Oxford University Press (OUP) Acta Medica Okayama 2730-6151 5 1 2025 Proliferation of a bloom-forming phytoplankton via uptake of polyphosphate-accumulating bacteria under phosphate-limiting conditions ycaf192 EN Seiya Fukuyama Institute of Plant Science and Resources, Okayama University Fumiko Usami Institute of Plant Science and Resources, Okayama University Ryuichi Hirota Graduate School of Integrated Sciences for Life, Hiroshima University Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Shizuka Ohara Graduate School of Integrated Sciences for Life, Hiroshima University Ken Kondo Research Institute of Environment, Agriculture and Fisheries , Osaka Prefecture Yuki Gomibuchi Department of Physics and Information Technology, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology Takuo Yasunaga Department of Physics and Information Technology, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology Toshimitsu Onduka Hatsukaichi Branch, Fisheries Technology Institute , Fisheries Research and Education Agency Akio Kuroda Graduate School of Integrated Sciences for Life, Hiroshima University Kazuhiko Koike Graduate School of Integrated Sciences for Life, Hiroshima University Shoko Ueki Institute of Plant Science and Resources, Okayama University Harmful algal blooms negatively impact the ecosystem and fisheries in affected areas. Eutrophication is a major factor contributing to bloom occurrence, and phosphorus is particularly important in limiting the growth of bloom-forming algae. Although algae efficiently utilize orthophosphate (Pi) as a phosphorous source over other molecular forms, Pi is often limited in the marine environment. While uptake and utilization of soluble inorganic and organic phosphorous by bloom-forming algae has been extensively studied, the details of geochemical and biological phosphorous cycling remain to be elucidated. Here, we report for the first time that the bloom-forming alga Heterosigma akashiwo can phagocytose bacteria and grow under phosphate-depleted conditions. The addition of Vibrio comitans to Pi-depleted H. akashiwo enabled the alga propagate to high cell densities, whereas other bacterial strains had only a minor effect. Importantly, V. comitans accumulates polyphosphate—a linear polymer of Pi—at high levels. The extent of algal proliferation induced by the addition of Vibrio species and polyphosphate-accumulating Escherichia coli correlated strongly with their polyphosphate content, indicating that bacterial polyphosphate served as an alternative PO43− source for H. akashiwo. The direct uptake of polyphosphate-accumulating bacteria through algal phagocytosis may represent a novel biological phosphorous-cycling pathway in marine ecosystems. The role of polyphosphate-accumulating marine bacteria as a hidden phosphorous source required for bloom formation warrants further investigation. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 1422-0067 26 17 2025 Augmentation of the Benzyl Isothiocyanate-Induced Antiproliferation by NBDHEX in the HCT-116 Human Colorectal Cancer Cell Line 8145 EN Ruitong Sun Graduate School of Environmental and Life Science, Okayama University Aina Yano Graduate School of Environmental and Life Science, Okayama University Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Shintaro Munemasa Graduate School of Environmental and Life Science, Okayama University Yoshiyuki Murata Graduate School of Environmental and Life Science, Okayama University Toshiyuki Nakamura Graduate School of Environmental and Life Science, Okayama University Yoshimasa Nakamura Graduate School of Environmental and Life Science, Okayama University Increased drug metabolism and elimination are prominent mechanisms mediating multidrug resistance (MDR) to not only chemotherapy drugs but also anti-cancer natural products, such as benzyl isothiocyanate (BITC). To evaluate the possibility of combined utilization of a certain compound to overcome this resistance, we focused on glutathione S-transferase (GST)-dependent metabolism of BITC. The pharmacological treatment of a pi-class GST-selective inhibitor, 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX), significantly increased BITC-induced toxicity in human colorectal cancer HCT-116 cells. However, NBDHEX unexpectedly increased the level of the BITC–glutathione (GSH) conjugate as well as BITC-modified proteins, suggesting that NBDHEX might increase BITC-modified protein accumulation by inhibiting BITC–GSH excretion instead of inhibiting GST. Furthermore, NBDHEX significantly potentiated BITC-induced apoptosis with the enhanced activation of apoptosis-related pathways, such as c-Jun N-terminal kinase and caspase-3 pathways. These results suggested that combination treatment with NBDHEX may be an effective way to overcome MDR with drug efflux and thus induce the biological activity of BITC at lower doses. No potential conflict of interest relevant to this article was reported.

ScienceMatters Acta Medica Okayama 2297-8240 2 9 2016 S-nitrosylation of laforin inhibits its phosphatase activity and is implicated in Lafora disease EN Rikako Toyota Yasuko Honjo Graduate School of Natural Science and Technology, Okayama University; Research Institute for Radiation Biology and Medicine, Hiroshima University Risa Imajo Graduate School of Natural Science and Technology, Okayama University; Research Institute for Radiation Biology and Medicine, Hiroshima University Ayano Satoh Graduate School of Natural Science and Technology, Okayama University; Research Institute for Radiation Biology and Medicine, Hiroshima University Recently, the relation between S-nitrosylation by nitric oxide (NO), which is over�produced under pathological conditions and neurodegenerative diseases, includingAlzheimer’s and Parkinson’s diseases, has become a focus of attention. Although mostcases of Parkinson’s disease are known to be caused by mutations in the Parkin gene, arecent finding has indicated that S-nitrosylation of Parkin affects its enzymatic activityand leads to the Parkinsonian phenotype. Therefore, it is important to understand thefunction of S-nitrosylated proteins in the pathogenesis of neurodegenerative diseases.Lafora disease (LD, OMIM 254780) is a neurodegenerative disease characterized by theaccumulation of insoluble glucans called Lafora bodies (LBs). LD is caused by mutationsin genes that encode the glucan phosphatase, Laforin, or the E3 ubiquitin ligase, Malin.In this study, we hypothesized that LD may be caused by S-nitrosylation of Laforin,which is similar to the finding that Parkinson’s disease is caused by S-nitrosylation ofParkin. To test this hypothesis, we first determined whether Laforin was S-nitrosylatedusing a biotin switch assay, and compared the three main functions of unmodified andS-nitrosylated Laforin, namely glucan- and Malin-binding activity and phosphataseactivity. Furthermore, we examined whether the numbers of LBs were changed byNO in the cells expressing wild-type Laforin. Here, we report for the first time thatS-nitrosylation of Laforin inhibited its phosphatase activity and that LB formation wasincreased by an NO donor. Our results suggest a possible hypothesis for LD pathogenesis; that is, the decrease in phosphatase activity of Laforin by S-nitrosylation leads toincreased LB formation. Therefore, LD may be caused not only by mutations in theLaforin or Malin genes, but also by the S-nitrosylation of Laforin. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0278-6915 197 2025 Fraglide-1 from traditional Chinese aromatic vinegar: A natural AhR antagonist for atopic dermatitis 115301 EN Kosuke Kato Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Miki Akamatsu Graduate School of Environmental and Life Science, Okayama University Saya Kakimaru Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mayuko Koreishi Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masahiro Takagi School of Materials Science, Japan Advanced Institute of Science and Technology Masahiro Miyashita Graduate School of Agriculture, Kyoto University Yoshiyuki Murata Graduate School of Environmental and Life Science, Okayama University Yoshimasa Nakamura Graduate School of Environmental and Life Science, Okayama University Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yoshio Tsujino Graduate School of Science, Technology and Innovation, Kobe University Traditional Chinese Zhenjiang aromatic vinegar (Kozu) contains Fraglide-1 (FG1), a bioactive lactone with demonstrated peroxisome proliferator-activated receptor gamma (PPARγ) agonist and antioxidant activities. This study explored FG1's novel ability to antagonize the aryl hydrocarbon receptor (AhR) signaling pathway, which regulates artemin expression and contributes to itching and inflammation in atopic dermatitis. Through molecular docking simulations and cell-based assays in human keratinocytes, we demonstrated FG1's potent antagonistic activity against AhR signaling. FG1 effectively suppressed FICZ-induced inflammatory responses, including artemin expression, with potency (half maximal inhibitory concentration, IC50 = 5.1 μM) comparable to the synthetic antagonist StemRegenin 1 (SR1) while demonstrating a superior safety profile (median lethal concentration, LC50 > 100 μM vs. 27.5 μM for SR1). These findings expand our understanding of bioactive compounds from traditional fermented foods and their regulatory effects on AhR signaling, providing a foundation for future studies on FG1's role in modulating skin inflammation. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 1756-4646 125 2025 Resveratrol, a food-derived polyphenol, promotes Melanosomal degradation in skin fibroblasts through coordinated activation of autophagy, lysosomal, and antioxidant pathways 106672 EN Saki Okamoto Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Saya Kakimaru Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mayuko Koreishi Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mika Sakamoto National Institute of Genetics, ROIS Yoshimasa Nakamura Graduate School of Environmental and Life Science, Okayama University Hideya Ando Department of Applied Chemistry and Biotechnology, Okayama University of Science Yoshio Tsujino Graduate School of Science, Technology, and Innovation, Kobe University Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Resveratrol, a polyphenol found in grapes and peanuts, is known for diverse biological activities, yet its effects on dermal hyperpigmentation (so-called dark spots) remain unexplored. We investigated resveratrol's ability to enhance melanosomal degradation in human dermal fibroblasts. At concentrations of 25-50 mu M, resveratrol increased autophagy as measured by microtubule-associated protein 1A/1B-light chain 3 (LC3)-II/LC3-I ratio and enhanced lysosomal activity as assessed by a lysosomal activity reporter system. RNA sequencing revealed upregulation of lysosomal and autophagy-related genes, including cathepsins. Furthermore, reporter assays showed resveratrol's activation of antioxidant response via nuclear factor erythroid 2-related factor 2 (NRF2)mediated, leading to upregulation of transcription factor EB/transcription factor E3 (TFEB/TFE3), master regulators of lysosomal function. In fibroblasts pre-loaded with melanosomes, resveratrol reduced melanosome content compared to control by day 3. The findings reveal the activation of interconnected autophagy, lysosomal, and antioxidant pathways by resveratrol, suggesting potential applications in functional foods targeting dermal hyperpigmentation. No potential conflict of interest relevant to this article was reported.

Nature Portfolio Acta Medica Okayama 2045-2322 14 1 2024 Development of a novel AAK1 inhibitor via Kinobeads-based screening 6723 EN Akari Yoshida Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Satomi Ohtsuka Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Fumiya Matsumoto Department of Science Education, Graduate School of Education, Okayama University Tomoyuki Miyagawa Department of Science Education, Graduate School of Education, Okayama University Rei Okino Department of Science Education, Graduate School of Education, Okayama University Yumeya Ikeda Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Natsume Tada Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Akira Gotoh Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masaki Magari Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Naoya Hatano Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ryo Morishita CellFree Sciences Co. Ltd Ayano Satoh Organelle Systems Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yukinari Sunatsuki Graduate School of Natural Science and Technology, Okayama University Ulf J. Nilsson Department of Chemistry, Lund University Teruhiko Ishikawa Department of Science Education, Graduate School of Education, Okayama University Hiroshi Tokumitsu Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University A chemical proteomics approach using Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor-immobilized sepharose (TIM-063-Kinobeads) identified main targets such as CaMKK alpha/1 and beta/2, and potential off-target kinases, including AP2-associated protein kinase 1 (AAK1), as TIM-063 interactants. Because TIM-063 interacted with the AAK1 catalytic domain and inhibited its enzymatic activity moderately (IC50 = 8.51 mu M), we attempted to identify potential AAK1 inhibitors from TIM-063-derivatives and found a novel AAK1 inhibitor, TIM-098a (11-amino-2-hydroxy-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) which is more potent (IC50 = 0.24 mu M) than TIM-063 without any inhibitory activity against CaMKK isoforms and a relative AAK1-selectivity among the Numb-associated kinases family. TIM-098a could inhibit AAK1 activity in transfected cultured cells (IC50 = 0.87 mu M), indicating cell-membrane permeability of the compound. Overexpression of AAK1 in HeLa cells significantly reduced the number of early endosomes, which was blocked by treatment with 10 mu M TIM-098a. These results indicate TIM-063-Kinobeads-based chemical proteomics is efficient for identifying off-target kinases and re-evaluating the kinase inhibitor (TIM-063), leading to the successful development of a novel inhibitory compound (TIM-098a) for AAK1, which could be a molecular probe for AAK1. TIM-098a may be a promising lead compound for a more potent, selective and therapeutically useful AAK1 inhibitor. No potential conflict of interest relevant to this article was reported.

Public Library of Science Acta Medica Okayama 1932-6203 19 1 2024 Aromatic oil from lavender as an atopic dermatitis suppressant e0296408 EN Haruna Sato Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kosuke Kato Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mayuko Koreishi Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yoshimasa Nakamura Graduate School of Environmental and Life Science, Okayama University Yoshio Tsujino Graduate School of Science, Technology, and Innovation, Kobe University Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University In atopic dermatitis (AD), nerves are abnormally stretched near the surface of the skin, making it sensitive to itching. Expression of neurotrophic factor Artemin (ARTN) involved in such nerve stretching is induced by the xenobiotic response (XRE) to air pollutants and UV radiation products. Therefore, AD can be monitored by the XRE response. Previously, we established a human keratinocyte cell line stably expressing a NanoLuc reporter gene downstream of XRE. We found that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan metabolite and known inducer of the XRE, increased reporter and Artemin mRNA expression, indicating that FICZ-treated cells could be a model for AD. Lavender essential oil has been used in folk medicine to treat AD, but the scientific basis for its use is unclear. In the present study, we investigated the efficacy of lavender essential oil and its major components, linalyl acetate and linalool, to suppress AD and sensitize skin using the established AD model cell line, and keratinocyte and dendritic cell activation assays. Our results indicated that lavender essential oil from L. angustifolia and linalyl acetate exerted a strong AD inhibitory effect and almost no skin sensitization. Our model is useful in that it can circumvent the practice of using animal studies to evaluate AD medicines. No potential conflict of interest relevant to this article was reported.

Spandidos Publications Acta Medica Okayama 2049-9434 16 1 2021 Evaluation of skin sensitization based on interleukin‑2 promoter activation in Jurkat cells 3 EN Taichi Nagahata Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yoshio Tsujino Graduate School of Science, Technology and Innovation, Kobe University Eiji Takayama Department of Oral Biochemistry, Asahi University School of Dentistry Haruka Hikasa Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Skin sensitization is an allergic reaction caused by certain chemical substances, and is an important factor to be taken into consideration when evaluating the safety of numerous types of products. Although animal testing has long been used to evaluate skin sensitization, the recent trend to regulate such testing has led to the development and use of alternative methods. Skin sensitization reactions are summarized in the form of an adverse outcome pathway consisting of four key events (KE), including covalent binding to skin proteins (KE1), keratinocyte activation (KE2), and dendritic cell activation (KE3). Equivalent alternative methods have been developed for KE1 to KE3, but no valid alternative has yet been developed for the evaluation of KE4 and T‑cell activation. Current alternative methods rely on data from KE1 to KE3 to predict the effect of chemicals on skin sensitization. The addition of KE4 data is expected to improve the accuracy and reproducibility of such predictions. The aim of this study was to establish an assay to evaluate KE4 T‑cell activation to supplement data on skin sensitization related to KE4. To evaluate T‑cell activation, the Jurkat T‑cell line stably expressing luciferase downstream of the pro‑inflammatory cytokine interleukin‑2 promoter was used. After exposure to known skin sensitizing agents and control substances, luciferase activity measurements revealed that this assay was valid for evaluating skin sensitization. However, two skin sensitizers known to have immunosuppressive effects on T‑cells reacted negatively in this assay. The results revealed that this assay simultaneously allows for monitoring of the skin sensitization and immuno‑suppressiveness of chemical substances and supplements KE4 T‑cell activation data, and may thus contribute to reducing the use of animal experiments. No potential conflict of interest relevant to this article was reported.

Humana New York Acta Medica Okayama 2022 An Electron Tomographic Analysis of Giantin-Deficient Golgi Proposes a New Function of the Golgin Protein Family 235 246 EN Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mitsuko Hayashi-Nishino Institute of Scientific and Industrial Research, Osaka University Kunihiko Nishino Institute of Scientific and Industrial Research, Osaka University The Golgi apparatus is an organelle that mediates modifications, sorting, and transport of proteins and lipids. Golgins are a group of proteins with coiled-coil structures that localize to the Golgi and are thought to function as tethers to facilitate the docking of vesicles, Rab GTPases, and cytoskeleton components to the Golgi stack. Giantin is the longest golgin and has been thought to function as a tether for COPI vesicles along with other golgins, such as p115 and GM130. Contrary to our expectation that the loss of the tether will result in an increase in untethered COPI vesicles in the cytoplasm, our electron microscopy observations showed that the fenestrae normally present in Golgi cisternae were reduced upon Giantin knockdown. We also found that this structural change is accompanied by altered secretion of cargo proteins and cell surface glycosylation. These results indicate that there exists a correlation between Golgi structural changes caused by the loss of Giantin and Golgi function. Here, we describe electron tomography methods for the detection of structural changes in the Golgi. No potential conflict of interest relevant to this article was reported.

Public Library of Science Acta Medica Okayama 1932-6203 18 2 2023 Fucosyltransferase 8 (FUT8) and core fucose expression in oxidative stress response e0281516 EN Yuki Kyunai Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University Mika Sakamoto National Institute of Genetics, ROIS Mayuko Koreishi Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yoshio Tsujino Graduate School of Science, Technology, and Innovation, Kobe University Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University GlycoMaple is a new tool to predict glycan structures based on the expression levels of 950 genes encoding glycan biosynthesis-related enzymes and proteins using RNA-seq data. The antioxidant response, protecting cells from oxidative stress, has been focused on because its activation may relieve pathological conditions, such as neurodegenerative diseases. Genes involved in the antioxidant response are defined within the GO:0006979 category, including 441 human genes. Fifteen genes overlap between the glycan biosynthesis-related genes defined by GlycoMaple and the antioxidant response genes defined by GO:0006979, one of which is FUT8. 5-Hydroxy-4-phenyl-butenolide (5H4PB) extracted from Chinese aromatic vinegar induces the expression of a series of antioxidant response genes that protect cells from oxidative stress via activation of the nuclear factor erythroid 2-related factor 2-antioxidant response element pathway. Here, we show that FUT8 is upregulated in both our RNA-seq data set of 5H4PB-treated cells and publicly available RNA-seq data set of cells treated with another antioxidant, sulforaphane. Applying our RNA-seq data set to GlycoMaple led to a prediction of an increase in the core fucose of N-glycan that was confirmed by flow cytometry using a fucose-binding lectin. These results suggest that FUT8 and core fucose expression may increase upon the antioxidant response. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 23 19 2022 Immune State Conversion of the Mesenteric Lymph Node in a Mouse Breast Cancer Model 11035 EN Tsukasa Shigehiro Research Institute for Biomedical Sciences, Tokyo University of Science Maho Ueno Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University Mayumi Kijihira Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University Ryotaro Takahashi Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Chiho Umemura Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Eman A. Taha Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Chisaki Kurosaka Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University Megumi Asayama Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University Hiroshi Murakami Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yoshimasa Nakamura Graduate School of Environmental and Life Science, Okayama University Junichiro Futami Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Junko Masuda Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University 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-gamma 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-gamma 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)-beta-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. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0947-6539 28 37 2022 Design and Synthesis of Glycosylated Cholera Toxin B Subunit as a Tracer of Glycoprotein Trafficking in Organelles of Living Cells e202201253 EN Yuta Maki Department of Chemistry, Graduate School of Science, Osaka University Kazuki Kawata Department of Chemistry, Graduate School of Science, Osaka University Yanbo Liu Department of Chemistry, Graduate School of Science, Osaka University Kang‐Ying Goo Department of Chemistry, Graduate School of Science, Osaka University Ryo Okamoto Department of Chemistry, Graduate School of Science, Osaka University Yasuhiro Kajihara Department of Chemistry, Graduate School of Science, Osaka University Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Glycosylation of proteins is known to be essential for changing biological activity and stability of glycoproteins on the cell surfaces and in body fluids. Delivering of homogeneous glycoproteins into the endoplasmic reticulum (ER) and the Golgi apparatus would enable us to investigate the function of asparagine-linked (N-) glycans in the organelles. In this work, we designed and synthesized an intentionally glycosylated cholera toxin B-subunit (CTB) to be transported to the organelles of mammalian cells. The heptasaccharide, the intermediate structure of various complex-type N-glycans, was introduced to the CTB. The synthesized monomeric glycosyl-CTB successfully entered mammalian cells and was transported to the Golgi and the ER, suggesting the potential use of synthetic CTB to deliver and investigate the functions of homogeneous N-glycans in specific organelles of living cells. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 23 3 2022 A Major Intestinal Catabolite of Quercetin Glycosides, 3-Hydroxyphenylacetic Acid, Protects the Hepatocytes from the Acetaldehyde-Induced Cytotoxicity through the Enhancement of the Total Aldehyde Dehydrogenase Activity 1762 EN Yujia Liu School of Biological Engineering, Dalian Polytechnic University Takumi Myojin Graduate School of Environmental and Life Science, Okayama University Kexin Li Graduate School of Environmental and Life Science, Okayama University Ayuki Kurita Graduate School of Environmental and Life Science, Okayama University Masayuki Seto Graduate School of Environmental and Life Science, Okayama University Ayano Motoyama Graduate School of Environmental and Life Science, Okayama University Xiaoyang Liu Graduate School of Environmental and Life Science, Okayama University Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Shintaro Munemasa Graduate School of Environmental and Life Science, Okayama University Yoshiyuki Murata Graduate School of Environmental and Life Science, Okayama University Toshiyuki Nakamura Graduate School of Environmental and Life Science, Okayama University Yoshimasa Nakamura Graduate School of Environmental and Life Science, Okayama University Aldehyde dehydrogenases (ALDHs) are the major enzyme superfamily for the aldehyde metabolism. Since the ALDH polymorphism leads to the accumulation of acetaldehyde, we considered that the enhancement of the liver ALDH activity by certain food ingredients could help prevent alcohol-induced chronic diseases. Here, we evaluated the modulating effects of 3-hydroxyphenylacetic acid (OPAC), the major metabolite of quercetin glycosides, on the ALDH activity and acetaldehyde-induced cytotoxicity in the cultured cell models. OPAC significantly enhanced the total ALDH activity not only in mouse hepatoma Hepa1c1c7 cells, but also in human hepatoma HepG2 cells. OPAC significantly increased not only the nuclear level of aryl hydrocarbon receptor (AhR), but also the AhR-dependent reporter gene expression, though not the nuclear factor erythroid-2-related factor 2 (Nrf2)-dependent one. The pretreatment of OPAC at the concentration required for the ALDH upregulation completely inhibited the acetaldehyde-induced cytotoxicity. Silencing AhR impaired the resistant effect of OPAC against acetaldehyde. These results strongly suggested that OPAC protects the cells from the acetaldehyde-induced cytotoxicity, mainly through the AhR-dependent and Nrf2-independent enhancement of the total ALDH activity. Our findings suggest that OPAC has a protective potential in hepatocyte models and could offer a new preventive possibility of quercetin glycosides for targeting alcohol-induced chronic diseases. No potential conflict of interest relevant to this article was reported.

Springer Nature Acta Medica Okayama 1226-4601 24 1 2021 Synthesis and characterization of conductive flexible cellulose carbon nanohorn sheets for human tissue applications 18 EN Karthik Paneer Selvam Graduate School of Natural Science and Technology, Okayama University Taichi Nagahata Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kosuke Kato Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mayuko Koreishi Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Toshiyuki Nakamura Graduate School of Environmental and Life Science, Okayama University Yoshimasa Nakamura Graduate School of Environmental and Life Science, Okayama University Takeshi Nishikawa Graduate School of Natural Science and Technology, Okayama University Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yasuhiko Hayashi Graduate School of Natural Science and Technology, Okayama University Background<br> Conductive sheets of cellulose and carbon nanomaterials and its human skin applications are an interesting research aspect as they have potential for applications for skin compatibility. Hence it is needed to explore the effects and shed light on these applications.<br> Method<br> To fabricate wearable, portable, flexible, lightweight, inexpensive, and biocompatible composite materials, carbon nanohorns (CNHs) and hydroxyethylcellulose (HEC) were used as precursors to prepare CNH-HEC (Cnh-cel) composite sheets. Cnh-cel sheets were prepared with different loading concentrations of CNHs (10, 20 50,100mg) in 200mg cellulose. To fabricate the bio-compatible sheets, a pristine composite of CNHs and HEC was prepared without any pretreatment of the materials.<br> Results<br> The obtained sheets possess a conductivity of 1.83x10(-10)S/m and bio-compatible with human skin. Analysis for skin-compatibility was performed for Cnh-cel sheets by h-CLAT in vitro skin sensitization tests to evaluate the activation of THP-1 cells. It was found that THP-1 cells were not activated by Cnh-cel; hence Cnh-cel is a safe biomaterial for human skin. It was also found that the composite allowed only a maximum loading of 100mg to retain the consistent geometry of free-standing sheets of <100<mu>m thickness. Since CNHs have a unique arrangement of aggregates (dahlia structure), the composite is homogeneous, as verified by transmission electron microscopy (TEM) and, scanning electron microscopy (SEM), and other functional properties investigated by Raman spectroscopy, Fourier transform infrared spectroscopy (FT-IR), conductivity measurement, tensile strength measurement, and skin sensitization.<br> Conclusion<br> It can be concluded that cellulose and CNHs sheets are conductive and compatible to human skin applications. No potential conflict of interest relevant to this article was reported.

Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Acta Medica Okayama 2021 3D model of control Golgi EN Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University 10.18926/61965 No potential conflict of interest relevant to this article was reported.

Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Acta Medica Okayama 2021 3D model of Giantin deficient Golgi EN Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University 10.18926/61964 No potential conflict of interest relevant to this article was reported.

Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Acta Medica Okayama 2021 Cover slide rack EN Yuki M Kyunai Faculty of Engineering, Department of Applied Chemistry and Biotechnology, Okayama University Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University 10.18926/61943 No potential conflict of interest relevant to this article was reported.

Nature Acta Medica Okayama 2045-2322 10 1 2020 Development of an experimental method of systematically estimating protein expression limits in HEK293 cells 4798 EN Yoshihiro Mori Graduate School of Natural Science and Technology, Okayama University Yuki Yoshida Sony Computer Science Laboratories Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Hisao Moriya Research Core for Interdisciplinary Sciences, Okayama University Protein overexpression sometimes causes cellular defects, although the underlying mechanism is still unknown. A protein's expression limit, which triggers cellular defects, is a useful indication of the underlying mechanism. In this study, we developed an experimental method of estimating the expression limits of target proteins in the human embryonic kidney cell line HEK293 by measuring the proteins' expression levels in cells that survived after the high-copy introduction of plasmid DNA by which the proteins were expressed under a strong cytomegalovirus promoter. The expression limits of nonfluorescent target proteins were indirectly estimated by measuring the levels of green fluorescent protein (GFP) connected to the target proteins with the self-cleaving sequence P2A. The expression limit of a model GFP was similar to 5.0% of the total protein, and sustained GFP overexpression caused cell death. The expression limits of GFPs with mitochondria-targeting signals and endoplasmic reticulum localization signals were 1.6% and 0.38%, respectively. The expression limits of four proteins involved in vesicular trafficking were far lower compared to a red fluorescent protein. The protein expression limit estimation method developed will be valuable for defining toxic proteins and consequences of protein overexpression. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2072-6694 12 5 2020 Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles 1260 EN Eman A. Taha Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Chiharu Sogawa Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuka Okusha Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hotaka Kawai Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences May Wathone Oo Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Abdellatif Elseoudi Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yanyin Lu Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Nagatsuka Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Satoshi Kubota Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ayano Satoh Department of Medical Bioengineering, Okayama University Graduate School of Natural Science and Technology Kuniaki Okamoto Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takanori Eguchi Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences The tumor organoid (tumoroid) model in three-dimensional (3D) culture systems has been developed to reflect more closely the in vivo tumors than 2D-cultured tumor cells. Notably, extracellular vesicles (EVs) are efficiently collectible from the culture supernatant of gel-free tumoroids. Matrix metalloproteinase (MMP) 3 is a multi-functional factor playing crucial roles in tumor progression. However, roles of MMP3 within tumor growth and EVs have not unveiled. Here, we investigated the protumorigenic roles of MMP3 on integrities of tumoroids and EVs. We generated MMP3-knockout (KO) cells using the CRISPR/Cas9 system from rapidly metastatic LuM1 tumor cells. Moreover, we established fluorescent cell lines with palmitoylation signal-fused fluorescent proteins (tdTomato and enhanced GFP). Then we confirmed the exchange of EVs between cellular populations and tumoroids. LuM1-tumoroids released large EVs (200-1000 nm) and small EVs (50-200 nm) while the knockout of MMP3 resulted in the additional release of broken EVs from tumoroids. The loss of MMP3 led to a significant reduction in tumoroid size and the development of the necrotic area within tumoroids. MMP3 and CD9 (a category-1 EV marker tetraspanin protein) were significantly down-regulated in MMP3-KO cells and their EV fraction. Moreover, CD63, another member of the tetraspanin family, was significantly reduced only in the EVs fractions of the MMP3-KO cells compared to their counterpart. These weakened phenotypes of MMP3-KO were markedly rescued by the addition of MMP3-rich EVs or conditioned medium (CM) collected from LuM1-tumoroids, which caused a dramatic rise in the expression of MMP3, CD9, and Ki-67 (a marker of proliferating cells) in the MMP3-null/CD9-low tumoroids. Notably, MMP3 enriched in tumoroids-derived EVs and CM deeply penetrated recipient MMP3-KO tumoroids, resulting in a remarkable enlargement of solid tumoroids, while MMP3-null EVs did not. These data demonstrate that EVs can mediate molecular transfer of MMP3, resulting in increasing the proliferation and tumorigenesis, indicating crucial roles of MMP3 in tumor progression. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2072-6694 12 1 2019 Hematopoietic Cells Derived from Cancer Stem Cells Generated from Mouse Induced Pluripotent Stem Cells 82 EN Ghmkin Hassan Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Said M. Afify Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Neha Nair Graduate School of Natural Science and Technology, Okayama University Kazuki Kumon Graduate School of Natural Science and Technology, Okayama University Amira Osman Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Juan Du Graduate School of Natural Science and Technology, Okayama University Hager Mansour Hagar A. Abu Quora Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Hend M. Nawara Graduate School of Natural Science and Technology, Okayama University Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Maram H. Zahra Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Nobuhiro Okada Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Akimasa Seno Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masaharu Seno Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Cancer stem cells (CSCs) represent the subpopulation of cancer cells with the ability to differentiate into other cell phenotypes and initiated tumorigenesis. Previously, we reported generating CSCs from mouse induced pluripotent stem cells (miPSCs). Here, we investigated the ability of the CSCs to differentiate into hematopoietic cells. First, the primary cells were isolated from malignant tumors that were formed by the CSCs. Non-adherent cells (NACs) that arose from adherent cells were collected and their viability, as well as the morphology and expression of hematopoietic cell markers, were analyzed. Moreover, NACs were injected into the tail vein of busulfan conditioned Balb/c nude mice. Finally, CSCs were induced to differentiate to macrophages while using IL3 and SCF. The round nucleated NACs were found to be viable, positive for hematopoietic lineage markers and CD34, and expressed hematopoietic markers, just like homing to the bone marrow. When NACs were injected into mice, Wright-Giemsa staining showed that the number of white blood cells got higher than those in the control mice after four weeks. CSCs also showed the ability to differentiate toward macrophages. CSCs were demonstrated to have the potential to provide progenies with hematopoietic markers, morphology, and homing ability to the bone marrow, which could give new insight into the tumor microenvironment according to the plasticity of CSCs. No potential conflict of interest relevant to this article was reported.

Nature Publishing Group Acta Medica Okayama 2045-2322 9 2019 Yeast screening system reveals the inhibitory mechanism of cancer cell proliferation by benzyl isothiocyanate through down-regulation of Mis12 8866 EN Naomi Abe-Kanoh Graduate School of Environmental and Life Science, Okayama University Narumi Kunisue Graduate School of Environmental and Life Science, Okayama University Takumi Myojin Graduate School of Environmental and Life Science, Okayama University Ayako Chino Research Core for Interdisciplinary Sciences, Okayama University Shintaro Munemasa Graduate School of Environmental and Life Science, Okayama University Yoshiyuki Murata Graduate School of Environmental and Life Science, Okayama University Ayano Satoh Graduate School of Natural Science and Technology, Okayama University Hisao Moriya Research Core for Interdisciplinary Sciences, Okayama University Yoshimasa Nakamura Graduate School of Environmental and Life Science, Okayama University, Okayama Benzyl isothiocyanate (BITC) is a naturally-occurring isothiocyanate derived from cruciferous vegetables. BITC has been reported to inhibit the proliferation of various cancer cells, which is believed to be important for the inhibition of tumorigenesis. However, the detailed mechanisms of action remain unclear. In this study, we employed a budding yeast Saccharomyces cerevisiae as a model organism for screening. Twelve genes including MTW1 were identified as the overexpression suppressors for the antiproliferative effect of BITC using the genome-wide multi-copy plasmid collection for S. cerevisiae. Overexpression of the kinetochore protein Mtw1 counteracts the antiproliferative effect of BITC in yeast. The inhibitory effect of BITC on the proliferation of human colon cancer HCT-116 cells was consistently suppressed by the overexpression of Mis12, a human orthologue of Mtw1, and enhanced by the knockdown of Mis12. We also found that BITC increased the phosphorylated and ubiquitinated Mis12 level with consequent reduction of Mis12, suggesting that BITC degrades Mis12 through an ubiquitin-proteasome system. Furthermore, cell cycle analysis showed that the change in the Mis12 level affected the cell cycle distribution and the sensitivity to the BITC-induced apoptosis. These results provide evidence that BITC suppresses cell proliferation through the post-transcriptional regulation of the kinetochore protein Mis12. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2296634X 7 2019 The Golgin Protein Giantin Regulates Interconnections Between Golgi Stacks 160 EN Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mitsuko Hayashi-Nishino Institute of Scientific and Industrial Research, Osaka University Takuto Shakuno Graduate School of Natural Science and Technology, Okayama University Junko Masuda Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mayuko Koreishi Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Runa Murakami Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yoshimasa Nakamura raduate School of Environmental and Life Science, Okayama University Toshiyuki Nakamura raduate School of Environmental and Life Science, Okayama University Naomi Abe-Kanoh raduate School of Environmental and Life Science, Okayama University Yasuko Honjo esearch Institute for Radiation Biology and Medicine, Hiroshima University Joerg Malsam Center for Biochemistry (BZH), Heidelberg University Sidney Yu School of Biomedical Sciences, The Chinese University of Hong Kong Kunihiko ishino Institute of Scientific and Industrial Research, Osaka University Golgins are a family of Golgi-localized long coiled-coil proteins. The major golgin function is thought to be the tethering of vesicles, membranes, and cytoskeletal elements to the Golgi. We previously showed that knockdown of one of the longest golgins, Giantin, altered the glycosylation patterns of cell surfaces and the kinetics of cargo transport, suggesting that Giantin maintains correct glycosylation through slowing down transport within the Golgi. Giantin knockdown also altered the sizes and numbers of mini Golgi stacks generated by microtubule de-polymerization, suggesting that it maintains the independence of individual Golgi stacks. Therefore, it is presumed that Golgi stacks lose their independence following Giantin knockdown, allowing easier and possibly increased transport among stacks and abnormal glycosylation. To gain structural insights into the independence of Golgi stacks, we herein performed electron tomography and 3D modeling of Golgi stacks in Giantin knockdown cells. Compared with control cells, Giantin-knockdown cells had fewer and smaller fenestrae within each cisterna. This was supported by data showing that the diffusion rate of Golgi membrane proteins is faster in Giantin-knockdown Golgi, indicating that Giantin knockdown structurally and functionally increases connectivity among Golgi cisternae and stacks. This increased connectivity suggests that contrary to the cis-golgin tether model, Giantin instead inhibits the tether and fusion of nearby Golgi cisternae and stacks, resulting in transport difficulties between stacks that may enable the correct glycosylation of proteins and lipids passing through the Golgi. No potential conflict of interest relevant to this article was reported.

FCCA Acta Medica Okayama 0915-7352 25 146 2013 Transport within the Golgi: for the Study of Glycoprotein Movement 241 244 EN Ayano Satoh Yasuko Iwakiri No potential conflict of interest relevant to this article was reported.

Elsevier Science Inc Acta Medica Okayama 0002-9440 182 3 2013 Absence of Nogo-B (Reticulon 4B) Facilitates Hepatic Stellate Cell Apoptosis and Diminishes Hepatic Fibrosis in Mice 786 795 EN Keitaro Tashiro Ayano Satoh Teruo Utsumi Chuhan Chung Yasuko Iwakiri Nogo-B (reticulon 4B) accentuates hepatic fibrosis and cirrhosis, but the mechanism remains unclear. The aim of this study was to identify the role of Nogo-B in hepatic stellate cell (HSC) apoptosis in cirrhotic livers. Cirrhosis was generated by carbon tetrachloride inhalation in wild-type (WT) and Nogo-A/B knockout (Nogo-B KO) mice. HSCs were isolated from WT and Nogo-B KO mice and cultured for activation and transformation to myofibroblasts (MF-HSCs). Human hepatic stellate cells (LX2 cells) were used to assess apoptotic responses of activated HSCs after silencing or overexpressing Nogo-B. Livers from cirrhotic Nogo-B KO mice showed significantly reduced fibrosis (P < 0.05) compared with WT mice. Apoptotic cells were more prominent in fibrotic areas of cirrhotic Nogo-B KO livers. Nogo-B KO MF-HSCs showed significantly increased Levels of apoptotic markers, cleaved poly (ADP-ribose) polymerase, and caspase-3 and -8 (P < 0.05) compared with WT MF-HSCs in response to staurosporine. Treatment with tunicamycin, an endoplasmic reticulum stress inducer, increased cleaved caspase-3 and -8 levels in Nogo-B KO MF-HSCs compared with WT MF-HSCs (P < 0.01). In LX2 cells, Nogo-B knockdown enhanced apoptosis in response to staurosporine, whereas Nogo-B overexpression inhibited apoptosis. The absence of Nogo-B enhances apoptosis of HSCs in experimental cirrhosis. Selective blockade of Nogo-B in HSCs may represent a potential therapeutic strategy to mitigate liver fibrosis. (Am J Pathol 2013, 182: 786-795; http://dx.doLorg/10.1016Aajpath.2012.11.032) No potential conflict of interest relevant to this article was reported.

Public Library Science Acta Medica Okayama 1932-6203 8 3 2013 The Golgin Tether Giantin Regulates the Secretory Pathway by Controlling Stack Organization within Golgi Apparatus EN Mayuko Koreishi Thomas J. Gniadek Sidney Yu Junko Masuda Yasuko Honjo Ayano Satoh Golgins are coiled-coil proteins that play a key role in the regulation of Golgi architecture and function. Giantin, the largest golgin in mammals, forms a complex with p115, rab1, GM130, and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), thereby facilitating vesicle tethering and fusion processes around the Golgi apparatus. Treatment with the microtubule destabilizing drug nocodazole transforms the Golgi ribbon into individual Golgi stacks. Here we show that siRNA-mediated depletion of giantin resulted in more dispersed Golgi stacks after nocodazole treatment than by control treatment, without changing the average cisternal length. Furthermore, depletion of giantin caused an increase in cargo transport that was associated with altered cell surface protein glycosylation. Drosophila S2 cells are known to have dispersed Golgi stacks and no giantin homolog. The exogenous expression of mammalian giantin cDNA in S2 cells resulted in clustered Golgi stacks, similar to the Golgi ribbon in mammalian cells. These results suggest that the spatial organization of the Golgi ribbon is mediated by giantin, which also plays a role in cargo transport and sugar modifications. No potential conflict of interest relevant to this article was reported.

Public Library Science Acta Medica Okayama 1932-6203 8 1 2013 CK2 Phosphorylates Sec31 and Regulates ER-To-Golgi Trafficking EN Mayuko Koreishi Sidney Yu Mayumi Oda Yasuko Honjo Ayano Satoh Protein export from the endoplasmic reticulum (ER) is an initial and rate-limiting step of molecular trafficking and secretion. This is mediated by coat protein II (COPII)-coated vesicles, whose formation requires small GTPase Sar1 and 6 Sec proteins including Sec23 and Sec31. Sec31 is a component of the outer layer of COPII coat and has been identified as a phosphoprotein. The initiation and promotion of COPII vesicle formation is regulated by Sar1; however, the mechanism regulating the completion of COPII vesicle formation followed by vesicle release is largely unknown. Hypothesizing that the Sec31 phosphorylation may be such a mechanism, we identified phosphorylation sites in the middle linker region of Sec31. Sec31 phosphorylation appeared to decrease its association with ER membranes and Sec23. Non-phosphorylatable mutant of Sec31 stayed longer at ER exit sites and bound more strongly to Sec23. We also found that CK2 is one of the kinases responsible for Sec31 phosphorylation because CK2 knockdown decreased Sec31 phosphorylation, whereas CK2 overexpression increased Sec31 phosphorylation. Furthermore, CK2 knockdown increased affinity of Sec31 for Sec23 and inhibited ER-to-Golgi trafficking. These results suggest that Sec31 phosphorylation by CK2 controls the duration of COPII vesicle formation, which regulates ER-to-Golgi trafficking. No potential conflict of interest relevant to this article was reported.

PUBLIC LIBRARY SCIENCE Acta Medica Okayama 1932-6203 7 4 2012 A Model of Cancer Stem Cells Derived from Mouse Induced Pluripotent Stem Cells e33544 EN Ling Chen Tomonari Kasai Yueguang Li Yuh Sugii Guoliang Jin Masashi Okada Arun Vaidyanath Akifumi Mizutani Ayano Satoh Takayuki Kudoh Mary J. C. Hendrix David S Salomon Li Fu Masaharu Seno Cancer stem cells (CSCs) are capable of continuous proliferation and self-renewal and are proposed to play significant roles in oncogenesis, tumor growth, metastasis and cancer recurrence. CSCs are considered derived from normal stem cells affected by the tumor microenvironment although the mechanism of development is not clear yet. In 2007, Yamanaka's group succeeded in generating Nanog mouse induced pluripotent stem (miPS) cells, in which green fluorescent protein (GFP) has been inserted into the 5'-untranslated region of the Nanog gene. Usually, iPS cells, just like embryonic stem cells, are considered to be induced into progenitor cells, which differentiate into various normal phenotypes depending on the normal niche. We hypothesized that CSCs could be derived from Nanog miPS cells in the conditioned culture medium of cancer cell lines, which is a mimic of carcinoma microenvironment. As a result, the Nanog miPS cells treated with the conditioned medium of mouse Lewis lung carcinoma acquired characteristics of CSCs, in that they formed spheroids expressing GFP in suspension culture, and had a high tumorigenicity in Balb/c nude mice exhibiting angiogenesis in vivo. In addition, these iPS-derived CSCs had a capacity of self-renewal and expressed the marker genes, Nanog, Rex1, Eras, Esg1 and Cripto, associated with stem cell properties and an undifferentiated state. Thus we concluded that a model of CSCs was originally developed from miPS cells and proposed the conditioned culture medium of cancer cell lines might perform as niche for producing CSCs. The model of CSCs and the procedure of their establishment will help study the genetic alterations and the secreted factors in the tumor microenvironment which convert miPS cells to CSCs. Furthermore, the identification of potentially bona fide markers of CSCs, which will help the development of novel anti-cancer therapies, might be possible though the CSC model. No potential conflict of interest relevant to this article was reported.

Dove Medical Press Acta Medica Okayama 2230-3170 2011 1 2011 Detection of in situ cleaved p115 with the cut specific antibodies in rapid protein inactivation system by tobacco etch viral protease cleavage 5 11 EN Mayuko Koreishi The Research Core for Interdisciplinary Sciences (RCIS), Okayama University Yasuko Honjo The Research Core for Interdisciplinary Sciences (RCIS), Okayama University Ayano Satoh The Research Core for Interdisciplinary Sciences (RCIS), Okayama University Gene perturbation methods are commonly used in the study of gene and protein function. The authors of this paper recently developed a rapid protein inactivation technique utilizing tobacco etch virus (TEV)-derived protease. TEV protease recognizes the ENLYFQG (Glu-Asn-Leu-Tyr-Phe-Gln-Gly) amino acid sequence and specifically cleaves between Q and G. The authors developed antibodies that recognize the cleaved TEV (ENLYFQ) sequence, both in vitro and in vivo, but do not bind to uncleaved TEV (ENLYFQG). Using these antibodies, in situ protein cleavage was successfully detected. These antibodies used in combination with the TEV protease may be a useful complement to other perturbation methods. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1932-6203 7 1 2012 TRAPPC9 Mediates the Interaction between p150^Glued and COPII Vesicles at the Target Membrane EN Min Zong Ayano Satoh Mei Kuen Yu Ka Yu Siu Wing Yan Ng Hsiao Chang Chaｎ Julian A. Tanner Sidney Yu Background: The transport of endoplasmic reticulum (ER)-derived COPII vesicles toward the ER-Golgi intermediate compartment (ERGIC) requires cytoplasmic dynein and is dependent on microtubules. p150^Glued, a subunit of dynactin, has been implicated in the transport of COPII vesicles via its interaction with COPII coat components Sec23 and Sec24. However, whether and how COPII vesicle tether, TRAPP (Transport protein particle), plays a role in the interaction between COPII vesicles and microtubules is currently unknown. Principle Findings: We address the functional relationship between COPII tether TRAPP and dynactin. Overexpressed TRAPP subunits interfered with microtubule architecture by competing p150^Glued away from the MTOC. TRAPP subunit TRAPPC9 bound directly to p150^Glued via the same carboxyl terminal domain of p150^Glued that binds Sec23 and Sec24. TRAPPC9 also inhibited the interaction between p150^Glued and Sec23/Sec24 both in vitro and in vivo, suggesting that TRAPPC9 serves to uncouple p150^Glued from the COPII coat, and to relay the vesicle-dynactin interaction at the target membrane. Conclusions: These findings provide a new perspective on the function of TRAPP as an adaptor between the ERGIC membrane and dynactin. By preserving the connection between dynactin and the tethered and/or fused vesicles, TRAPP allows nascent ERGIC to continue the movement along the microtubules as they mature into the cis-Golgi. No potential conflict of interest relevant to this article was reported.

The American Society for Cell Biology Acta Medica Okayama 1059-1524 20 19 2009 mTrs130 Is a Component of a Mammalian TRAPPII Complex, a Rab1 GEF That Binds to COPI-coated Vesicles 4205 4215 EN Akinori Yamasaki Shekar Menon Sidney Yu Jemima Barrowman Timo Meerloo Viola Oorschot Judith Klumperman Ayano Satoh Susan Ferro-Novick The GTPase Rab1 regulates endoplasmic reticulum-Golgi and early Golgi traffic. The guanine nucleotide exchange factor (GEF) or factors that activate Rab1 at these stages of the secretory pathway are currently unknown. Trs130p is a subunit of the yeast TRAPPII (transport protein particle II) complex, a multisubunit tethering complex that is a GEF for the Rab1 homologue Ypt1p. Here, we show that mammalian Trs130 (mTrs130) is a component of an analogous TRAPP complex in mammalian cells, and we describe for the first time the role that this complex plays in membrane traffic. mTRAPPII is enriched on COPI (Coat Protein I)-coated vesicles and buds, but not Golgi cisternae, and it specifically activates Rab1. In addition, we find that mTRAPPII binds to γ1COP, a COPI coat adaptor subunit. The depletion of mTrs130 by short hairpin RNA leads to an increase of vesicles in the vicinity of the Golgi and the accumulation of cargo in an early Golgi compartment. We propose that mTRAPPII is a Rab1 GEF that tethers COPI-coated vesicles to early Golgi membranes. No potential conflict of interest relevant to this article was reported.