Publications by authors named "Ikuo Wada"

159 Publications

IZUMO family member 3, IZUMO3, is involved in male fertility through the acrosome formation.

Mol Reprod Dev 2021 Jul 10;88(7):479-481. Epub 2021 Jun 10.

Department of Cell Science, School of Medicine, Institute of Biomedical Sciences, Fukushima Medical University, Fukushima, Fukushima, Japan.

Many factors are involved in acrosome biogenesis in order for appropriate acrosome formation to occur. Here, we demonstrate that IZUMO family member 3, IZUMO3, plays an important role in acrosome biogenesis, as proven by gene disruption experiments. A loss of IZUMO3 in round spermatids affects acrosomal granule positioning due to lack of acrosomal granule contact with the inner acrosomal membrane, leading to the formation of grossly malformed spermatozoa associated with male subfertility. Thus, we suggest that mammalian spermiogenesis needs an elaborate acrosome biogenesis through IZUMO3 involvement.
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http://dx.doi.org/10.1002/mrd.23520DOI Listing
July 2021

Phosphorylation of human phospholipase A1 DDHD1 at newly identified phosphosites affects its subcellular localization.

J Biol Chem 2021 Jul 3;297(1):100851. Epub 2021 Jun 3.

Faculty of Pharma-Science, Teikyo University, Itabashi-Ku, Tokyo, Japan. Electronic address:

Phospholipase A1 (PLA1) hydrolyzes the fatty acids of glycerophospholipids, which are structural components of the cellular membrane. Genetic mutations in DDHD1, an intracellular PLA1, result in hereditary spastic paraplegia (HSP) in humans. However, the regulation of DDHD1 activity has not yet been elucidated in detail. In the present study, we examined the phosphorylation of DDHD1 and identified the responsible protein kinases. We performed MALDI-TOF MS/MS analysis and Phos-tag SDS-PAGE in alanine-substitution mutants in HEK293 cells and revealed multiple phosphorylation sites in human DDHD1, primarily Ser8, Ser11, Ser723, and Ser727. The treatment of cells with a protein phosphatase inhibitor induced the hyperphosphorylation of DDHD1, suggesting that multisite phosphorylation occurred not only at these major, but also at minor sites. Site-specific kinase-substrate prediction algorithms and in vitro kinase analyses indicated that cyclin-dependent kinase CDK1/cyclin A2 phosphorylated Ser8, Ser11, and Ser727 in DDHD1 with a preference for Ser11 and that CDK5/p35 also phosphorylated Ser11 and Ser727 with a preference for Ser11. In addition, casein kinase CK2α1 was found to phosphorylate Ser104, although this was not a major phosphorylation site in cultivated HEK293 cells. The evaluation of the effects of phosphorylation revealed that the phosphorylation mimic mutants S11/727E exhibit only 20% reduction in PLA1 activity. However, the phosphorylation mimics were mainly localized to focal adhesions, whereas the phosphorylation-resistant mutants S11/727A were not. This suggested that phosphorylation alters the subcellular localization of DDHD1 without greatly affecting its PLA1 activity.
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http://dx.doi.org/10.1016/j.jbc.2021.100851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8234217PMC
July 2021

Evolutionarily conserved sperm factors, DCST1 and DCST2, are required for gamete fusion.

Elife 2021 04 19;10. Epub 2021 Apr 19.

Department of Cell Science, Institute of Biomedical Sciences, School of Medicine, Fukushima Medical University, Fukushima, Japan.

To trigger gamete fusion, spermatozoa need to activate the molecular machinery in which sperm IZUMO1 and oocyte JUNO (IZUMO1R) interaction plays a critical role in mammals. Although a set of factors involved in this process has recently been identified, no common factor that can function in both vertebrates and invertebrates has yet been reported. Here, we first demonstrate that the evolutionarily conserved factors dendrocyte expressed seven transmembrane protein domain-containing 1 (DCST1) and dendrocyte expressed seven transmembrane protein domain-containing 2 (DCST2) are essential for sperm-egg fusion in mice, as proven by gene disruption and complementation experiments. We also found that the protein stability of another gamete fusion-related sperm factor, SPACA6, is differently regulated by DCST1/2 and IZUMO1. Thus, we suggest that spermatozoa ensure proper fertilization in mammals by integrating various molecular pathways, including an evolutionarily conserved system that has developed as a result of nearly one billion years of evolution.
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http://dx.doi.org/10.7554/eLife.66313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8055269PMC
April 2021

SEL1L degradation intermediates stimulate cytosolic aggregation of polyglutamine-expanded protein.

FEBS J 2021 Feb 11. Epub 2021 Feb 11.

Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Japan.

Misfolded proteins in the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD). In mammalian cells, the HRD1-SEL1L membrane ubiquitin ligase complex plays a central role in this process. However, SEL1L is inherently unstable, and excess SEL1L is also degraded by ERAD. Accordingly, when proteasome activity is inhibited, multiple degradation intermediates of SEL1L appear in the cytosol. In this study, we searched for factors that inhibit SEL1L degradation and identified OS-9 and XTP3-B, two ER lectins that regulate glycoprotein ERAD. SEL1L degradation was characterized by a ladder of degradation products, and the C-terminal Pro-rich region of SEL1L was responsible for generation of this pattern. In the cytosol, these degradation intermediates stimulated aggregation of polyglutamine-expanded Huntingtin protein (Htt-polyQ-GFP) by interacting with aggregation-prone proteins, including Htt-polyQ-GFP. Collectively, our findings indicate that peptide fragments of ER proteins generated during ERAD may affect protein aggregation in the cytosol, revealing the interconnection of protein homeostasis across subcellular compartments.
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http://dx.doi.org/10.1111/febs.15761DOI Listing
February 2021

Integrative immunogenomic analysis of gastric cancer dictates novel immunological classification and the functional status of tumor-infiltrating cells.

Clin Transl Immunology 2020 17;9(10):e1194. Epub 2020 Oct 17.

Department of Immunotherapeutics The University of Tokyo Hospital Tokyo Japan.

Objectives: A better understanding of antitumor immunity will help predict the prognosis of gastric cancer patients and tailor the appropriate therapies in each patient. Therefore, we propose a novel immunological classification of gastric cancer.

Methods: We performed whole-exome sequencing (WES), RNA-Seq and flow cytometry in 29 gastric cancer patients who received surgery. The TCGA data set of 323 gastric cancer patients and RNA-Seq data of 45 patients who received pembrolizumab (Kim . 2018; : 1449-1458) were also analysed.

Results: Immunogram analysis of cancer-immunity interaction of gastric cancer revealed immune signatures of four main types, designated Hot1, Hot2, Intermediate and Cold. Immunologically hot tumors displayed a dysfunctional T-cell signature, while cold tumors had an exclusion signature. tumor-infiltrating lymphocyte analysis documented T-cell dysfunction with the expression of checkpoint molecules and impaired cytokine production. The T-cell function was more profoundly damaged in Hot1 than Hot2 tumors. Patients in Hot2 subtypes had better survival in our cohort and TCGA cohort. Although these immunological subtypes overlapped to some degree with the molecular subtypes in the TCGA, intratumoral immune responses cannot be predicted solely based on histological or molecular subtyping of gastric cancer. Molecular and immunological classifications complement each other to predict the responses to anti-PD-1 therapy and have the potential to be a biomarker for the treatment of gastric cancer.

Conclusion: The immunological classification of gastric cancer resulted in four subtypes. Hot tumors were further divided into two subtypes, between which the functional status of T cells was different.
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http://dx.doi.org/10.1002/cti2.1194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568758PMC
October 2020

Unveiling a novel function of CD9 in surface compartmentalization of oocytes.

Development 2020 08 14;147(15). Epub 2020 Aug 14.

Department of Cell Science, Institute of Biomedical Sciences, School of Medicine, Fukushima Medical University, 1 Hikarigaoka, Fukushima City, Fukushima 960-1295, Japan.

Gamete fusion is an indispensable process for bearing offspring. In mammals, sperm IZUMO1-oocyte JUNO recognition essentially carries out the primary step of this process. In oocytes, CD9 is also known to play a crucial role in gamete fusion. In particular, microvilli biogenesis through CD9 involvement appears to be a key event for successful gamete fusion, because CD9-disrupted oocytes produce short and sparse microvillous structures, resulting in almost no fusion ability with spermatozoa. In order to determine how CD9 and JUNO cooperate in gamete fusion, we analyzed the molecular profiles of each molecule in CD9- and JUNO-disrupted oocytes. Consequently, we found that CD9 is crucial for the exclusion of GPI-anchored proteins, such as JUNO and CD55, from the cortical actin cap region, suggesting strict molecular organization of the unique surface of this region. Through distinct surface compartmentalization due to CD9 governing, GPI-anchored proteins are confined to the appropriate fusion site of the oocyte.
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http://dx.doi.org/10.1242/dev.189985DOI Listing
August 2020

Visualization of Procollagen IV Reveals ER-to-Golgi Transport by ERGIC-independent Carriers.

Cell Struct Funct 2020 Jul 18;45(2):107-119. Epub 2020 Jun 18.

Laboratory of Molecular and Cellular Biology, Institute for Frontier Life and Medical Sciences, Kyoto University.

Collagen is the most abundant protein in animal tissues and is critical for their proper organization. Nascent procollagens in the endoplasmic reticulum (ER) are considered too large to be loaded into coat protein complex II (COPII) vesicles, which have a diameter of 60-80 nm, for exit from the ER and transport to the Golgi complex. To study the transport mechanism of procollagen IV, which generates basement membranes, we introduced a cysteine-free GFP tag at the N-terminus of the triple helical region of the α1(IV) chain (cfSGFP2-col4a1), and examined the dynamics of this protein in HT-1080 cells, which produce endogenous collagen IV. cfSGFP2-col4a1 was transported from the ER to the Golgi by vesicles, which were a similar size as small cargo carriers. However, mCherry-ERGIC53 was recruited to α-antitrypsin-containing vesicles, but not to cfSGFP2-col4a1-containing vesicles. Knockdown analysis revealed that Sar1 and SLY1/SCFD1 were required for transport of cfSGFP2-col4a1. TANGO1, CUL3, and KLHL12 were not necessary for the ER-to-Golgi trafficking of procollagen IV. Our data suggest that procollagen IV is exported from the ER via an enlarged COPII coat carrier and is transported to the Golgi by unique transport vesicles without recruitment of ER-Golgi intermediate compartment membranes.Key words: collagen, procollagen IV, endoplasmic reticulum, ER-to-Golgi transport, ERGIC.
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http://dx.doi.org/10.1247/csf.20025DOI Listing
July 2020

Evaluation of the Cytotoxicity of Various Hand Disinfectants and Ozonated Water to Human Keratinocytes in a Cultured Epidermal Model.

Adv Skin Wound Care 2020 Jun;33(6):313-318

At the Fukushima Medical University, Fukushima, Japan, Jun Kashiwazaki, DDS, is Research Scientist, Department of Infection Control; Kiwamu Nakamura, MD, is Associate Professor, Department of Infection Control; Yasuka Hara, MD, is Research Scientist, Department of Infection Control; Rie Harada, MMT, is Research Scientist, Department of Infection Control; Ikuo Wada, PhD, is Professor, Department of Cell Science, Institute of Biomedical Sciences; and Keiji Kanemitsu, MD, is Professor, Department of Infection Control. The authors have disclosed no financial relationships related to this article. Submitted April 10, 2019; accepted July 9, 2019.

Objective: To evaluate the cytotoxicity of various hand disinfectants and ozonated water to human keratinocytes using a cultured epidermal model.

Design: Using a test protocol from the Organization for Economic Co-operation and Development, investigators applied hand disinfectants containing either 83% ethanol, 0.2% benzalkonium chloride, 0.5% povidone-iodine, 1% chlorhexidine, 1% chlorhexidine ethanol, or ozonated water to a cultured human epidermal model. Surface morphology and histologic changes were evaluated by scanning electron microscopy and hematoxylin-eosin staining.

Main Outcome Measures: Production of inflammatory cytokine interleukin 1α by keratinocytes and cell death rate.

Main Results: Electron microscopic analysis revealed the creation of small holes on the stratum corneum, and hematoxylin-eosin staining revealed perinuclear vacuolation of keratinocytes and cells with a condensed nucleus. Interleukin 1α was detected in the culture supernatants. More than 80% of keratinocytes did not survive after a 15-minute application of disinfectants. However, no significant damage was detected with ozonated water.

Conclusions: Ozonated water did far less damage to keratinocytes than the tested disinfectants. Although the ability of ozonated water to disinfect hands of medical staff members requires further study, it might serve as an alternative with minimum cytotoxicity.
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http://dx.doi.org/10.1097/01.ASW.0000658592.51430.eaDOI Listing
June 2020

Integral role of receptor for advanced glycation end products (RAGE) in nondiabetic atherosclerosis.

Fukushima J Med Sci 2019 ;65(3):109-121

Department of Cardiology and Hematology, Fukushima Medical University.

An advanced glycation end products (AGE)/a receptor for AGE (RAGE) axis plays a central role in the pathogenesis of diabetic vascular remodeling. This study was conducted to clarify the role of RAGE in nondiabetic atherosclerosis. We used the aortic and coronary atherosclerotic lesions of Watanabe heritable hyperlipidemic (WHHL) rabbits prone to myocardial infarction (WHHLMI) at 1 to 14 months. Immunohistochemistry demonstrated the significant expression of RAGE as early as at 1 month with the stronger expression at 3 and 7 months, which was remarkably diminished at 14 months. RAGE expression was concordant with AGE accumulation. The major original sources of RAGE expression were macrophages and smooth muscle cells in addition to endothelial cells, and RAGE expression was distributed in the areas of phospholipid products, a component of oxidized LDL and nitrotyrosine. The concentrations of serum AGE did not alter significantly with aging. These findings suggested the expression of RAGE was induced by hyperlipidemia and oxidative stress independent of diabetes in WHHLMI rabbits. Additionally, our in vitro study showed that silencing of RAGE tended to attenuate oxidized-LDL-triggered PAI-1 expression in human cultured macrophages, as well as oxidized-LDL-induced tissue factor expression in peritoneal macrophages, suggesting a possible role of RAGE in prothrombogenic molecular regulation. In conclusion, the present study provides in vivo evidence that RAGE plays an integral role in the initiation and progression of nondiabetic atherosclerosis, suggesting that RAGE may be a novel target for treating not only diabetic but also nondiabetic vascular complications.
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http://dx.doi.org/10.5387/fms.2019-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012590PMC
May 2020

Radiographic analysis of subclinical appearances of the hip joint among patients with labral tears.

J Orthop Surg Res 2019 Nov 14;14(1):369. Epub 2019 Nov 14.

Department of Orthopaedic Surgery, Nagoya City University Graduate School of Medical Sciences, 1, Kawasumi, Mizuho-cho, Mizuho-ku Nagoya, Aichi, 467-8601, Japan.

Objective: Labral tears can be complicated by hip diseases, including osteoarthritis or femoral acetabular impingement. To accurately plan hip arthroscopy or subsequent conversion to total hip arthroplasty, the presence of bony abnormalities in the hip joint must be evaluated. This study aimed to elucidate the utility of multiplanar reconstruction computed tomography (mCT) for the detection of subclinical coincidence of osteoarthritis or femoral acetabular impingement with a labrum tear.

Materials And Methods: We retrospectively analysed 34 patients (36 hips) with labrum tears without apparent osteoarthritis or hip dysplasia from 2012 to 2015. The joint spaces were calculated using radiographs or mCT, and the detection rates of degenerative cyst and herniation pit were compared.

Results: Narrow joint spaces (< 2 mm) were more clearly detected in mCT (p < 0.05, chi-square analysis) than in radiographs. The detection rate of cysts in the acetabulum was 8.3% using radiographs and 36.1% using mCT (p < 0.001, chi-square analysis). Additionally, the detection of herniation pit was 8.3% and 25.0% using radiographs and mCT, respectively (p = 0.053, chi-square analysis).

Conclusion: We performed the radiographic analysis of patients with labral tears using radiographs and mCT. The mCT allowed for fine detection of narrow joint spaces and subtle subclinical appearances. The results of this study may provide surgeons with more appropriate strategies for the treatment of labral tears.
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http://dx.doi.org/10.1186/s13018-019-1435-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854768PMC
November 2019

SDF2-like protein 1 (SDF2L1) regulates the endoplasmic reticulum localization and chaperone activity of ERdj3 protein.

J Biol Chem 2019 12 17;294(50):19335-19348. Epub 2019 Oct 17.

Laboratory of Molecular and Cellular Biology, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto 606-8507, Japan

Molecular chaperones facilitate protein folding by associating with nascent polypeptides, thereby preventing protein misfolding and aggregation. Endoplasmic reticulum (ER) chaperone BiP, the sole HSP70 chaperone in the ER, is regulated by HSP40 chaperones, including ER-resident protein ERdj3 (DNAJB11). ERdj3 lacks an ER retrieval signal, is secreted under ER stress conditions, and functions as a chaperone in the extracellular space, but how its secretion is regulated remains unclear. We recently showed that ERdj3 forms a complex with ER-resident stromal cell-derived factor 2 (SDF2) and SDF2L1 (SDF2-like protein 1) and thereby prevents protein aggregation during the BiP chaperone cycle. However, the contribution of the ERdj3-SDF2L1 complex to protein quality control is poorly understood. Here, we analyzed the intracellular localization and chaperone activity of ERdj3 in complex with SDF2L1. We found that ERdj3 was retained in the ER by associating with SDF2/SDF2L1. analyses revealed that the ERdj3 dimer incorporated two SDF2L1 molecules; otherwise, ERdj3 alone formed a homotetramer. The ERdj3-SDF2L1 complex suppressed ER protein aggregation, and this suppression did not require substrate transfer to BiP. The ERdj3-SDF2L1 complex inhibited aggregation of denatured GSH -transferase (GST) and maintained GST in a soluble oligomeric state. Both and , the chaperone activities of the ERdj3-SDF2L1 complex were higher than those of ERdj3 alone. These findings suggest that, under normal conditions, ERdj3 functions as an ER chaperone in complex with SDF2/SDF2L1 but is secreted into the extracellular space when it cannot form this complex.
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http://dx.doi.org/10.1074/jbc.RA119.009603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6916475PMC
December 2019

Sperm IZUMO1-Dependent Gamete Fusion Influences Male Fertility in Mice.

Int J Mol Sci 2019 Sep 27;20(19). Epub 2019 Sep 27.

Department of Cell Science, Institute of Biomedical Sciences, School of Medicine, Fukushima Medical University, 1 Hikarigaoka, Fukushima City, Fukushima 960-1295, Japan.

Sperm-egg fusion is accomplished through the interaction of a specific set of membrane proteins in each gamete: sperm IZUMO1 and oocyte JUNO. Recently, we found that alternative splicing of the gene generates a novel IZUMO1 isoform (IZUMO1_v2). Here, we obtained four mouse lines, having graded different levels of IZUMO1 protein by combining an original IZUMO1 (IZUMO1_v1) knockout with IZUMO1-null (both IZUMO1_v1 and _v2 disrupted) genetic background, in order to determine how the quantity of IZUMO1 influences male fertility. Subsequently, we clarified that the signal intensity from two quantitative assays, western blot and immunostaining analyses with a monoclonal antibody against mouse IZUMO1, were strongly correlated with average litter size. These results suggest that evaluating IZUMO1 protein levels is useful for predicting fecundity, and is a suitable test for male fertility.
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http://dx.doi.org/10.3390/ijms20194809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801368PMC
September 2019

Nigrostriatal Dopaminergic Dysfunction and Altered Functional Connectivity in REM Sleep Behavior Disorder With Mild Motor Impairment.

Front Neurol 2019 26;10:802. Epub 2019 Jul 26.

Department of Neurology, Nagoya City University Graduate School of Medical Science, Aichi, Japan.

Rapid eye movement sleep behavior disorder is parasomnia characterized by symptoms of dream enactment and loss of muscle atonia during rapid eye movement sleep. Mild motor impairment is present in some patients with rapid eye movement sleep behavior disorder and presumed to be a risk factor for conversion to synucleinopathies. The purpose of this study is to identify patients with mild motor impairment by evaluating finger tapping and to investigate its pathophysiology. Twenty-three patients with rapid eye movement sleep behavior disorder and 20 healthy control subjects were recruited in the present study. We accurately evaluated finger tapping including amplitude, peak open, and close speed with a magnetic sensing device and identified patients with mild motor impairment. Moreover, we performed I-2β-carbomethoxy-3β-(4-iodophenyl) nortropane SPECT and resting state functional MRI. I-2β-carbomethoxy-3β-(4-iodophenyl) nortropane uptake for each bilateral caudate, anterior putamen, and posterior putamen was calculated and the resting state functional connectivity of sensorimotor network was analyzed. Using finger tapping parameters, we identified eight patients with mild motor impairment. In patients with mild motor impairment, all finger tapping parameters were significantly impaired when compared to patients with normal motor function, while they exhibited no significant differences in Unified Parkinson's Disease Rating Scale part III score. I-2β-carbomethoxy-3β-(4-iodophenyl) nortropane uptake in the right posterior putamen, bilateral anterior putamen, and caudate was significantly lower when compared to healthy controls or patients with rapid eye movement sleep behavior disorder with normal motor function. These patients also exhibited decreased cortico-striatal functional connectivity and increased cortico-cerebellar functional connectivity when compared to healthy controls or patients with normal motor function. Our results show that mild motor impairment in rapid eye movement sleep behavior disorder evaluated by finger tapping task presented mild nigrostriatal dopaminergic dysfunction as well as alterations in resting state sensorimotor network. Although longitudinal follow up is necessary, such patients may have higher risk of short-term conversion to synucleinopathies.
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http://dx.doi.org/10.3389/fneur.2019.00802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6677031PMC
July 2019

Impaired Motor Skill Acquisition Using Mirror Visual Feedback Improved by Transcranial Direct Current Stimulation (tDCS) in Patients With Parkinson's Disease.

Front Neurosci 2019 19;13:602. Epub 2019 Jun 19.

Department of Rehabilitation Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.

Recent non-invasive brain stimulation techniques in combination with motor training can enhance neuroplasticity and learning. It is reasonable to assume that such neuroplasticity-based interventions constitute a useful rehabilitative tool for patients with Parkinson's Disease (PD). Regarding motor skill training, many kinds of tasks that do not involve real motor movements have been applied to PD patients. The purpose of this study is to elucidate whether motor skill training using mirror visual feedback (MVF) is useful to patients with PD in order to improve untrained hand performance dependent on the time course of training; and whether MVF combined with anodal transcranial direct current stimulation (tDCS) over primary motor cortex (M1) causes an additional effect based on increased motor cortical excitability. Eighteen right-handed patients with PD in the off-medication state and 10 age-matched healthy subjects (HS) performed four sessions of right-hand ball rotation using MVF (intervention) on two separate days, 1 week apart (day 1 and day 2). HS subjects received only sham stimulation. The intervention included four sessions of motor-skill training using MVF for 20 min comprised of four sets of training for 30 s each. PD patients were randomly divided into two intervention groups without or with anodal tDCS over the right M1 contralateral to the untrained hand. As the behavior evaluation, the number of ball rotations of the left hand was counted before (pre) and immediately after (post) intervention on both days (pre day 1, post day 1, pre day 2, and post day 2). Motor evoked potential (MEP), input-output function, and cortical silent period were recorded to evaluate the motor cortical excitatory and inhibitory system in M1 pre day 1 and post day 2. The number of ball rotations of the left hand and the facilitation of MEP by intervention were significantly impaired in patients with PD compared to HS. In contrast, if anodal tDCS was applied to right M1 of patients with PD, the number of ball rotations in accordance with I-O function at 150% intensity was significantly increased after day 1 and retained until day 2. This finding may help provide a new strategy for neurorehabilitation improving task-specific motor memory without real motor movements in PD.
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http://dx.doi.org/10.3389/fnins.2019.00602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6593084PMC
June 2019

Cecal cancer with essential thrombocythemia treated by laparoscopic ileocecal resection: a case report.

Surg Case Rep 2019 Jun 21;5(1):101. Epub 2019 Jun 21.

Department of Surgical Oncology, Faculty of Medical Sciences, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan.

Background: Essential thrombocythemia (ET) is a myeloproliferative disorder characterized by thrombocytosis and a propensity for both thrombotic and hemorrhagic events. ET rarely occurs simultaneously with colorectal cancer. Here, we report a case of colorectal cancer in an ET patient treated using laparoscopic ileocecal resection.

Case Presentation: A 40-year-old woman was admitted to our hospital after presenting with liver dysfunction. She had been previously diagnosed with ET; aspirin and anagrelide had been prescribed. Subsequent examination at our hospital revealed cecal cancer. Distant metastasis was absent; laparoscopic ileocecal resection was performed. Anagrelide was discontinued only on the surgery day. She was discharged on the seventh postoperative day without thrombosis or hemorrhage. However, when capecitabine and oxaliplatin were administered as adjuvant chemotherapy with continued anagrelide administration, she experienced hepatic dysfunction and thrombocytopenia; thus, anagrelide was discontinued. Five days later, her platelet count recovered. Subsequently, anagrelide and aspirin administration was resumed, without any adjuvant chemotherapy. Her liver function normalized gradually in 4 months. One-year post operation, she is well without tumor recurrence or new metastasis.

Conclusions: To our knowledge, this is the first report of laparoscopic colectomy performed on an ET patient receiving anagrelide. Our report shows that complications such as bleeding or thrombosis can be avoided by anagrelide administration. Contrastingly, thrombocytopenia due to anagrelide intake should be considered when chemotherapy that could cause bone marrow suppression is administered.
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http://dx.doi.org/10.1186/s40792-019-0660-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588662PMC
June 2019

Reply to letter to the editor by Watanabe.

J Orthop Sci 2019 07 29;24(4):762. Epub 2019 Apr 29.

Department of Rehabilitation Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan. Electronic address:

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http://dx.doi.org/10.1016/j.jos.2019.04.005DOI Listing
July 2019

Integrative genome analysis identified the KANNO blood group antigen as prion protein.

Transfusion 2019 07 24;59(7):2429-2435. Epub 2019 Apr 24.

Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Background: Anti-KANNO, a broadly reactive RBC alloantibody, is found among some Japanese pregnant women, but the genetic basis of the corresponding antigen remains unclear.

Study Design And Methods: We integrated a statistical approach to identify the coding gene for KANNO antigen by conducting a genome-wide association study (GWAS) on four KANNO-negative individuals and 415 healthy Japanese. We also applied whole-exome sequencing to them and performed a replication study to confirm the identified genome variation using independent 14 KANNO-negative individuals. A monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA) assay was used to locate KANNO antigen on RBC-specific membrane protein. In vivo and in vitro binding assays of anti-KANNO were further applied to the cells expressing a candidate protein.

Results: The GWAS revealed a genome-wide significant association of chromosome 20p13 locus (p = 2.76E-08; odds ratio > 1000 [95% confidence interval = 48-23,674]). The identified single-nucleotide polymorphism located in an intronic region of the prion protein (PRNP) gene. Whole-exome sequencing revealed a missense variant in the PRNP gene (rs1800014, E219K), which is in linkage disequilibrium with the single-nucleotide polymorphism identified in the GWAS. All 18 KANNO-negative individuals possessed the homozygous genotype of the missense variant. The MAIEA assay using anti-KANNO and mouse antihuman prion protein showed a clear difference between KANNO-positive and KANNO-negative RBCs. Anti-KANNO showed direct binding to CHO-K1 cells expressing wild-type PRNP but not to those expressing E219K PRNP.

Conclusion: We first identified the coding gene of the high-frequency antigen KANNO located in PRNP and the missense variation (E219K) that affects the seropositivity of the KANNO antigen, which were confirmed by PRNP overexpressed cells.
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http://dx.doi.org/10.1111/trf.15319DOI Listing
July 2019

Both Svp26 and Mnn6 are required for the efficient ER exit of Mnn4 in Saccharomyces cerevisiae.

J Gen Appl Microbiol 2019 Dec 6;65(5):215-224. Epub 2019 Mar 6.

Department of Biotechnology, The University of Tokyo.

Incorporation of membrane and secretory proteins into COPII vesicles are facilitated either by the direct interaction of cargo proteins with COPII coat proteins, or by ER exit adaptor proteins which mediate the interaction of cargo proteins with COPII coat proteins. Svp26 is one of the ER exit adaptor proteins in the yeast Saccharomyces cerevisiae. The ER exit of several type II membrane proteins have been reported to be facilitated by Svp26. We demonstrate here that the efficient incorporation of Mnn4, a type II membrane protein required for mannosyl phosphate transfer to glycoprotein-linked oligosaccharides, into COPII vesicles is also dependent on the function of Svp26. We show that Mnn4 is localized to the Golgi. In addition to Mnn4, Mnn6 is known to be also required for the transfer of mannosyl phosphate to the glycans. We show, by indirect immunofluorescence, that Mnn6 localizes to the ER. As in the case with Svp26, deletion of the MNN6 gene results in the accumulation of Mnn4 in ER. In vitro COPII vesicle budding assays show that Svp26 and Mnn6 facilitate the incorporation of Mnn4 into COPII vesicles. In contrast to Svp26, which is itself efficiently captured into the COPII vesicles, Mnn6 was not incorporated into the COPII vesicles. Mnn4 and Mnn6 have the DXD motif which is often found in the many glycosyltransferases and functions to coordinate a divalent cation essential for the reaction. Alcian blue dye binding assay shows that substitution of the first D in this motif present in Mnn4 by A impairs the Mnn4 function. In contrast, amino acid substitutions in DXD motifs present in Mnn6 did not affect the function of Mnn6. These results suggest that Mnn4 may be directly involved in the mannosyl phosphate transfer reaction.
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http://dx.doi.org/10.2323/jgam.2018.10.002DOI Listing
December 2019

Alternative splicing of the Izumo1 gene ensures triggering gamete fusion in mice.

Sci Rep 2019 02 28;9(1):3151. Epub 2019 Feb 28.

Department of Cell Science, Institute of Biomedical Sciences, School of Medicine, Fukushima Medical University, 1 Hikarigaoka, Fukushima City, Fukushima, 960-1295, Japan.

IZUMO1 is a sperm acrosomal membrane protein that is essential for mammalian fertilization through recognition of JUNO on the oocyte surface and accompanying IZUMO1-JUNO complex formation. Here, we report a new Izumo1 gene splicing variant (IZUMO1_v2) with a unique 52-amino-acid-long signal sequence transcribed from Exon 1b. Although the mRNA amount of Izumo1_v2 is 76 times lower than that of the original Izumo1 (IZUMO1_v1) in the testis, the cell-oocyte assay indicates that IZUMO1_v2-expressing COS-7 cells have the ability to attach to the oocyte equivalent of IZUMO1_v1. To clarify the physiological function of IZUMO1_v2, we produced an IZUMO1_v1-specific knockout mouse line with a nine-base deletion adjacent to the initial methionine codon of IZUMO1_v1 by the CRISPR/Cas9 system. The IZUMO1_v1 knockout male mice carry 0.19-fold lower level of IZUMO1 protein in the spermatozoon; however, reduction in fertility was only minimally affected compared to the wild-type mice, suggesting that only a small fraction of IZUMO1 is sufficient for triggering sperm-egg fusion. We propose that the alternative splicing generating IZUMO1_v2 might function as a fail-safe in mouse for when splicing is disturbed.
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http://dx.doi.org/10.1038/s41598-019-40130-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395798PMC
February 2019

Differences in postural stability and dynamic visual acuity among healthy young adults in relation to sports activity: a cross sectional study.

J Phys Ther Sci 2019 Jan 10;31(1):53-56. Epub 2019 Jan 10.

Department of Rehabilitation Medicine, Graduate School of Medical Sciences, Nagoya-City University: 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan.

[Purpose] Sports activity has been shown to improve postural stability and vestibular function in healthy older adults. The hypothesis was that healthy young adults undertaking sports activity will also have better postural stability and vestibular function compared with healthy young adults who do not undertake sports activity. The purpose of this study was to investigate the differences in postural stability and vestibular function between healthy young adults who undertake sports activity and those who do not undertake such activity. [Participants and Methods] Thirty-nine healthy young adults were recruited and divided into sports and non-sports groups on the basis of their response to a questionnaire concerning regular participation in sports activities over the past 12 months. In both groups, postural stability was measured during quiet standing and standing during head rotation, and dynamic visual acuity was assessed during head rotation. [Results] The results showed significant differences in postural stability during head rotation and dynamic visual acuity between the two groups, whereas no significant differences were found in postural stability during quiet standing. [Conclusion] The results suggest that healthy young adults who undertake sports activity have better postural stability during head rotation and better dynamic visual acuity. The causal effect of these differences is not clear and further investigation is warranted.
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http://dx.doi.org/10.1589/jpts.31.53DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6348173PMC
January 2019

[A Case of Resection of Advanced Pancreatic Adenosquamous Carcinoma in which Multidisciplinary Treatment Was Effective].

Gan To Kagaku Ryoho 2019 Jan;46(1):178-180

Dept. of Surgery, Tokyo Metropolitan Bokutoh Hospital.

A 72-year-old man with general fatigue was referred, and CT and MRI revealed a pancreatic mass with necrosis that was suspected of invading the stomach, splenic artery, celiac artery, liver, and portal vein. Upper gastrointestinal endoscopy showed an extrinsic mass with ulcer formation in the posterior wall of the upper gastric corpus and irregular mucosa in the lower esophagus incidentally. Biopsy showed squamous cell carcinoma from both lesions, leading to the diagnosis of pancreatic adenosquamous carcinoma and early esophageal cancer. We performed distal pancreatectomy with splenectomy, total gastrectomy, partial hepatectomy, superior mesenteric-portal vein resection, and reconstruction. The pathological results revealed pancreatic adenosquamous carcinoma and infiltration of cancer cells at the dissected peripancreatic margin. Therefore, we administered radiotherapy(50.4 Gy to the retroperitoneal region)in postoperative month 2. Endoscopic mucosal resection was performed for the early stage esophageal cancer lesion in postoperative month 5. Three courses of S-1 were administered as adjuvant therapy since postoperative month 7, and he is currently alive without recurrence 1 year and 8 months after surgery. Multidisciplinary treatment can be effective for locally advanced pancreatic adenosquamous carcinoma.
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January 2019

Svp26 facilitates ER exit of mannosyltransferases Mnt2 and Mnt3 in Saccharomyces cerevisiae.

J Gen Appl Microbiol 2019 Sep 31;65(4):180-187. Epub 2019 Jan 31.

Department of Biotechnology, The University of Tokyo.

After being translocated into the ER lumen, membrane and secretory proteins are transported from the ER to the early Golgi by COPII vesicles. Incorporation of these cargo proteins into COPII vesicles are facilitated either by direct interaction of cargo proteins with COPII coat proteins or by ER exit adaptor proteins which mediate the interaction of cargo proteins with COPII coat proteins. Svp26 is one of the ER exit adaptor proteins in yeast Saccharomyces cerevisiae. ER exit of several type II membrane proteins have been reported to be facilitated by Svp26. We demonstrate here that efficient incorporation of Mnt2 and Mnt3 into COPII vesicles is also dependent on the function of Svp26. Mnt2 and Mnt3 are Golgi-localized α-1,3-mannosyltransferases with type II membrane topology involved in protein O-glycosylation. Immunoisolation of the yeast Golgi subcompartments quantitatively showed that Mnt2 and Mnt3 are more abundant in the early Golgi fraction than in the late Golgi fraction. Subcellular fractionation and fluorescence microscopy showed that deletion of the SVP26 gene results in the accumulation of Mnt2 and Mnt3 in ER. Using an in vitro COPII vesicle formation assay, we further demonstrate that Svp26 facilitates incorporation of Mnt2 and Mnt3 into COPII vesicles. Finally, we showed that Mnt2 and Mnt3 were co-immunoprecipitated with Svp26 from digitonin-solubilized membranes. These results indicate that Svp26 functions as an ER exit adaptor protein of Mnt2 and Mnt3.
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http://dx.doi.org/10.2323/jgam.2018.09.001DOI Listing
September 2019

Objective measures of physical activity in patients with chronic unilateral vestibular hypofunction, and its relationship to handicap, anxiety and postural stability.

Auris Nasus Larynx 2019 Feb 30;46(1):70-77. Epub 2018 Jun 30.

Department of Rehabilitation Medicine, Graduate School of Medical Sciences, Nagoya City University, 1, Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan.

Objective: Dizziness is one of the most common symptoms in the general population. Patients with dizziness experience balance problems and anxiety, which can lead to decreased physical activity levels and participation in their daily activities. Moreover, recovery of vestibular function from vestibular injury requires physical activity. Although there are reports that decreased physical activity is associated with handicap, anxiety, postural instability and reduced recovery of vestibular function in patients with chronic dizziness, these data were collected by self-report questionnaires. Therefore, the objective data of physical activity and the relationships between physical activity, handicap, anxiety and postural stability in patients with chronic dizziness are not clear. The purpose of this research was to objectively measure the physical activity of patients with chronic dizziness in daily living as well as handicap, anxiety and postural stability compared to healthy adults. Additionally, we aimed to investigate the relationships between physical activity, handicap, anxiety and postural stability in patients with chronic dizziness.

Methods: Twenty-eight patients with chronic dizziness of more than 3 months caused by unilateral vestibular hypofunction (patient group) and twenty-eight age-matched community dwelling healthy adults (healthy group) participated in this study. The amount of physical activity including time of sedentary behavior, light physical activity, moderate to vigorous physical activity and total physical activity using tri-axial accelerometer, self-perceived handicap and anxiety using questionnaires, and postural stability were measured using computerized dynamic posturography.

Results: The results showed worse handicap, anxiety and postural stability in the patient group compared to the healthy group. Objective measures of physical activity revealed that the patient group had significantly longer time of sedentary behavior, shorter time of light physical activity, and shorter time of total physical activity compared to the healthy group; however, time of moderate to vigorous physical activity was not significantly different between groups. Moreover, there were correlations between physical activity and postural stability in the patient group, while there were no correlations between physical activity, handicap or anxiety in the patient group.

Conclusion: These results suggest that objectively measured physical activity of the patients with chronic unilateral vestibular hypofunction is lower compared to the healthy adults, and less active patients showed decreased postural stability. However, the details of physical activity and causal effect between physical activity and postural stability were not clear and further investigation is needed.
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http://dx.doi.org/10.1016/j.anl.2018.06.010DOI Listing
February 2019

Pathology and management of flexible flat foot in children.

J Orthop Sci 2019 Jan 23;24(1):9-13. Epub 2018 Oct 23.

Department of Rehabilitation Medicine, Nagoya City University, Graduate School of Medical Sciences, Nagoya, Japan. Electronic address:

We describe the pathology and treatment of flexible flat foot in children. The flexible flat foot is seen in the overly flexible foot and usually involves hypermobility of the subtalar joint. It typically occurs in childhood and may continue to adulthood. The arch develops spontaneously during the first decade of life in most children and comes within the normal range observed in adult feet. We prescribed orthoses for the treatment of flexible flat foot patients. Lateral weight-bearing radiographs and ultrasonography were helpful for the evaluation of the flat foot. Bleck recommended the UCBL shoe insert in cases of flexible flat foot if the standing or lateral rentgenogram demonstrates a talar plantar flexion angle (TPF) of 45° or greater. Bordelon suggested that cases of flexible flat foot should be treated if the standing or lateral roentgenogram demonstrates a Meary's talo-1st metatarsal angle (T1-MTA) of -15°or greater. However, the radiograph of a young child's foot poses some difficulties in making an accurate evaluation, because of the radiolucent cartilage zone. In this situation, a sagittal image obtained by ultrasonography has proved to be a powerful aid to evaluate the type of the flat foot. We classified the flat foot into three types: talo-navicular sag (T-N sag), naviculo-cuneiform sag (NC sag) and talo-navicular and naviculo-cuneiform sag (Mixed sag) following the criteria of Tachdjian. We recommended the NC sag and Mixed sag groups to be treated by using orthoses, while we kept a status of watchful waiting for the T-N sag group. However, we should consider the increasing complaints of children and their parents during the orthotic treatment. A through discussion between the parents of patients and the pediatric orthopedic doctors is necessary before orthotic treatment is started.
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http://dx.doi.org/10.1016/j.jos.2018.09.018DOI Listing
January 2019

Complex formation of sphingomyelin synthase 1 with glucosylceramide synthase increases sphingomyelin and decreases glucosylceramide levels.

J Biol Chem 2018 11 21;293(45):17505-17522. Epub 2018 Sep 21.

From the Faculty of Pharma-Science, Teikyo University, Tokyo 173- 8605, Japan and

Sphingolipids, including sphingomyelin (SM) and glucosylceramide (GlcCer), are generated by the addition of a polar head group to ceramide (Cer). Sphingomyelin synthase 1 (SMS1) and glucosylceramide synthase (GCS) are key enzymes that catalyze the conversion of Cer to SM and GlcCer, respectively. GlcCer synthesis has been postulated to occur mainly in -Golgi, and SM synthesis is thought to occur in /-Golgi; however, SMS1 and GCS are known to partially co-localize in cisternae, especially in -Golgi. Here, we report that SMS1 and GCS can form a heteromeric complex, in which the N terminus of SMS1 and the C terminus of GCS are in close proximity. Deletion of the N-terminal sterile α-motif of SMS1 reduced the stability of the SMS1-GCS complex, resulting in a significant reduction in SM synthesis In contrast, chemical-induced heterodimerization augmented SMS1 activity, depending on an increase in the amount and stability of the complex. Fusion of the SMS1 N terminus to the GCS C terminus via linkers of different lengths increased SM synthesis and decreased GlcCer synthesis These results suggest that formation of the SMS1-GCS heteromeric complex increases SM synthesis and decreases GlcCer synthesis. Importantly, this regulation of relative Cer levels by the SMS1-GCS complex was confirmed by CRISPR/Cas9-mediated knockout of SMS1 or GCS combined with pharmacological inhibition of Cer transport protein in HEK293T cells. Our findings suggest that complex formation between SMS1 and GCS is part of a critical mechanism controlling the metabolic fate of Cer in the Golgi.
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http://dx.doi.org/10.1074/jbc.RA118.002048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6231140PMC
November 2018

Preoperative radiologic predictors of successful soft tissue release surgery for hip subluxation among cerebral palsy patients: A STROBE compliant study.

Medicine (Baltimore) 2018 Aug;97(33):e11847

Department of Orthopedic Surgery Department of Molecular and Cellular Biology Department of Integrative Anatomy Department of Rehabilitation Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.

Paralytic hip subluxation is a common problem in children with cerebral palsy. Although surgical procedures such as soft tissue release and osteotomy have been advocated for its prevention, the exact indications of such procedures remain unclear. We attempted to evaluate preoperative radiographic parameters and identify prognostic factors in children with cerebral palsy. We retrospectively investigated 43 hips in 27 children with cerebral palsy who had undergone soft tissue release surgery for hip subluxation. We evaluated the age at the time of surgery and the radiographic parameters such as the center-edge angle (CEA), the migration percentage (MP), and the acetabular index (AI) at 3 time points: preoperation, 1 year after surgery, and at final follow-up. The outcome measure was determined by the MP value at final follow-up. Student t test was used to compare the quantitative variables between 2 groups (good vs poor outcome). Then the multiple regression analysis was applied to determine the prognostic factors upon soft tissue release surgery. Children with good outcome exhibited higher CEA (average value of -1.43° vs -13.2° in those with poor outcome), lower MP (53.9% vs 71.3%), and lower AI (28.1° vs 35.3°). Upon multiple regression analysis, we found that the age at the time of surgery, preoperative CEA, and preoperative MP did not appear to be independent prognostic factors. The only independent factor that affected prognosis after soft tissue release surgery was the preoperative AI. The preoperative AI values <34° were associated with the good outcome with specificity of 87% and sensitivity of 60% according to the receiver operating characteristic curve analysis. These findings indicate that the outcome of soft tissue release surgery can be predicted by the preoperative AI value.
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http://dx.doi.org/10.1097/MD.0000000000011847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112945PMC
August 2018

Monitoring dimeric status of IZUMO1 during the acrosome reaction in living spermatozoon.

Cell Cycle 2018 25;17(11):1279-1285. Epub 2018 Jul 25.

a Department of Cell Science , Institute of Biomedical Sciences, School of Medicine, Fukushima Medical University , Fukushima City , Japan.

The acrosome reaction (AR) is indispensable for successful spermatozoon-oocyte fusion. Recent studies have indicated that sperm IZUMO1 gradually gathers in the equatorial segment (EQ), which is the initiation site of sperm-egg fusion, after the AR. In addition, by examining the binding process of oocytes and Izumo1-expressing cultured cells to reconstitute the early steps of fertilization, we previously demonstrated that robust IZUMO1-dependent adhesion specifically occurs at the contact site along with the dimerization of IZUMO1. However, when IZUMO1 dimerizes after the AR in living spermatozoon is unknown. Here, we report dynamics of IZUMO1 dimerization during the AR in spermatozoa by combining transgenic mice and time-lapse imaging using a set of bimolecular fluorescence complementation (BiFC) probes. Surprisingly, dimeric IZUMO1 was already formed at the acrosomal cap region before the AR and redistributed into the EQ after the AR. We categorized the translocation of the dimer into two types: Type 1, the near-simultaneous appearance of BiFC signals with IZUMO1-mCherry; and Type 2, the delayed formation of dimer in the EQ. Those findings suggest that, before encountering oocytes, spermatozoa are prepared to boost their affinity with JUNO.
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http://dx.doi.org/10.1080/15384101.2018.1489181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6110575PMC
October 2019

ER-resident protein 46 (ERp46) triggers the mannose-trimming activity of ER degradation-enhancing α-mannosidase-like protein 3 (EDEM3).

J Biol Chem 2018 07 21;293(27):10663-10674. Epub 2018 May 21.

From the Laboratory of Molecular and Cellular Biology, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto 606-8507,

Protein folding in the cell is regulated by several quality-control mechanisms. Correct folding of glycoproteins in the endoplasmic reticulum (ER) is tightly monitored by the recognition of glycan signals by lectins in the ER-associated degradation (ERAD) pathway. In mammals, mannose trimming from -glycans is crucial for disposal of misfolded glycoproteins. The mannosidases responsible for this process are ER mannosidase I and ER degradation-enhancing α-mannosidase-like proteins (EDEMs). However, the molecular mechanism of mannose removal by EDEMs remains unclear, partly owing to the difficulty of reconstituting mannosidase activity Here, our analysis of EDEM3-mediated mannose-trimming activity on a misfolded glycoprotein revealed that ERp46, an ER-resident oxidoreductase, associates stably with EDEM3. This interaction, which depended on the redox activity of ERp46, involved formation of a disulfide bond between the cysteine residues of the ERp46 redox-active sites and the EDEM3 α-mannosidase domain. In a defined system consisting of recombinant proteins purified from HEK293 cells, the mannose-trimming activity of EDEM3 toward the model misfolded substrate, the glycoprotein T-cell receptor α locus (TCRα), was reconstituted only when ERp46 had established a covalent interaction with EDEM3. On the basis of these findings, we propose that disposal of misfolded glycoproteins through mannose trimming is tightly connected to redox-mediated regulation in the ER.
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http://dx.doi.org/10.1074/jbc.RA118.003129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6036223PMC
July 2018

Phosphorylation of SNAP-23 at Ser95 causes a structural alteration and negatively regulates Fc receptor-mediated phagosome formation and maturation in macrophages.

Mol Biol Cell 2018 07 17;29(13):1753-1762. Epub 2018 May 17.

Division of Molecular Biology, School of Life Sciences, Faculty of Medicine, Tottori University, Yonago, Tottori 683-8503, Japan.

SNAP-23 is a plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) involved in Fc receptor (FcR)-mediated phagocytosis. However, the regulatory mechanism underlying its function remains elusive. Using phosphorylation-specific antibodies, SNAP-23 was found to be phosphorylated at Ser95 in macrophages. To understand the role of this phosphorylation, we established macrophage lines overexpressing the nonphosphorylatable S95A or the phosphomimicking S95D mutation. The efficiency of phagosome formation and maturation was severely reduced in SNAP-23-S95D-overexpressing cells. To examine whether phosphorylation at Ser95 affected SNAP-23 structure, we constructed intramolecular Förster resonance energy transfer (FRET) probes of SNAP-23 designed to evaluate the approximation of the N termini of the two SNARE motifs. Interestingly, a high FRET efficiency was detected on the membrane when the S95D probe was used, indicating that phosphorylation at Ser95 caused a dynamic structural shift to the closed form. Coexpression of IκB kinase (IKK) 2 enhanced the FRET efficiency of the wild-type probe on the phagosome membrane. Furthermore, the enhanced phagosomal FRET signal in interferon-γ-activated macrophages was largely dependent on IKK2, and this kinase mediated a delay in phagosome-lysosome fusion. These results suggested that SNAP-23 phosphorylation at Ser95 played an important role in the regulation of SNARE-dependent membrane fusion during FcR-mediated phagocytosis.
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http://dx.doi.org/10.1091/mbc.E17-08-0523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6080709PMC
July 2018

[A Case of Double Cancer of Intrahepatic Cholangiocarcinoma and Gastric Carcinoma with Difficult Diagnosis of the Primary Tumor with Peritoneal Metastasis].

Gan To Kagaku Ryoho 2018 Apr;45(4):746-748

Dept. of Surgery, Tokyo Metropolitan Bokutoh Hospital.

Double cancer of intrahepatic cholangiocarcinoma and gastric cancer is rare. A 62-year-old man underwent gastrectomy for gastric cancer. The pathological findings were tub1>tub2, m, ly0, v0, n0, Stage I A. Two years and a month later, a liver tumor(diameter of 3 cm)and a pelvic mass(diameter of 2.5 cm)were observed. Metastasis from gastric cancer was suspected and chemotherapy(SOX)was administered. However, after 5 courses, CT revealed worseningof the liver tumor (diameter of 12 cm)and pelvic mass(diameter of 3 cm). Intrahepatic cholangiocarcinoma and its peritoneal metastasis were also suspected. There was a limit to treatment with chemotherapy, and it was difficult to judge whether to target gastric cancer or intrahepatic cholangiocarcinoma for chemotherapy. In addition, the lesions were localized in the right lobe of the liver and the pelvis. Therefore, we decided to perform resection. As a second-stage operation, pelvic mass extraction and portal vein embolization were performed first. The pathological result of the pelvic mass assessment was mucinous carcinoma. Subsequently, expansive right hepatectomy was performed. The pathological findings were also suggestive of mucinous carcinoma, which was finally diagnosed as intrahepatic cholangiocarcinoma and peritoneal dissemination. Six months after the surgery, several recurrent nodules were observed in the pelvis and GEM plus CDDP was initiated. Currently, 1 year after surgery, there are no restrictions in the activities of daily life of the patient and he is treated on an outpatient basis.
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April 2018
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