unit composed of a single hTERT promoter, located downstream of the cDNA plus a polyA signal), which demonstrated gene expression comparable to that of the C-TSC system. The improved adenovirus REIC/Dkk-3 product with the C-T cassette, named Ad-C-T-REIC, exhibited a higher expression level of REIC/Dkk3, similar to that of Ad-C-TSC-REIC. Notably, the vector mitigated the cell death of donor HEK293 cells, resulting in a higher rate of production of its adenovirus. These results indicated that Ad-C-T-REIC has the potential to be a useful tool for application in cancer gene therapy.

DOI： 10.3892/ol.2017.6201

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

The intracellular delivery of functionally active transcription factor proteins is emerging as a promising technique for artificial regulation of cellular functions. However, in addition to the cell membrane, which acts as a barrier to macromolecules, the aggregation-favored properties of structurally flexible transcription factor proteins limit the application of this method. In-cell folding technique can be used to overcome these issues. This technique solubilizes denatured protein by reversible alkyl-disulfide cationization (S-cationization), and simultaneously endows efficient intracellular delivery and folding to the biologically active conformation in the reducing environment of the cytosol. Because cationized protein is internalized into cells by adsorption-mediated endocytosis, endosomal escape is crucial for this technique. In this study, we utilized a sensitive luciferase reporter gene assay to quantitatively evaluate in-cell folding of the artificial transcription factor GAIA-VP16. Although the cationic moiety of S-cationized protein was slightly affected, co-transduction of amphipathic peptide Endo-PORTER dramatically improved in-cell folding efficiency. Live cell imaging of fluorescent-labeled GAL4-VP16 revealed that some of the proteins diffused into the cytosol and nucleus through co-transduction with Endo-PORTER. Real-time monitoring of light output of luciferase revealed the kinetics of in-cell folding, supporting that endosomal-release assisted by Endo-PORTER was stimulated by endosome acidification. Because this method can transduce proteins uniformly and repeatedly into living cells, S-cationized transcription factor proteins are widely applicable for the artificial regulation of cellular functions. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2016.11.011

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S100A11, a small Ca2+ binding protein, acts extracellularly as a mediator of cancer progression. That raises the question of how a protein that lacks the classical secretory signal is able to be secreted outside cells without being damaged. Some insights into this question have been obtained, and there has been accumulating evidence indicating a pivotal role of a non-classical vesicle-mediated pathway using lysosomes or peroxisomes for the protein secretion. To obtain a more precise insight into the secretory mechanism of S100A11, we first screened representative cancer cells exhibiting significantly active secretion of S100A11. From the results of profiling, we turned our attention to aggressive cancer mesothelioma cells. In mesothelioma cells, we found that abundant dimeric S100A11 was produced selectively in the peroxisome after transportation of monomeric S100A11 through an interaction with PEX14, a peroxisome membrane protein, resulting in peroxisomal secretion of dimerized S100A11. In an extracellular environment in vitro, dimerized S100A11 promoted mesothelial cell invasion indirectly with the help of fibroblast cells. Overall, the results indicate that the peroxisome functions as an essential vesicle for the production of dimerized S100A11 and the subsequent secretion of the protein from mesothelioma cells and that peroxisome-mediated secretion of dimerized S100A11 might play a critical role in mesothelioma progression in a tumor microenvironment.

DOI： 10.1007/s12307-016-0185-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The dynamic interaction between tumor cells and their microenvironment induces a proinflammatory milieu that drives cancer development and progression. The S100A8/A9 complex has been implicated in chronic inflammation, tumor development, and progression. The cancer microenvironment contributes to the up-regulation of this protein complex in many invasive tumors, which is associated with the formation of pre-metastatic niches and poor prognosis. Changing adhesive preference of cancer cells is at the core of the metastatic process that governs the reciprocal interactions of cancer cells with the extracellular matrices and neighboring stromal cells. Cell adhesion molecules (CAMs) have been confirmed to have high-level expression in various highly invasive tumors. The expression and function of CAMs are profoundly influenced by the extracellular milieu. S100A8/A9 mediates its effects by binding to cell surface receptors, such as heparan sulfate, TLR4 and RAGE on immune and tumor cells. RAGE has recently been identified as an adhesion molecule and has considerably high identity and similarity to ALCAM and MCAM, which are frequently over-expressed on metastatic malignant melanoma cells. In this study, we demonstrated that ALCAM and MCAM also function as S100A8/A9 receptors as does RAGE and induce malignant melanoma progression by NF-kappa B activation and ROS formation. Notably, MCAM not only activated NF-kappa B more prominently than ALCAM and RAGE did but also mediated intracellular signaling for the formation of lung metastasis. MCAM is known to be involved in malignant melanoma development and progression through several mechanisms. Therefore, MCAM is a potential effective target in malignant melanoma treatment.

DOI： 10.1007/s10585-016-9801-2

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

It is now known that multicomponent protein assemblies strictly regulate many protein functions. The S100 protein family is known to play various physiological roles, which are associated with alternative complex formations. To prepare sufficient amounts of heterodimeric S100A8 and S100A9 proteins, we developed a method for bicistronic coexpression from a single-vector system using Escherichia coli cells as a host. The complex formation between S100A8 and S100A9 appears to be dependent on the thermodynamic stability of the protein during expression. The stable S100A8/A9 heterodimer complex spontaneously formed during coexpression, and biologically active samples were purified by cation-exchange chromatography. Semi-stable homodimers of S100A8 and S100A9 were also formed when expressed individually. These results suggest that the assembly of S100 protein complexes might be regulated by expression levels of partner proteins in vivo. Because protein assembly occurs rapidly after protein synthesis, coexpression of relevant proteins is crucial for the design of multicomponent recombinant protein expression systems.

DOI： 10.1016/j.bbrep.2016.03.009

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Rhodococcus jostii RHA1 (RHA1) degrades polychlorinated biphenyl (PCB) via co-metabolism with biphenyl. To identify the novel open reading frames (ORFs) that contribute to PCB/biphenyl metabolism in RHA1, we compared chromatin immunoprecipitation chip and transcriptomic data. Six novel ORFs involved in PCB/biphenyl metabolism were identified. Gene deletion mutants of these 6 ORFs were made and were tested for their ability to grow on biphenyl. Interestingly, only the ro10225 deletion mutant showed deficient growth on biphenyl. Analysis of Ro10225 protein function showed that growth of the ro10225 deletion mutant on biphenyl was recovered when exogenous recombinant Ro10225 protein was added to the culture medium. Although Ro10225 protein has no putative secretion signal sequence, partially degraded Ro10225 protein was detected in conditioned medium from wild-type RHA1 grown on biphenyl. This Ro10225 fragment appeared to form a complex with another PCB/biphenyl oxidation enzyme. These results indicated that Ro10225 protein is essential for the formation of the PCB/biphenyl dioxygenase complex in RHA1.

DOI： 10.1080/09168451.2015.1127134

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Humoral immune responses against tumor-associated antigens (TAAs) or cancer/testis antigens (CTAs) aberrantly expressed in tumor cells are frequently observed in cancer patients. Recent clinical studies have elucidated that anticancer immune responses with increased levels of anti-TAA/CTA antibodies improve cancer survival rates. Thus, these antibody levels are promising biomarkers for diagnosing the efficiency of cancer immunotherapy. Full-length antigens are favored for detecting anti-TAA/CTA antibodies because candidate antigen proteins contain multiple epitopes throughout their structures. In this study, we developed a methodology to prepare purified water-soluble and full-length antigens by using cysteine sulfhydryl group cationization (S-cationization) chemistry. S-Cationized antigens can be prepared from bacterial inclusion bodies, and they exhibit improved protein solubility but preserved antigenicity. Anti-TAA/CTA antibodies detected in cancer patients appeared to recognize linear epitopes, as well as conformational epitopes, and because the frequency of cysteine side-residues on the epitope-paratope interface was low, any adverse effects of S-cationization were virtually negligible for antibody binding. Furthermore, S-cationized antigen-immobilized Luminex beads could be successfully used in highly sensitive quantitative-multiplexed assays. Indeed, patients with a more broadly induced serum anti-TAA/CTA antibody level showed improved progression-free survival after immunotherapy. The comprehensive anti-TAA/CTA assay system, which uses S-cationized full-length and water-soluble recombinant antigens, may be a useful diagnostic tool for assessing the efficiency of cancer immunotherapy.

DOI： 10.1021/acs.bioconjchem.5b00328

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

Reduced expression in immortalized cells (REIC)/Dickkopf (Dkk)-3 is a tumor-suppressor gene and has been studied as a promising therapeutic gene for cancer gene therapy. Intratumoral injection of an adenovirus vector carrying the human REIC/Dkk-3 gene (Ad-REIC) elicits cancer cell-specific apoptosis and anticancer immune responses. The cytokine-like effect of secretory REIC/Dkk-3 on the induction of dendritic cell (DC)-like cell differentiation from monocytes plays a role in systemic anticancer immunity. In the present study, we generated recombinant full-length and N-terminally truncated REIC/Dkk-3 to characterize the biological activity of the protein. During the purification procedure, we identified a 17 kDa cysteine-rich stable product (C17-REIC) showing limited degradation. Further analysis showed that the C17-REIC domain was sufficient for the induction of DC-like cell differentiation from monocytes. Concomitant with the differentiation of DCs, the REIC/Dkk-3 protein induced the phosphorylation of glycogen synthase kinase 3 beta (GSK-3 beta) and signal transducers and activators of transcription (STAT) at a level comparable to that of granulocyte/macrophage colony-stimulating factor. In a mouse model of subcutaneous renal adenocarcinoma, intraperitoneal injection of full-length and C17-REIC proteins exerted anticancer effects in parallel with the activation of immunocompetent cells such as DCs and cytotoxic T lymphocytes in peripheral blood. Taken together, our results indicate that the stable cysteine-rich core region of REIC/Dkk-3 is responsible for the induction of anticancer immune responses. Because REIC/Dkk-3 is a naturally circulating serum protein, the upregulation REIC/Dkk-3 protein expression could be a promising option for cancer therapy.

DOI： 10.3892/or.2015.3885

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

DOI： 10.1271/kagakutoseibutsu.53.245

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Other Link： https://jlc.jst.go.jp/DN/JLC/20009210033?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

Immunotherapy is one of the attractive treatment strategies for advanced prostate cancer. The US Food and Drug Administration (FDA) previously approved the therapeutic vaccine, sipuleucel-T, which is composed of autologous antigen-presenting cells cultured with a fusion protein [prostatic acid phosphatase (PAP) and granulocyte-macrophage colony-stimulating factor (GMCSF)]. Although sipuleucel-T has been shown to prolong the median survival of patients for 4.1 months, more robust therapeutic effects may be expected by modifying the vaccination protocol. In the present study, we aimed to develop and validate a novel vaccination strategy using multiple PAP-fused cytokines for prostate cancer treatment. Using a super gene expression (SGE) system that we previously established to amplify the production of a recombinant protein, significant amounts of PAP-fused cytokines [human GMCSF, interleukin-2 (IL2), IL4, IL7 and mouse GMCSF and IL4] were obtained. We examined the activity of the fusion proteins in vitro to validate their cytokine functions. A significant upregulation of dendritic cell differentiation from monocytes was achieved by PAP-GMCSF when used with the other PAP-fused cytokines. The PAP-fused human IL2 significantly increased the proliferation of lymphocytes, as determined by flow cytometry. We also investigated the in vivo therapeutic effects of multiple PAP-fused cytokines in a mouse prostate cancer model bearing prostate-specific antigen (PSA)- and PAP-expressing tumors. The simultaneous intraperitoneal administration of PAP-GMCSF, -IL2, -IL4 and -IL7 significantly prevented tumor induction and inhibited the tumor growth in the PAP-expressing tumors, yet not in the PSA-expressing tumors. The in vivo therapeutic effects with the multiple PAP-fused cytokines were superior to the effects of PAP-GMCSF alone. We thus demonstrated the advantages of the combined use of multiple PAP-fused cytokines including PAP-GMCSF, and propose a promising prostatic antigen-vaccination strategy to enhance the therapeutic effects.

DOI： 10.3892/or.2015.3770

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Preventing protein aggregation is a major goal of biotechnology. Since protein aggregates are mainly comprised of unfolded proteins, protecting against denaturation is likely to assist solubility in an aqueous medium. Contrary to this concept, we found denatured total cellular protein mixture from mammalian cell kept high solubility in pure water when the mixture was nucleic acids free. The lysates were prepared from total cellular protein pellet extracted by using guanidinium thiocyanate-phenol-chloroform mixture of TRIzol, denatured and reduced total protein mixtures remained soluble after extensive dialysis against pure water. The total cell protein lysates contained fully disordered proteins that readily formed large aggregates upon contact with nucleic acids or salts. These findings suggested that the highly flexible mixtures of disordered proteins, which have fully ionized side chains, are protected against aggregation. Interestingly, this unusual solubility is characteristic of protein mixtures from higher eukaryotes, whereas most prokaryotic protein mixtures were aggregated under identical conditions. This unusual solubility of unfolded protein mixtures could have implications for the study of intrinsically disordered proteins in a variety of cells.

DOI： 10.1371/journal.pone.0113295

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Background: RAGE receptor plays a critical role in many inflammatory disorders. Results: Functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated cell survival. Conclusion: DAP10 membrane adaptor is critically involved in RAGE-mediated survival signaling upon S100A8/A9 binding. Significance: This is the first report demonstrating that RAGE-mediated survival signaling is critically regulated by DAP10 interaction.
The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes.

DOI： 10.1074/jbc.M114.573071

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：HUMANA PRESS INC  

For expression of genes in mammalian cells, various vectors have been developed using promoters including CMV, EF-1 alpha, and CAG promoters and have been widely used. However, such expression vectors sometimes fail to attain sufficient expression levels depending on the nature of cargo genes and/or on host cell types. In the present study, we aimed to develop a potent promoter system that enables high expression levels of cargo genes ubiquitously in many different cell types. We found that insertion of an additional promoter downstream of a cargo gene greatly enhanced the expression levels. Among the constructs we tested, C-TSC cassette (C: CMV-RU5' located upstream; TSC: another promoter unit composed of triple tandem promoters, hTERT, SV40, and CMV, located downstream of the cDNA plus a polyadenylation signal) had the most potent capability, showing far higher efficiency than that of potent conventional vector systems. The results indicate that the new expression system is useful for production of recombinant proteins in mammalian cells and for application as a gene therapeutic measure.

DOI： 10.1007/s12033-014-9738-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

Gene expression systems with various promoters, including the cytomegalovirus (CMV) promoter, have been developed to increase the gene expression in a variety of normal and cancer cells. In particular, in the clinical trials of cancer gene therapy, a more efficient and robust gene expression system is required to achieve sufficient therapeutic outcomes. By inserting the triple translational enhancer sequences of human telomerase reverse transcriptase (hTERT), Simian virus 40 (SV40) and CMV downstream of the sequence of the BGH polyA, we were able to develop a novel gene expression system that significantly enhances the expression of the genes of interest. We termed this novel gene expression cassette the super gene expression (SGE) system, and herein verify the utility of the SGE cassette for a replication-deficient adenoviral vector. We newly developed an adenoviral vector expressing the tumor suppressor, reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3), based on the CMV promoter-driven SGE system (Ad-SGE-REIC) and compared the therapeutic utility of Ad-SGE-REIC with that of the conventional adenoviral vectors (Ad-CMV-REIC or Ad-CAG-REIC). The results demonstrated that the CMV promoter-SGE system allows for more potent gene expression, and that the Ad-SGE-REIC is superior to conventional adenoviral systems in terms of the REIC protein expression and therapeutic effects. Since the SGE cassette can be applied for the expression of various therapeutic genes using various vector systems, we believe that this novel system will become an innovative tool in the field of gene expression and gene therapy.

DOI： 10.3892/or.2013.2958

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

The receptor for advanced glycation end products (RAGE) is a multi-ligand cell surface receptor and a member of the immunoglobulin superfamily. RAGE is involved in a wide range of inflammatory, degenerative and hyper-proliferative disorders which span over different organs by engaging diverse ligands, including advanced glycation end products, S100 family proteins, high-mobility group protein B1 (HMGB1) and amyloid beta. We previously demonstrated that the cytoplasmic domain of RAGE is phosphorylated upon the binding of ligands, enabling the recruitment of two distinct pairs of adaptor proteins, Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) and myeloid differentiation protein 88 (MyD88). This engagement allows the activation of downstream effector molecules, and thereby mediates a wide variety of cellular processes, such as inflammatory responses, apoptotic cell death, migration and cell growth. Therefore, inhibition of the binding of TIRAP to RAGE may abrogate intracellular signaling from ligand-activated RAGE. In the present study, we developed inhibitor peptides for RAGE signaling (RAGE-I) by mimicking the phosphorylatable cytosolic domain of RAGE. RAGE-I was efficiently delivered into the cells by polyethylenimine (PEI) cationization. We demonstrated that RAGE-I specifically bound to TIRAP and abrogated the activation of Cdc42 induced by ligand-activated RAGE. Furthermore, we were able to reduce neuronal cell death induced by an excess amount of S100B and to inhibit the migration and invasion of glioma cells in vitro. Our results indicate that RAGE-I provides a powerful tool for therapeutics to block RAGE-mediated multiple signaling.

DOI： 10.3892/ijmm.2013.1467

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Protein cationization techniques are powerful protein transduction methods for mammalian cells. As we demonstrated previously, cationized proteins with limited conjugation to polyethylenimine have excellent ability to enter into cells by adsorption-mediated endocytosis [Futami, J., et al. (2005) J. Biosci. Bioeng. 99, 95-103]. In this study, we show that proteins with extensive and uniform cationization covering the protein surface reach the cytoplasm and nucleus more effectively than proteins with limited cationic polymers or proteins that are fused to cationic peptides. Although extensive modification of carboxylates results in loss of protein function, chicken avidin retains biotin-binding ability even after extensive amidation of carboxylates. Using this cationized avidin carrier system, the protein transduction ability of variously cationized avidins was investigated using biotinylated protein as a probe. The results revealed that cationized avidins bind rapidly to the cell surface followed by endocytotic uptake. Small amounts of uniformly cationized avidin showed direct penetration into the cytoplasm within a 15 min incubation. This penetration route seemed to be energy dependent and functioned under cellular physiological conditions. A biotinylated exogenous transcription factor protein that penetrated cells was demonstrated to induce target gene expression in living cells.

DOI： 10.1021/bc300030d

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Accumulating evidence indicates that dysfunction of mitochondria is a common feature of Parkinson disease. Functional loss of a familial Parkinson disease-linked gene, BRPK/PINK1 (PINK1), results in deterioration of mitochondrial functions and eventual neuronal cell death. A mitochondrial chaperone protein has been shown to be a substrate of PINK1 kinase activity. In this study, we demonstrated that PINK1 has another action point in the cytoplasm. Phosphorylation of Akt at Ser-473 was enhanced by overexpression of PINK1, and the Akt activation was crucial for protection of SH-SY5Y cells from various cytotoxic agents, including oxidative stress. Enhanced Akt phosphorylation was not due to activation of phosphatidylinositol 3-kinase but due to activation of mammalian target of rapamycin complex 2 (mTORC2) by PINK1. Rictor, a specific component of mTORC2, was phosphorylated by overexpression of PINK1. Furthermore, overexpression of PINK1 enhanced cell motility. These results indicate that PINK1 exerts its cytoprotective function not only in mitochondria but also in the cytoplasm through activation of mTORC2.

DOI： 10.1074/jbc.M110.179390

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

The Wnt canonical signaling pathway is essential for the early development of eukaryotic organisms and plays a key role in cell proliferation, differentiation, and oncogenesis. Moreover, the Wnt canonical signaling pathway contributes to the self-renewal of mouse hematopoietic stem cells (HSCs). Here, we demonstrate artificial activation of the Wnt canonical signaling pathway by beta-catenin protein transduction. Constitutively active beta-catenin protein was introduced into human embryonic kidney HEK-293 cells using a polyethylenimine (PEI) cationization method, or with the BioPORTER protein transduction reagent. We have previously shown that modification with PEI effectively causes proteins to be internalized by living mammalian cells. PEI-cationized, constitutively active beta-catenin protein was added to HEK-293 cells, and induction of several Wnt/beta-catenin target genes was detected by real-time PCR. However, using BioPORTER to introduce active beta-catenin did not activate the Wnt canonical signaling pathway. Introduction of eGFPNuc (enhanced green fluorescent protein variant containing a nuclear localization signal) into HEK-293 cells using the BioPORTER reagent caused significant cell death, as determined by propidium iodide staining. In contrast, the PEI-modified eGFPNuc did not impair survival of HEK-293 cells. These results indicate that the Wnt canonical signaling pathway could be successfully activated by transduction of PEI-cationized active beta-catenin, and the PEI-cationization method is an effective and safe technology for protein transduction into mammalian cells.

DOI： 10.1002/biot.200900132

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

To develop a novel dendritic cell (DC)-based vaccine for inducing antigen-specific CD8(+) T cell responses by cross-presentation, we tested a novel antigen delivery system that introduces Soluble antigens into the cytosol of cells by an endocytosis-mediated mechanism which avoids damaging the plasma membrane ("Endo-Porter"(TM)). Proteins released from endosomes into the cytoplasm are degraded by the proteasome, and fragmented antigenic peptides are presented to the classical cytosolic MHC class I pathway. DCs pulsed with OVA protein in the presence of Endo-Porter efficiently stimulate OVA peptide-specific CD8(+) T (OT-I) cells. Although this agent diverts some of the endocytosed antigens away from the classical MHC class II-restricted presentation pathway to the class I pathway, the activation of CD4(+) T cells was found not to be hampered by Endo-Porter-mediated antigen delivery. On the contrary, it was rather augmented, probably due to the increased uptake of antigen. Because specific CD4(+) T cell help is required to license DCs for cross-priming, Endo-Porter-mediated antigen delivery is a promising approach for developing more efficient cancer vaccines targeting both CD4(+) and CD8(+) T cells. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2010.01.019

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

The REIC/Dkk-3 gene has been reported to be a tumor suppressor and the expression is significantly down-regulated in a broad range of cancer cell types. The protein is secretory, but the physiological function remains unclear. This study demonstrated that recombinant REIC/Dkk-3 protein induced the differentiation of human CD14(+) monocytes into a novel cell type ((REIC/Dkx-3)Mo). (REIC/Dkk-3)Mo resembles immature dendritic cells generated with IL-4 and GM-CSF. Both these cell populations exhibit similar proportions of CD11c(+), CD40(+), CD86(+) and HLA-DR(+) cells and endocytic capacity, but (REIC/Dkk-3)Mo is negative for CD1a antigen. An analysis of the signal transducers and activators of transcription (STAT) pathways revealed that REIC/Dkk-3 induces phosphorylation of STAT 1 and STAT 3. Furthermore, intratumoral administration of REIC/Dkk-3 protein significantly suppressed tumor growth with CD11c(+) and CD8(+) (dendritic and killer T cell marker, respectively) cell accumulation and enhanced anticancer cytolytic activity of splenocytes. These data indicated a cytokine-like role of REIC/Dkk-3 protein in monocyte differentiation that might be exploited therapeutically.

DOI： 10.3892/ijo_00000191

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The glutathione S-transferase (GST)-fused protein expression system has been extensively used to generate a large quantity of proteins and has served for functional analysis in vitro. In this study, we developed a novel approach for the efficient intracellular delivery of GST-fused proteins into living cells to expand their usefulness up to in vivo use. Since protein cationization techniques are powerful strategies for efficient intracellular uptake by adsorptive-mediated endocytosis, GST-fused proteins were cationized by forming a complex with a polycationic polyethylenimine (PEI)-glutathione conjugate. On screening of protein transduction, optimized PEI-glutathione conjugate for protein transduction was characterized by a partly oligomerized mixture of PEI with average molecular masses of 600 (PEI600) modified with multiple glutathiones, which could have sufficient avidity for GST. Furthermore, enhanced endosomal escape of transduced GST-fused proteins was observed when they were delivered with a glutathione-conjugated PEI600 derivative possessing a hydroxybutenyl moiety. These results were confirmed by both intracellular confocal imaging of GST-fused green fluorescent protein and activation of an endogenous growth signal transduction pathway by a GST-fused constitutively active mutant of a kinase protein. These PEI-glutathione conjugates seem to be convenient molecular tools for protein transduction of widely used GST-fused proteins.

DOI： 10.1093/jb/mvn087

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Authorship：Lead author   Language：English   Publisher：BENTHAM SCIENCE PUBL LTD  

The cytotoxic properties of naturally occurring or engineered RNases correlate well with their efficiency of cellular internalization and digestion level of cellular RNA. Cationized RNases are considered to adsorb to the anionic cellular surface by Coulombic interactions, and then become efficiently internalized into cells by an endocytosis-like pathway. The design of cytotoxic RNases by chemical modification of surface carboxylic residues is one of the powerful strategies for enhancing cellular internalization and is accompanied with a decreased sensitivity for the cytoplasmic RNase inhibitor. Although chemically modified cationized RNases showed decreased ribonucleolytic activity, improved endocytosis and decreased affinity to the endogenous RNase inhibitor conclusively contribute to their ability to digest cellular RNA. Furthermore, the cytotoxicity of cationized RNases can be drastically enhanced by co-endocytosis with an endosome-destabilizing peptide. Since efficient cellular internalization of proteins into living cells is an important technology for biotechnology, studies concerning the design of cytotoxic RNases provided general perceptions for protein-based drug design.

DOI： 10.2174/138920108784567326

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Polyethylenimine (PEI) cationization is a powerful strategy for protein transduction into cells. In this study, we attempted the artificial regulation of cell proliferation by protein transduction of the N-terminal domain (1-132 amino acids) of the simian virus 40 large T-antigen (SVLT-N), which inactivates retinoblastoma family proteins but not p53. To deliver SVLT-N into cells, we employed an indirect cationization method by forming a complex of biotynylated SVLT-N through disulfide bonds (biotin-SS-SVLT-N) and PEI-cationized avidin (PEI600-avidin). Using this complex, SVLT-N was transduced into the nucleus of confluent and quiescent Balb/c 3T3 cells and was found to be complexed with a cellular target protein, pRb. Furthermore, SVLT-N transduction induced cell proliferation in spite of confluent conditions. Because SVLT-N thus transduced into cells gradually degraded and was not detectable after a 4-d incubation, transiently transformed cells were obtained by this method. These results suggest that oncogene protein transduction technology has great potential for in vitro regulation of cell proliferation.

DOI： 10.1263/jbb.105.34

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We previously revealed a novel signal pathway involving S100A11 for inhibition of the growth of normal human keratinocytes (NHK) caused by high Ca++ or transforming growth factor beta. Exposure to either agent resulted in transfer of S100A11 to nuclei, where it induced p21(WAF1). In contrast, S100A11 has been shown to be overexpressed in many human cancers. To address this apparent discrepancy, we analyzed possible new functions of S100A11, and we provide herein evidence that 1) S100A11 is actively secreted by NHK; 2) extracellular S100A11 acts on NHK to enhance the production of epidermal growth factor family proteins, resulting in growth stimulation; 3) receptor for advanced glycation end products, nuclear factor-kappa B, Akt, and cAMP response element-binding protein are involved in the S100A11-triggered signal transduction; and 4) production and secretion of S100A11 are markedly enhanced in human squamous cancer cells. These findings indicate that S100A11 plays a dual role in growth regulation of epithelial cells.

DOI： 10.1091/mbc.E07-07-0682

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Regulation of cell growth and apoptosis is one of the pleiotropic functions of annexin A1 (ANXA1). Although previous reports on the overexpression of ANXA1 in many human cancers and on growth suppression and/or induction of apoptosis by ANXA1 may indicate the tumor-suppressive nature of ANXA1, molecular mechanisms of the function of ANXA1 remain largely unknown. Here we provide evidence that ANXA1 mechanistically links the epidermal growth factor-triggered growth signal pathway with cytosolic phospholipase A(2) (cPLA(2)), an initiator enzyme of the arachidonic acid cascade, through interaction with S100A11 in normal human keratinocytes (NHK). Ca2+ -dependent binding of S100A11 to ANXA1 facilitated the binding of the latter to cPLA2, resulting in inhibition of cPLA2 activity, which is essential for the growth of NHK. On exposure of NHK to epidermal growth factor, ANXA1 was cleaved solely at Trp(12), and this cleavage was executed by cathepsin D. In squamous cancer cells, this pathway was shown to be constitutively activated. The newly found mechanistic intersection may be a promising target for establishing new measures against human cancer and other cell growth disorders.

DOI： 10.1074/jbc.M707538200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper-like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild-type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L-RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by Xray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L-RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L-and 2L-RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L-RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L- RNase 1) was also evaluated. N4L- RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper-like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.

DOI： 10.1110/ps.072851407

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Authorship：Lead author,　Corresponding author   Language：English   Publisher：INFORMA HEALTHCARE  

The development of a method for the efficient intracellular delivery of inherently non-permeable proteins is needed for manipulation of cellular phenotypes or the discovery of protein-based drugs. It has been demonstrated that proteins artificially cationized by chemical conjugation show efficient intracellular delivery via adsorptive-mediated endocytosis and then can exert their biological activity in cells. Studies have also revealed that cationic peptides known as cell-penetrating peptides (CPPs) provide a means to deliver molecules into mammalian cells. Although the internalization mechanisms remain controversial, it is now becoming clear that the main port of entry into cells by CPPs also involves adsorptive-mediated endocytosis rather than the direct penetration of the plasma membrane. As the mammalian cell membrane possesses an abundance of negatively charged glycoproteins and glycosphingolipids, cationization of proteins is a reasonable choice to endow them with the ability for intracellular delivery. Cationization of proteins is usually accompanied by drastic changes in protein properties, structure and biological activities. Recently developed sophisticated protein chemistry can minimize these side effects. Therefore, protein cationization techniques will hopefully prove to be powerful tools for innovative research and drug discovery. In this review, techniques for cationization of proteins and their intracellular delivery, as well as some of their potential therapeutic applications, are discussed.

DOI： 10.1517/17460441.2.2.261

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

The protein no longer terminates here. Non-natural amino acids were attached onto a tRNA and were then transferred with the aid of L/F-tRNA-protein transferase to the N termini of proteins possessing N-terminal Lys or Arg components. By this new technique, 1- and 2-naphthylalanine or 3-nitrotyrosine were coupled to the N termini of several proteins. A novel and convenient method for adding single Lys units at the N termini of various proteins was also developed.

DOI： 10.1002/cbic.200600181

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Cationization is a powerful strategy for internalizing a protein into living cells. On the other hand, a reversibly cationized denatured protein through disulfide bonds is not only soluble in water but also able to fold to the native conformation in vitro. When these advantages in cationization were combined, we developed a novel method to deliver a denatured protein into cells and simultaneously let it fold to express its function within cells. This "in-cell folding" method enhances the utility of recombinant proteins expressed in Escherichia coli as inclusion bodies; that is, the recombinant proteins in inclusion bodies are solubilized by reversible cationization through cysteine residues by disulfide bonds with aminopropyl methanethiosulfonate or pyridyldithiopropionylpolyethylenimine and then incubated with cells without an in vitro folding procedure. As a model protein, we investigated human tumor-suppressor p53. Treatment of p53-null Saos-2 cells with reversibly cationized p53 revealed that all events examined as indications of the activation of p53 in cells, such as reduction of disulfide bonds followed by tetramer formation, localization into the nucleus, induction of p53 target genes, and induction of apoptosis of cells, occurred. These results suggest that reversible cationization of a denatured protein through cysteine residues is an alternative method for delivery of a functional protein into cells. This method would be very useful when a native folded protein is not readily available.

DOI： 10.1021/bi052642a

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT INST ANTICANCER RESEARCH  

Background: We previously showed the usefulness of a fused protein of human pancreatic ribonuclease1 (hRNase1) with human epidermal growth factor (hEGF) for molecular targeting of EGF receptor (EGFR)-overexpressing cells. In this study, the mechanisms underlying the inhibition of cell growth by RNase-EGF fused proteins was confirmed. Materials and Methods: Des. 1-7 hRNase1 was genetically fused to hEGF. The fused proteins were expressed and isolated from Escherichia coli. The internalization of hRNase1-hEGF was confirmed by confocal fluorescence microscopy. The growth-inhibitory effect of the fused proteins was evaluated by MTT assay. Results: FITC-labelled hRNase1-hEGF was internalized into EGFR-overexpressing A431 cells. The internalization was not observed in A 431 cells pre-treated with hEGF and EGFR-deficient H69 cells. The growth-inhibitory effect of des.1-7 hRNase1-hEGF against A431 cells was statistically significantly more pronounced than that of hRNase1-hEGF. Conclusion: RNase-EGF fused proteins are internalized through EGFR and inhibit cell growth by exerting their ribonucleolytic activity in the cytosol.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Proteins and peptides are useful research and therapeutic tools, however applications are limited because delivery to the desired location is not easily achievable. There are two hurdles in protein/peptide delivery to the brain: the blood-brain barrier and intracellular penetration. Penetration to both brain and the intracellular space can be achieved by adjusting hydrophilicity, and small molecule pharmacological agents have been successfully developed using this approach. But with proteins and peptides, it is difficult to modify the hydrophilicity without influencing biological functions. Trans-acting factor protein from the human immunodeficiency virus contains a highly conserved cationic peptide sequence necessary for transduction across the cell membrane. While trans-acting factor peptide has been used for in vitro protein transduction, its in vivo application is very limited because it is rapidly degraded by proteolysis. Polyethylenimine is a chemically synthesized small molecule cationization agent; the charge density is greater than a peptide-based cationic cluster such as trans-acting factor, and it is resistant to proteolysis in vivo. We first tested intracellular protein transduction following direct brain injection in mice using polyethylenimine-conjugated green fluorescence protein and beta-galactosidase (molecular weights 29 and 540 kDa, respectively). Polyethylenimine-conjugates penetrated to the intracellular space immediately surrounding the injection site within one hour. We further tested polyethylenimine-mediated protein transduction following intranasal administration, which bypasses the blood-brain barrier. Polyethylenimine-conjugates in pH 7.5 solution did not reach the brain, probably because the polyethylenimine-conjugates penetrated into the intracellular space where first exposed to the tissue, i.e. at the nasal mucosae. We temporarily reduced the electrostatic interaction between cationized polyethylenimine-conjugates and cellular surfaces by adjusting the pH to 4.5; solution rapidly reached the brain and penetrated to the intracellular space. This study suggests that polyethylenimine is a useful protein transduction agent in the brain in vivo, and adjusting cationic charge interaction can determine the extent of brain penetration. (c) 2006 IBRO. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuroscience.2006.01.041

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S180_4

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There is a rapid global rise in obesity, and the link between obesity and diabetes remains somewhat obscure. We identified an adipocytokine, designated as visceral adipose tissue-derived serpin (vaspin), which is a member of serine protease inhibitor family. Vaspin cDNA was isolated by from visceral white adipose tissues (WATs) of Otsuka Long-Evans Tokushima fatty (OLETIF) rat, an animal model of abdominal obesity with type 2 diabetes. Rat, mouse, and human vaspins are made up of 392,394, and 395 amino acids, respectively; exhibit approximate to 40% homology with alpha(1)-antitrypsin; and are related to serine protease inhibitor family. Vaspin was barely detectable in rats at 6 wk and was highly expressed in adipocytes of visceral WATs at 30 wk, the age when obesity, body weight, and insulin levels peak in OLETF rats. The tissue expression of vaspin and its serum levels decrease with worsening of diabetes and body weight loss at 50 wk. The expression and serum levels were normalized with the treatment of insulin or insulin-sensitizing agent, pioglitazone, in OLETF rats. Administration of vaspin to obese CRL:CD-1 (ICR) (ICR) mice fed with high-fat high-sucrose chow improved glucose tolerance and insulin sensitivity reflected by normalized serum glucose levels. It also led to the reversal of altered expression of genes relevant to insulin resistance, e.g., leptin, resistin, TNF alpha, glucose transporter-4, and adiponectin. In DNA chip analyses, vaspin treatment resulted in the reversal of expression in approximate to 50% of the high-fat high-sucrose-induced genes in WATs. These findings indicate that vaspin exerts an insulin-sensitizing effect targeted toward WATs in states of obesity.

DOI： 10.1073/pnas.0504703102

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

Previously, we have reported that cationized-proteins covalently modified with polyethylenimine (PEI) (direct PEI-cationization) efficiently enter cells and function in the cytosol [Futami et al. (2005) J. Biosci. Bioeng. 99,95-1031. However, it may be more convenient if a protein could be delivered into cells just by mixing the protein with a PEI-cationized carrier protein having a specific affinity (indirect PEI-cationization). Thus, we prepared PEI-cationized avidin (PEI-avidin), streptavidin (PEI-streptavidin), and protein G (PEI-protein G), and examined whether they could deliver biotinylated proteins and antibodies into living cells. PEI-avidin (and/or PEI-streptavidin) carried biotinylated GFPs into various mammalian cells very efficiently. A GFP variant containing a nuclear localization signal was found to arrive even in the nucleus. The addition of a biotinylated RNase A derivative mixed with PEI-streptavidin to a culture medium of 3T3-SV-40 cells resulted in remarkable cell growth inhibition, suggesting that the biotinylated RNase A derivative entered cells and digested intracellular RNA molecules. Furthermore, the addition of a fluorescein-labeled antiS100C (beta-actin binding protein) antibody mixed with PEI-protein G to human fibroblasts resulted in the appearance of a fluorescence image of actin-like filamentous structures in the cells. These results indicate that indirect PEI-cationization using non-covalent interaction is as effective as the direct PEI-cationization for the transduction of proteins into living cells and for expression of their functions in the cytosol. Thus, PEI-cationized proteins having a specific affinity for certain molecules such as PEI-streptavidin, PEI-avidin and PEI-protein G are concluded to be widely applicable protein transduction carrier molecules.

DOI： 10.1093/jb/mvi081

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

In the post-genomic era, there is pressing need for development of protein manipulation methodology to analyze functions of proteins in living cells. For this purpose, techniques to deliver functional proteins into living cells are currently being evaluated as alternative approaches to the introduction of transcriptionally active DNA. Here, we describe a novel method for efficient protein transduction into living cells in which a protein is simply cationized with polyethylenimine (PEI) by limited chemical conjugation. PEI-cationized proteins appear to adhere to the cell surface by ionic charge interaction and then internalize into cells in a receptor- and transporter-independent fashion. Since PEI is an organic macromolecule with a high cationic-charge density, limited coupling with PEI results in endowment of sufficient cationic charge to proteins without causing serious decline in their fundamental functions. A number of PEI-cationized proteins, such as ribonuclease (RNase), green fluorescent protein (GFP) and immunoglobulin (IgG), efficiently entered cells and functioned in the cytosol. Our results suggest that protein cationization techniques using PEI will be useful for the development of protein transduction technology.

DOI： 10.1263/jbb.99.095

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Decoy oligonucleotides have been used for functional sequestering of transcription factors. Efficient introduction into cells is a prerequisite for the oligonucleotides to exert their blocking function. Lipofection is the most widely used technique for that purpose because of its convenience and relatively high efficiency. However, the transduction efficiency of lipofection largely depends on cell types and experimental conditions and the introduced nucleotides are not specifically directed to nuclei where they exert their major function. In the present study, we designed a new system for transporting oligonucleotides into cell nuclei. The vehicle is composed of glutathione-S-transferase, 7 arginine residues, the DNA- binding domain of GAL4 and a nuclear localization signal, which are linked with flexible glycine stretches. The p53-responsive element linked to the GAL4 upstream activating sequence was efficiently transferred by the vehicle protein into nuclei of primary cultures of neuronal cells, embryonic stem cells and various human normal cells. Transcriptional activation of p21(WAF1/CIP1) and Bax by p53 on exposure to cisplatin was completely blocked by introducing the p53 decoy oligonucleotide. Thus, the system developed in the present study can be a convenient and powerful tool for specifically disrupting the function of DNA- binding proteins in culture.

DOI： 10.1093/nar/gni088

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Basic fibroblast growth factor (bFGF) was inserted in the middle of human ribonuclease 1 (RNase1) sequence at an RNase inhibitor (RI)-binding site (Gly89) by a new gene fusion technique, insertional-fusion. The resultant insertional-fusion protein (CL-RFN89) was active both as bFGF and as RNase. Furthermore, it acquired an additional ability of evading RI through steric blockade of RI-binding caused by fused bFGF domain. As a result, CL-RFN89 showed stronger growth inhibition on B16/BL6 melanoma cells than an RI-sensitive tandem fusion protein. Thus, the insertional-fusion technique increases accessible positions for gene fusion on RNase, resulting in construction of a potent cytotoxic RNase. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2004.05.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

There have been some attempts to develop immunotoxins utilizing human RNase as a cytotoxic domain of antitumor agents. We have recently shown that only human RNase 3 (eosinophil cationic protein, ECP) among five human pancreatic-type RNases excels in binding to the cell surface and has a growth inhibition effect on several cancer cell lines, even though the RNase activity of RNase 3 is completely inhibited by the ubiquitously expressed cytosolic RNase inhibitor. This phenomenon may be explained by that RNase 3 is very stable against proteolytic degradation because RNase 3 internalized through endocytosis could have a longer life time in the cytosol, resulting in the accumulation of enough of it to exceed the concentration of RNase inhibitor, which allows the degradation of cytosolic RNA molecules. Thus, we compared the stabilities of human pancreatic-type RNases (RNases 1-5) and bovine RNase A by means of guanidium chloride-induced denaturation experiments based on the assumption of a two-state transition for unfolding. It was demonstrated that RNase 3 is extraordinarily stabler than either RNase A or the other human RNases (by more than 25 kJ/mol). Thus, our data suggest that in addition to its specific affinity for certain cancer cell lines, the stability of RNase 3 contributes to its unique cytotoxic effect and that it is important to stabilize a human RNase moiety through protein engineering for the design of human RNase-based immunotoxins.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

Cationization of a protein is considered to be a powerful strategy for internalizing a functional protein into cells. Cationized proteins appear to adsorb to the cell surface by electrostatic interactions, then enter the cell in a receptor- and transporter-independent fashion. Thus, in principle, all cell types appear to take up cationized proteins. Since ribonucleases (RNases) have a latent cytotoxic potential, cationized RNases could be useful cancer chemotherapeutics. In this study, we investigated the effect of the degree of cationization on the cytotoxicity of RNase A by modifying carboxyl groups with ethylenediamine. We found that there is an optimum degree of modification for cytotoxicity, in which 5 to 7 out of 11 carboxyl groups in RNase A are modified, toward MCF-7 and 3T3-SV-40 cells. More interestingly, the cytotoxicity of cationized RNase As correlates well with the value of [RNase activity] x [estimated concentration of RNase free from RNase inhibitor], mimicking the practical enzymatic activity of cationized RNase As in cytosol. The results indicate that cationization of a protein to an optimum level is important for maintaining protein function in the cytosol. Sophisticated protein cationization techniques will help to advance protein transduction technology.

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Language：Japanese   Publisher：(一社)日本外科学会  

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Carboxyl groups of bovine RNase A were amidated with ethylenediamine (to convert negative charges of carboxylate anions to positive ones), 2-aminoethanol (to eliminate negative charges), and taurine (to keep negative charges), respectively, by a carbodiimide reaction. Human RNase 1 was also modified with ethylenediamine. Surprisingly, the modified RNases were all cytotoxic toward 3T3-SV-40 cells despite their decreased ribonucleolytic activity. However, their enzymatic activity was not completely eliminated by the presence of excess cytosolic RNase inhibitor (RI). As for native RNase A and RNase 1 which were not cytotoxic, they were completely inactivated by RI. More interestingly, within the cytotoxic RNase derivatives. cytotoxicity correlated well with the net positive charge. RNase 1 and RNase A modified with ethylenediamine were more cytotoxic than naturally occurring cytotoxic bovine seminal RNase. An experiment using the fluorescence-labeled RNase derivatives indicated that the more cationic RNases were more efficiently adsorbed to the cells. Thus, it is suggested that the modification of carboxyl groups could change complementarity of RNase to RI and as a result endow RNase cytotoxicity and that cationization enhances the efficiency of cellular uptake of RNase so as to strengthen its cytotoxicity. The finding that an extracellular human enzyme such as RNase 1 could be effectively internalized into the cell by cationization suggests that cationization is a simple strategy for efficient delivery of a protein into cells and may open the way of the development of new therapeutics.

DOI： 10.1021/bi010248g

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MUNKSGAARD INT PUBL LTD  

Human pancreatic ribonuclease 1 (RNase 1) is considered to be the human counterpart of bovine pancreatic RNase A. Truncation of seven amino-acid residues from the aminoterminal sequence resulted in RNase 1 Delta N7, which has a reduced ribonucleolytic activity and a lower affinity for the human placental RNase inhibitor (PRI). This RNase 1 variant has been cloned, heterologously overexpressed, purified and crystallized. Its crystal structure has been determined and refined using data to 1.9 Angstrom resolution. The molecule displays the alpha + beta folding topology typical of members of the RNase A superfamily. The main distinct features found in RNase 1 Delta N7 are basically located in three loops affecting the fitting of the enzyme to the active site of subtilisin and the shape of the B2 subsite. These changes, taken with the lack of the catalytically active residue Lys7, may explain the reduced affinity of RNase 1 Delta N7 for PRI and the low ribonucleolytic activity of the protein when compared with the native enzyme.

DOI： 10.1107/S0907444901001147

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

In order to stabilize human RNase 1 by introduction of an intramolecular cross-link, a mutant protein (4-118CL RNase 1), in which Arg4 and Val118 are replaced with cysteine residues and linked by a disulfide bond, was designed and expressed in Escherichia coli as inclusion bodies. The 4-118CL RNase 1 that refolded under redox conditions was a monomer without free SH groups and retained 11% of the activity of the wild-type recombinant RNase 1, indicating that the mutant enzyme was correctly folded with the formation of an additional disulfide bond between Cys4 and Cys118, From guanidium chloride denaturation experiments based on the assumption of a two-state transition for unfolding, it was demonstrated that the introduction of the present cross-link increased the thermodynamic stability of RNase 1 by 2.0 kcal/mol. This value was lower than that, 5.4 kcal/mol, theoretically calculated from the reduction of chain entropy of the unfolded state due to the introduction of the cross-link. These results suggest that the present cross-link also destabilized the folded state of RNase 1 by 3.4 kcal/mol. Along with the increase in the thermodynamic stability, the stability of RNase 1 against trypsin digestion was also significantly increased by the introduction of this cross-link. It is likely, although not proven, that stabilized human RNases are favorable for clinical use, because human RNase-based immunotoxins should have long half-lives as to proteolytic degradation after endocytosis.

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We investigated how the folding yield of disulfide-containing globular proteins having positive net charges from crude bacterial inclusion bodies was affected by additives in the folding buffer. In screening folding conditions for human ribonucleases and its derivative, we found that addition of salt (about 0.4 M) to a folding buffer increased the folding yield. This suggested that electrostatic interaction between polyanionic impurities such as nucleic acids and cationic unfolded protein led to the formation of aggregates under the low-salt conditions. Since inclusion bodies were found to contain nucleic acids regardless of the electrostatic nature of the expressed protein, the electrostatic interaction between phosphate moieties of nucleic acids and basic amino acid residues of a denatured protein may be large enough to cause aggregation, and therefore the addition of salt in a folding buffer may generally be useful for promotion of protein folding from crude inclusion bodies. We further systematically investigated additives such as glycerol, guanidium chloride, and urea that are known to act as chemical chaperons, and found that these additives, together with salt, synergistically improved folding yield. This study, suggesting that the addition of salt into the folding buffer is one of the crucial points to be considered, may pave the way for a systematic investigation of the folding conditions of disulfide-containing foreign proteins from crude bacterial inclusion bodies.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Pancreatic-type RNases are considered to have cytotoxic potential due to their ability to degrade RNA molecules when they enter the cytosol. However, most of these RNases show little cytotoxicity because cells have no active uptake mechanism for these RNases and because the ubiquitous cytoplasmic RNase inhibitor is considered to play a protective role against the endocytotic leak of RNases from the outside of cells. To study the cytotoxic potential of RNase toward malignant cells targeting growth factor receptors, the C-terminus of human RNase 1 was fused to the N-terminus of human basic fibroblast growth factor (bFGF). This RNase-FGF fused protein effectively inhibited the growth of mouse melanoma cell line B16/BL6 with high levels of cell surface FGF receptor. This effect appeared to result from prolongation of the overall cell cycle rather than the killing of cells or specific arrest in a particular phase of the cell cycle. Thus, human RNase 1 fused to a ligand of cell surface molecules, such as the FGF receptor, is shown to be an effective candidate for a selective cell targeting agent with low toxic effects on normal cell types.

DOI： 10.1093/protein/12.11.1013

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Recombinant human ribonuclease 1 (RNasel) was chemically linked to recombinant human epidermal growth factor (EGF). The cytotoxicity of this conjugate was assayed using MTT assay. The EGF-RNase conjugate showed dose-dependent cytotoxicity against breast and squamous cell carcinomas overexpressing the EGF receptor (EGFR). The cytotoxicity of the conjugate correlated positively with the level of EGFR expression by each cell line. These results suggest that the EGF-RNase conjugate is a more effective anticancer agent with less immunogenicity and toxicity than conventional chimeric breast cancer toxins.

DOI： 10.1007/BF02966516

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT INC PUBL  

The tissue-specific expression of five human pancreatic-type RNases and RNase inhibitor was analyzed by Northern hybridization against poly(A)(+)RNA prepared from 16 normal tissues. The widespread expression of RNase 1 was observed in almost all of the tissues. RNase 4 and angiogenin showed a similar distribution of expression abundantly present in the liver. This suggested the identity of the cell types producing these two molecules. However, no relativity appeared to be present between the vascularization of the tissues and the angiogenin expression. A narrow range of expression of the eosinophil-derived neurotoxin gene was observed. This localization seems related to the phagocytic cells in the tissues. The undetectable level of the eosinophil cationic protein mRNA in normal tissues suggests that the differentiation of eosinophils, triggered by inflammation and/or atopy, is required. The expression of RNase inhibitor was found to be ubiquitous. The regulatory function of inhibitor against RNases in the cell should be considered in studying the physiological significance of the pancreatic-type RNase family.

DOI： 10.1089/dna.1997.16.413

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

Human pancreatic ribonuclease I (hRNase 1) in the mature form has been produced in E.coli using T7 expression system. The recombinant hRNase 1 protein was solubilized from the inclusion bodies, refolded in glutathione redox system, and purified through chromatographic procedures by utilizing cation-exchange and reversed-phase columns. The ribonucleolytic activity of recombinant hRNase 1 was examined on yeast RNA and cytidylyl-3',5'-adenosine revealing the distinctive ribonucleolytic activity. The activity was perfectly inhibited by human placental RNase inhibitor. Truncation of 7 amino acid residues in the amino-terminal sequence resulted in much reduction in ribonucleolytic activity and in affinity to human placental RNase inhibitor with the disintegration of secondary structures of the protein observed by circular dichroism spectra. The present study has revealed the important contribution of the amino-terminal sequence of hRNase I to the characteristics of the protein. (C) 1995 Academic Press, Inc.

DOI： 10.1006/bbrc.1995.2638

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Language：English   Publisher：ELSEVIER SCIENCE BV  

A cDNA coding for human ribonuclease 4 was isolated from a pancreas cDNA library and sequenced. This cDNA (996 bp) includes an entire open reading frame encoding mature protein (119 aa) following signal peptide (28 aa). Expression of mature protein in Escherichia coli showed an apparent molecular mass of about 16 kDa, which was slightly lower than the mature form of human RNase 1, in SDS-PAGE.

DOI： 10.1016/0167-4781(95)00040-N

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A cDNA coding for human pancreatic ribonuclease was isolated from a pancreas cDNA library and sequenced. This cDNA (1620 bp) includes an entire open reading frame encoding mature protein (128 aa) following a signal peptide (28 aa) as well as 5'- and 3'-untranslated regions.

DOI： 10.1016/0167-4781(94)90208-9

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Presentation type：Poster presentation  

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Presentation type：Symposium, workshop panel (nominated)  

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Presentation type：Symposium, workshop panel (nominated)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (invited, special)  

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Presentation type：Oral presentation (general)  

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Presentation type：Poster presentation  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Symposium, workshop panel (public)  

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Presentation type：Symposium, workshop panel (nominated)  

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Presentation type：Oral presentation (invited, special)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Applicant：国立大学法人 岡山大学

Application no：特願2012-507073  Date applied：2011.3.24

Publication no：WO2011-118731  Date published：2011929

Patent/Registration no：特許5713006  Date registered：2015.3.20 

特許第5713006号

J-GLOBAL

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Applicant：株式会社日本触媒

Application no：特願2007-046025  Date applied：2007.2.26

Announcement no：特開2008-115150  Date announced：2008.5.22

Patent/Registration no：特許第5097412号  Date registered：2012.9.28 

特許第５０９７４１２号

J-GLOBAL

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Applicant：株式会社日本触媒

Application no：特願2005-356631  Date applied：2005.12.9

Announcement no：特開2007-159429  Date announced：2007.6.28

J-GLOBAL

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Applicant：株式会社日本触媒

Application no：特願2005-147250  Date applied：2005.5.19

Announcement no：特開2006-006316  Date announced：2006.1.12

Patent/Registration no：特許4885476  Date registered：2011.12.16  Date issued：2011.12.16

特許第４８８５４７６号

J-GLOBAL

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Applicant：株式会社日本触媒

Application no：特願2003-356571  Date applied：2003.10.16

Announcement no：特開2005-120017  Date announced：2005.5.12

J-GLOBAL

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Applicant：株式会社日本触媒

Application no：特願2003-149359  Date applied：2003.5.27

Announcement no：特開2004-350526  Date announced：2004.12.16

J-GLOBAL

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Applicant：株式会社日本触媒

Application no：特願2002-342486  Date applied：2002.11.26

Announcement no：特開2004-049214  Date announced：2004.2.19

J-GLOBAL

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Applicant：株式会社日本触媒

Application no：特願2002-287280  Date applied：2002.9.30

Announcement no：特開2004-121041  Date announced：2004.4.22

Patent/Registration no：特許第3996028号  Date registered：2007.8.10 

J-GLOBAL

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Country：Japan

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