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ABSTRACT: BACKGROUND: Although Helicobacter pylori eradication is a first-line treatment of gastric MALT lymphoma, roughly 25% of patients do not respond to treatment. CD4(+) FOXP3(+) regulatory T (Treg) cells regulate immune responses in physiological conditions and various inflammatory conditions, including H. pylori-associated diseases. Our goal was to determine how Treg cells affect responsiveness to H. pylori eradication therapy. MATERIALS AND METHODS: We performed dual immunohistochemistry for CD4 and FOXP3 to evaluate the prevalence of FOXP3(+) Treg cells in the stomach of 63 patients with MALT lymphoma and 55 patients with chronic active gastritis. Receiver operating characteristic analysis was carried out to determine the best cut-off point in differentiating H. pylori eradication responders from nonresponders. RESULTS: Both the FOXP3(+) /CD4(+) cell ratio and the absolute number of FOXP3(+) cells per high-power field in MALT lymphoma were significantly greater in H. pylori eradication responders compared with nonresponders, suggesting that Treg cells function in regression mechanisms of MALT lymphomas. Cut-off points with good sensitivities and specificities were obtained to predict eradication outcome. CONCLUSIONS: A high number of Treg cells or a high ratio of Treg cells to the total number of CD4(+) T cells in gastric MALT lymphoma could predict responsiveness to eradication therapy.
Helicobacter 04/2013; · 3.15 Impact Factor
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ABSTRACT: Tumors of the heart and the great vessels are very rare disease, and there are many disorders such as tumors originated from the heart and great vessels, metastatic tumors, and tumor-like lesions which do not fit into the usual concept of tumor or neoplasm; thus, it is very difficult to classify these tumors. We proposed a new classification of cardiovascular tumors for clinical use based on the accumulated biological analyses and clinical data of the reported literatures and our own study as benign tumors, malignant tumors, ectopic hyperplasia/ectopic tumors/others, and tumors of great vessels, with reference to the series of Atlas of tumor pathology of the Armed Forces Institute of Pathology and the recent World Health Organization classification of cardiac tumors issued in 2004. More than 50 disorders have been reported as tumors originated from the cardiovascular system, and various metastatic tumors from nearby organs, distant lesions, and intravascular extension tumors to the heart were reported. Based on the new classification, we reviewed epidemiology and incidence of cardiovascular tumors. Metastatic tumors are more frequent than tumors originated from the heart and great vessels, and cardiac myxoma is the most frequent tumors in all cardiac tumors.
General Thoracic and Cardiovascular Surgery 03/2013;
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ABSTRACT: N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) is the sulfotransferase responsible for biosynthesis of highly sulfated chondroitin sulfate CS-E. Although involvements of CS-E in neuronal cell functions have been extensively analyzed, the role of GalNAc4S-6ST in astrocytic tumor progression remains unknown. Here, we reveal that GalNAc4S-6ST transcripts were detected in astrocytic tumors derived from all 30 patients examined using quantitative reverse transcription-PCR analysis. Patients with high GalNAc4S-6ST mRNA expression had significantly worse outcome compared with patients with low expression, and multivariate survival analysis disclosed that GalNAc4S-6ST is an independent poor prognostic factor for astrocytic tumors. We then tested whether CS-E enhanced haptotaxic migration of glioblastoma U251-MG cells that endogenously express both the CS-E's scaffold tyrosine phosphatase ζ (PTPζ) and GalNAc4S-6ST, in the presence of CS-E's preferred ligands, pleiotrophin (PTN) or midkine (MK), using a modified Boyden chamber method. Haptotaxic stimulation of cell migration by PTN was most robust on control siRNA-transfected U251-MG cells, while that enhancing effect was cancelled following transduction of GalNAc4S-6ST siRNA. Similar results were obtained using MK, suggesting that both PTN and MK enhance migration of U251-MG cells by binding to CS-E. We also found that PTPζ as well as PTN and MK were frequently expressed in astrocytic tumor cells. Thus, our findings indicate that GalNAc4S-6ST mRNA expressed by astrocytic tumor cells is associated with poor patient prognosis likely by enhancing CS-E-mediated tumor cell motility in the presence of PTN and/or MK.
PLoS ONE 01/2013; 8(1):e54278. · 4.09 Impact Factor
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ABSTRACT: Hyaluronan (HA) is a primary component of the extracellular matrix of cells, and it is involved in the pathogenesis of atherosclerosis. The purpose of this study was to investigate the role of HA in neointimal formation after vascular injury and determine its tissue-specific role in vascular smooth muscle cells (VSMCs) by using a cre-lox conditional transgenic (cTg) strategy.
HA was found to be expressed in neointimal lesions in humans with atherosclerosis and after wire-mediated vascular injury in mice. Inhibition of HA synthesis using 4-methylumbelliferone markedly inhibited neointimal formation after injury. In vitro experiments revealed that low-molecular-weight HA (LMW-HA) induced VSMC activation, including migration, proliferation, and production of inflammatory cytokines, and reactive oxygen species (ROS). The migration and proliferation of VSMCs were mediated by the CD44/RhoA and CD44/ERK1/2 pathways, respectively. Because HA synthase 2 (HAS2) is predominantly expressed in injured arteries, we generated cTg mice that overexpress the murine HAS2 gene specifically in VSMCs (cHAS2/CreSM22α mice) and showed that HA overexpression markedly enhanced neointimal formation after cuff-mediated vascular injury. Further, HA-overexpressing VSMCs isolated from cHAS2/CreSM22α mice showed augmented migration, proliferation, and production of inflammatory cytokines and ROS.
VSMC-derived HA promotes neointimal formation after vascular injury, and HA may be a potential therapeutic target for cardiovascular disease.
PLoS ONE 01/2013; 8(3):e58760. · 4.09 Impact Factor
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ABSTRACT: Primary malignant pericardial mesothelioma is an exceptionally rare tumor, and making an antemortem diagnosis of this disease is notoriously difficult. We herein report the case of a 61-year-old woman with pericardial mesothelioma who presented with shortness of breath and peripheral edema of the lower limbs. Chest computed tomography (CT) showed an anterior mass and thickened pericardium with multiple pericardial nodules. A biopsy of the mediastinal mass was performed using right thoracotomy, and the histological findings indicated a sarcomatoid tumor. The patient was treated with chemotherapy; however, she but died three months after diagnosis. An autopsy confirmed a final diagnosis of sarcomatoid type primary malignant pericardial mesothelioma following extensive immunohistopathological examinations.
Internal Medicine 01/2013; 52(2):249-53. · 0.94 Impact Factor
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Misa Suzuki-Anekoji,
Atsushi Suzuki,
Sz-Wei Wu,
Kiyohiko Angata,
Keith K Murai,
Kazuhiro Sugihara,
Tomoya O Akama,
Kay-Hoo Khoo, Jun Nakayama,
Michiko N Fukuda,
Minoru Fukuda
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ABSTRACT: Chst10 adds sulfate on glucuronic acid (GlcA) to form a carbohydrate antigen, HNK-1, in glycoproteins and glycolipids. To determine the role of Chst10 in vivo, we generated systemic Chst10-deficient mutant mice. While Chst10-/- were born and grew to adulthood with no gross defects, they were subfertile. Uteri from Chst10-/- females at the pro-estrus stage were larger than those from wild-type females and exhibited a thick uterine endometrium. Serum estrogen levels in Chst10-/- females were higher than those from wild-type females, suggesting impaired down-regulation of estrogen. Since steroid hormones are often conjugated to GlcA, we hypothesized that Chst10 sulfates glucuronidated steroid hormone to regulate steroid hormone in vivo. Enzymatic activity assays and structural analysis of Chst10 products by HPLC and mass spectrometry revealed that Chst10 indeed sulfates glucuronidated estrogen, testosterone and other steroid hormones. We also identified an HPLC peak corresponding to sulfated and glucuronidated estradiol in serum from wild-type but not from Chst10 null female mice. Estrogen response element reporter assays revealed that Chst10-modified estrogen likely did not bind to its receptor. These results suggest that subfertility exhibited by female mice following Chst10 loss results from dysregulation of estrogen. Given that Chst10 transfers sulfates to several steroid hormones, Chst10 likely functions in wide-spread regulation of steroid hormones in vivo.
Journal of Biological Chemistry 12/2012; · 4.77 Impact Factor
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ABSTRACT: AIMS:: Warthin's tumour is composed of bilayered oncocytic epithelium and organised lymphoid stroma, which resembles mucosa-associated lymphoid tissue (MALT); however, the histogenesis of the lymphoid stroma is not fully understood. We hypothesised that lymphocytes consisting of the stroma are recruited via high endothelial venules (HEVs) by the mechanism operating in normal lymphocyte homing in secondary lymphoid organs. The aim of this study was to determine immunohistochemically the molecules expressed on these HEVs. METHODS:: Tissue sections of Warthin's tumour (n = 10) were immunostained for vascular addressin-related antigens including peripheral lymph node addressin (PNAd) and mucosal addressin cell adhesion molecule 1 (MAdCAM-1). An L-selectin·IgM chimera in situ binding assay was also carried out. Triple immunostaining for PNAd, CD3, and CD20/CD79α was performed to determine which lymphocyte subsets are closely associated with these HEVs. RESULTS:: HEVs in the lymphoid stroma of Warthin's tumour express PNAd, which is detected by MECA-79 as well as recently developed monoclonal antibodies S1 and S2. These HEVs were bound by L-selectin·IgM chimeras in a calcium-dependent manner, and numbers of lymphocytes, particularly T cells, attached to these HEVs. CONCLUSIONS:: The lymphoid stroma of Warthin's tumour is most likely developed by lymphocytes recruited via HEVs.
Pathology 12/2012; · 2.38 Impact Factor
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ABSTRACT: OBJECTIVES: Type 1 autoimmune pancreatitis (AIP) is histologically characterized by dense lymphoplasmacytic infiltration and marked storiform fibrosis, manifestations associated with pancreatic ducts. Such periductal lymphocyte recruitment is thought to be elicited by dysregulation of mechanisms governing physiological lymphocyte homing. The present study was undertaken to determine whether vascular addressins including peripheral lymph node addressin and mucosal addressin cell adhesion molecule 1 (MAdCAM-1) play a role in type 1 AIP histogenesis. METHODS: Tissue sections of type 1 AIP and tumor-associated non-AIP chronic pancreatitis, as well as normal pancreas, were subjected to immunohistochemical analysis using vascular addressin-related antibodies. RESULTS: The number of periductal mouse endothelial cell antigen 79-positive high endothelial venule (HEV)-like vessels was increased in type 1 AIP relative to that seen in non-AIP chronic pancreatitis, whereas the number of MAdCAM-1-positive HEV-like vessels did not differ between the 2 conditions. Mouse endothelial cell antigen 79 antigens are expressed on duct-forming epithelial cells not only in pancreas but also in salivary glands, which often harbor extrapancreatic lesions in type 1 AIP. CONCLUSIONS: Type 1 AIP can be characterized by periductal induction of MECA-79-positive HEV-like vessels. MECA-79-positive 6-sulfo sialyl Lewis X-related carbohydrate antigens expressed on duct-forming epithelial cells could be associated with type 1 AIP pathogenesis.
Pancreas 06/2012; · 2.39 Impact Factor
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ABSTRACT: Under normal and pathological conditions, lymphocyte migration into the gastrointestinal mucosa to form gut-associated lymphoid tissue is mediated by the L-selectin ligand peripheral lymph node addressin and the integrin α4β7 ligand mucosal addressin cell adhesion molecule 1 (MAdCAM-1) expressed on high endothelial venules (HEVs) and HEV-like vessels. In this review, we discuss these two distinct lymphocyte homing systems involved in the pathogenesis of chronic inflammatory gastrointestinal diseases with reference to our and others' previously published works. We also describe a recently developed recombinant integrin α4β7 heterodimeric IgG chimera that can be used as an immunohistochemical reagent to stain functional MAdCAM-1.
Seminars in Immunopathology 05/2012; 34(3):401-13. · 6.27 Impact Factor
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ABSTRACT: Previously, we reported that near-infrared irradiation that simulates solar near-infrared irradiation with pre- and parallel-irradiational cooling can non-thermally induce cytocidal effects in cancer cells. To explore these effects, we assessed cell viability, DNA damage response pathways, and the percentage of mitotic cancer cells after near-infrared treatment. Further, we evaluated the anti-cancer effects of near-infrared irradiation compared with doxorubicin in xenografts in nude mice by measuring tumor volume and assessing protein phosphorylation by immunoblot analysis. The cell viability of A549 lung adenocarcinoma cells was significantly decreased after three rounds of near-infrared irradiation at 20 J/cm(2). Apoptotic cells were observed in near-infrared treated cells. Moreover, near-infrared treatment increased the phosphorylation of ataxia-telangiectasia mutated (ATM) at Ser(1981), H2AX at Ser(139), Chk1 at Ser(317), structural maintenance of chromosome (SMC) 1 at Ser(966), and p53 at Ser(15) in A549 cells compared with control. Notably, near-infrared treatment induced the formation of nucleic foci of γH2AX. The percentage of mitotic A549 cells, as measured by histone H3 phosphorylation, decreased significantly after three rounds of near-infrared irradiation at 20 J/cm(2). Both near-infrared and doxorubicin inhibited the tumor growth of MDA-MB435 melanoma cell xenografts in nude mice and increased the phosphorylation of p53 at Ser(15), Chk1 at Ser(317), SMC1 at Ser(966), and H2AX at Ser(139) compared with control mice. These results indicate that near-infrared irradiation can non-thermally induce cytocidal effects in cancer cells as a result of activation of the DNA damage response pathway. The near-infrared irradiation schedule used here reduces discomfort and side effects. Therefore, this strategy may have potential application in the treatment of cancer.
Cancer Science 04/2012; 103(8):1467-73. · 3.33 Impact Factor
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Fumitoshi Karasawa,
Akira Shiota,
Yukinobu Goso,
Motohiro Kobayashi,
Yoshiko Sato,
Junya Masumoto,
Maiko Fujiwara,
Shuichi Yokosawa,
Takashi Muraki,
Shinichi Miyagawa,
Masatsugu Ueda,
Michiko N Fukuda,
Minoru Fukuda,
Kazuhiko Ishihara, Jun Nakayama
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ABSTRACT: Gastric gland mucin secreted from the lower portion of the gastric mucosa contains unique O-linked oligosaccharides (O-glycans) having terminal α1,4-linked N-acetylglucosamine residues (αGlcNAc). Previously, we identified human α1,4-N-acetylglucosaminyltransferase (α4GnT), which is responsible for the O-glycan biosynthesis and characterized αGlcNAc function in suppressing Helicobacter pylori in vitro. In the present study, we engineered A4gnt(-/-) mice to better understand its role in vivo. A4gnt(-/-) mice showed complete lack of αGlcNAc expression in gastric gland mucin. Surprisingly, all the mutant mice developed gastric adenocarcinoma through a hyperplasia-dysplasia-carcinoma sequence in the absence of H. pylori infection. Microarray and quantitative RT-PCR analysis revealed upregulation of genes encoding inflammatory chemokine ligands, proinflammatory cytokines, and growth factors, such as Ccl2, Il-11, and Hgf in the gastric mucosa of A4gnt(-/-) mice. Further supporting an important role for this O-glycan in cancer progression, we also observed significantly reduced αGlcNAc in human gastric adenocarcinoma and adenoma. Our results demonstrate that the absence of αGlcNAc triggers gastric tumorigenesis through inflammation-associated pathways in vivo. Thus, αGlcNAc-terminated gastric mucin plays dual roles in preventing gastric cancer by inhibiting H. pylori infection and also suppressing tumor-promoting inflammation.
The Journal of clinical investigation 03/2012; 122(3):923-34. · 15.39 Impact Factor
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ABSTRACT: Two members of the N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) family, GlcNAc6ST-1 and GlcNAc6ST-2, function in the biosynthesis of 6-sulfo sialyl Lewis X-capped glycoproteins expressed on high endothelial venules (HEVs) in secondary lymphoid organs. Thus, both enzymes play a critical role in L-selectin-expressing lymphocyte homing. Human GlcNAc6ST-1 is encoded by a 1593-bp open reading frame exhibiting two 5' in-frame methionine codons spaced 141 bp apart. Both resemble the consensus sequence for translation initiation. Thus, it has been hypothesized that both long and short forms of GlcNAc6ST-1 may be present, although endogenous expression of either form has not been confirmed in humans. Here, the authors developed an antibody recognizing amino acid residues between the first two human GlcNAc6ST-1 methionines. This antibody specifically recognizes the long form of the enzyme, a finding validated by Western blot analysis and immunofluorescence cytochemistry of HeLa cells misexpressing long and/or short forms of human GlcNAc6ST-1. Using this antibody, the authors carried out immunofluorescence histochemistry of human lymph node tissue sections and found endogenous expression of the long form of the enzyme in human tissue, predominantly in the trans-Golgi network of endothelial cells that form HEVs.
Journal of Histochemistry and Cytochemistry 01/2012; 60(5):397-407. · 2.72 Impact Factor
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Shingo Hatakeyama,
Kazuhiro Sugihara,
Toshiaki K Shibata, Jun Nakayama,
Tomoya O Akama,
Naoaki Tamura,
Shuk-Man Wong,
Andrey A Bobkov,
Yutaka Takano,
Chikara Ohyama,
Minoru Fukuda,
Michiko N Fukuda
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ABSTRACT: Although numerous carbohydrates play significant roles in mammalian cells, carbohydrate-based drug discovery has not been explored due to the technical difficulty of chemically synthesizing complex carbohydrate structures. Previously, we identified a series of carbohydrate mimetic peptides and found that a 7-mer peptide, designated I-peptide, inhibits hematogenous carbohydrate-dependent cancer cell colonization. During analysis of the endothelial surface receptor for I-peptide, we found that I-peptide bound to annexin 1 (Anxa1). Because Anxa1 is a highly specific tumor vasculature surface marker, we hypothesized that an I-peptide-like peptide could target anticancer drugs to the tumor vasculature. This study identifies IFLLWQR peptide, designated IF7, as homing to tumors. When synthetic IF7 peptide was conjugated to fluorescent Alexa 488 (A488) and injected intravenously into tumor-bearing mice, IF7-A488 targeted tumors within minutes. IF7 conjugated to the potent anticancer drug SN-38 and injected intravenously into nude mice carrying human colon HCT116 tumors efficiently suppressed tumor growth at low dosages with no apparent side effects. These results suggest that IF7 serves as an efficient drug delivery vehicle by targeting Anxa1 expressed on the surface of tumor vasculature. Given its extremely specific tumor-targeting activity, IF7 may represent a clinically relevant vehicle for anticancer drugs.
Proceedings of the National Academy of Sciences 11/2011; 108(49):19587-92. · 9.68 Impact Factor
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M Tony Hollingsworth,
Gerald W Hart,
James C Paulson,
Elizabeth Stansell,
Kevin Canis,
I-Cheuh Huang,
Maria Panico,
Howard Morris,
Stuart Haslam,
Michael Farzan, [......],
Kazunori Hamamura,
Takenosuke Yoshida,
Kaoru Akita,
Tetsuya Okajima,
Keiko Furukawa,
Takeshi Urano,
Koichi Furukawa,
L Renee Ruhaak,
Suzanne Miyamoto,
Carlito B Lebrilla
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ABSTRACT: Cell surface mucins configure the cell surface by presenting extended protein backbones that are heavily O-glycosylated. The glycopeptide structures establish physicochemical properties at the cell surface that enable and block the formation of biologically important molecular complexes. Some mucins, such as MUC1, associate with receptor tyrosine kinases and other cell surface receptors, and engage in signal transduction in order to communicate information regarding conditions at the cell surface to the nucleus. In that context, the MUC1 cytoplasmic tail (MUC1CT) receives phosphorylation signals from receptor tyrosine kinases and serine/threonine kinases, which enables its association with different signaling complexes that conduct these signals to the nucleus and perhaps other subcellular organelles. We have detected the MUC1CT at promoters of over 500 genes, in association with several different transcription factors, and have shown that promoter occupancy can vary under different growth factor conditions. However, the full biochemical nature of the nuclear forms of MUC1 and its function at these promoter regions remain undefined. I will present evidence that nuclear forms of the MUC1CT include extracellular and cytoplasmic tail domains. In addition, I will discuss evidence for a hypothesis that the MUC1CT possesses a novel catalytic function that enables remodeling of the transcription factor occupancy of promoters, and thereby engages in regulation of gene expression.
Glycobiology 11/2011; 21(11):1454-531. · 3.58 Impact Factor
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Tatsuya Kobayashi,
Keiichi Sakai,
Tsuyoshi Tada,
Yasuyuki Sekiguchi,
Yosuke Hara,
Kunihiko Kodama,
Tetsuya Goto,
Yuichiro Tanaka,
Kenji Sano, Jun Nakayama,
Kazuhiro Hongo
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ABSTRACT: We report a 67-year-old woman who was diagnosed with a gliosarcoma at a second operation after diagnosis of a fibrillary astrocytoma 5 months previously. Initially, she underwent a CT-guided stereotactic biopsy. Histological examination showed fibrillary astrocytoma (World Health Organization [WHO] grade II). Loss of heterozygosity (LOH) on 1p, 10q, and 19q was not detected. She received chemotherapy, but no radiotherapy. Five months after the biopsy, MRI revealed rapid tumor growth. Tissue obtained from partial removal of the tumor revealed gliosarcoma (WHO grade IV), and LOH on 10q and 19q was detected. The history, histopathology, and genetic alterations of this patient are discussed.
Journal of Clinical Neuroscience 09/2011; 18(9):1251-4. · 1.25 Impact Factor
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ABSTRACT: α-Dystroglycan (DG) carries glycan chains that bind to laminin and thus function in homeostasis of not only skeletal muscle but also of various epithelial cells. Loss of glycosylation has been suggested to play important roles in tumor development, particularly in detachment and migration of carcinoma cells. We previously reported that glycosylation of α-DG, but not levels of α-DG core protein itself, is reduced in prostate carcinoma. In this study, we investigate the association between reduction of laminin-binding glycans on α-DG and the degree of tumor cell differentiation and/or infiltrative properties, as assessed by the Gleason grading system.
Immunohistochemical analysis of 146 biopsy specimens of prostate adenocarcinoma with various Gleason scores was carried out employing IIH6 and 6C1 antibodies, which recognize laminin-binding glycans on α-DG and α-DG core proteins, respectively. Double immunofluorescence staining was performed to evaluate colocalization of α-DG and laminin, and to determine which types of epithelial cells express laminin-binding glycans on α-DG.
Reduction of α-DG glycosylation, rather than loss of α-DG core protein, was correlated with higher Gleason patterns. Reduction was most conspicuous at the interface between carcinoma cells and the basement membrane. In addition, in non-neoplastic prostate glands, laminin-binding glycans were expressed predominantly on the basolateral surface of basal cells.
Reduced expression of laminin-binding glycans on α-DG may contribute to formation of highly infiltrative behavior of prostate carcinoma cells. Substantial reduction of laminin-binding glycans in carcinoma tissue could be partly ascribed to disappearance of pre-existing basal cells.
The Prostate 08/2011; 71(11):1151-7. · 3.48 Impact Factor
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ABSTRACT: Astrocytic tumor is the most prevalent primary brain tumor. However, the role of cell surface carbohydrates in astrocytic tumor invasion is not known. In a previous study, we showed that polysialic acid facilitates astrocytic tumor invasion and thereby tumor progression. Here, we examined the role of HNK-1 glycan in astrocytic tumor invasion. A Kaplan-Meier analysis of 45 patients revealed that higher HNK-1 expression levels were positively associated with increased survival of patients. To determine the role of HNK-1 glycan, we transfected C6 glioma cells, which lack HNK-1 glycan expression, with β1,3-glucuronyltransferase-P cDNA, generating HNK-1-positive cells. When these cells were injected into the mouse brain, the resultant tumors were 60% smaller than tumors emerging from injection of the mock-transfected HNK-1-negative C6 cells. HNK-1-positive C6 cells also grew more slowly than mock-transfected C6 cells in anchorage-dependent and anchorage-independent assays. C6-HNK-1 cells migrated well after treatment of anti-β1 integrin antibody, whereas the same treatment inhibited cell migration of mock-transfected C6 cells. Similarly, α-dystroglycan containing HNK-1 glycan is different from those containing the laminin-binding glycans, supporting the above conclusion that C6-HNK-1 cells migrate independently from β1-integrin-mediated signaling. Moreover, HNK-1-positive cells exhibited attenuated activation of ERK 1/2 compared with mock-transfected C6 cells, whereas focal adhesion kinase activation was equivalent in both cell types. Overall, these results indicate that HNK-1 glycan functions as a tumor suppressor.
Journal of Biological Chemistry 07/2011; 286(37):32824-33. · 4.77 Impact Factor
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Hiroe Kitahara,
Junya Masumoto,
Alan L Parker,
Fukuto Maruta,
Naoki Kubo,
Akira Shimizu,
Noriyuki Akita,
Shiro Miwa,
Naoya Kobayashi, Jun Nakayama,
Shinichi Miyagawa
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ABSTRACT: Cholangiocarcinoma (CCA) is a common carcinoma of the liver, and the majority of patients with CCA have a poor prognosis due to the lack of effective nonsurgical therapies in addition to its rapid progression and inoperability at the time of diagnosis. The development of novel nonsurgical therapeutics that efficiently target CCA could significantly improve the prognosis for patients presenting with CCA. Here, we describe the iterative production and characterization of a novel peptide, designated COP35 (CCA-binding oligopeptide 35), which binds selectively to human CCA, identified by bacteriophage biopanning using the intrahepatic CCA cell line RBE and the normal cholangiocyte cell line MMNK-1. COP35 was found to augment the growth inhibitory effects of 5-fluorouracil (5-FU) against RBE cells. Utilizing pull-down assay and liquid chromatography, we identify the clathrin heavy chain accompanied by GRP78/BiP as a COP35-binding partner. In summary, we identify COP35 as a possible candidate for peptide-targeted therapies for CCA.
Molecular Cancer Research 06/2011; 9(6):688-701. · 4.29 Impact Factor
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ABSTRACT: Lymphocyte homing is regulated by a multistep process mediated by sequential adhesive interactions between circulating lymphocytes and high endothelial venules (HEVs). In gut-associated lymphoid tissue (GALT), the initial interactive step, "tethering and rolling," is partly mediated by integrin α4β7 expressed on GALT-homing lymphocytes and its ligand MAdCAM-1, which is exclusively expressed on HEVs in GALT. To probe functional MAdCAM-1 in tissue sections, we developed a soluble integrin α4β7 heterodimeric IgG chimera by joining the extracellular region of mouse integrin α4 and β7 subunits to a human IgG Fc domain. Western blot analysis revealed that co-transfection of HEK 293T cells with expression vectors encoding integrin α4•IgG and β7•IgG results in the formation of α4β7•IgG heterodimeric chimeras. This complex preferentially binds to CHO cells expressing MAdCAM-1 and, to a lesser extent, to cells expressing VCAM-1, but not to cells expressing ICAM-1. Moreover, α4β7•IgG specifically binds to HEVs in GALT in situ in a divalent cation-dependent fashion and inhibits lymphocyte binding to HEVs in GALT. These findings indicate that α4β7•IgG can be used as a probe for functional MAdCAM-1 expressed on HEVs in GALT and could potentially serve as an anti-inflammatory drug inhibiting GALT-specific lymphocyte migration.
Journal of Histochemistry and Cytochemistry 03/2011; 59(6):572-83. · 2.72 Impact Factor
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Masaya Fujita,
Akiko Tsuchida,
Akiko Hirata,
Natsumi Kobayashi,
Kohtaro Goto,
Kenji Osumi,
Yuriko Hirose, Jun Nakayama,
Takashi Yamanoi,
Hisashi Ashida,
Mamoru Mizuno
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ABSTRACT: In mammals, α-linked GlcNAc is primarily found in heparan sulfate/heparin and gastric gland mucous cell type mucin. α-N-Acetylglucosaminidases (αGNases) belonging to glycoside hydrolase family 89 are widely distributed from bacteria to higher
eukaryotes. Human lysosomal αGNase is well known to degrade heparin and heparan sulfate. Here, we reveal the substrate specificity
of αGNase (AgnC) from Clostridium perfringens strain 13, a bacterial homolog of human αGNase, by chemically synthesizing a series of disaccharide substrates containing
α-linked GlcNAc. AgnC was found to release GlcNAc from GlcNAcα1,4Galβ1pMP and GlcNAcα1pNP substrates (where pMP and pNP represent p-methoxyphenyl and p-nitrophenyl, respectively). AgnC also released GlcNAc from porcine gastric mucin and cell surface mucin. Because AgnC showed
no activity against any of the GlcNAcα1,2Galβ1pMP, GlcNAcα1,3Galβ1pMP, GlcNAcα1,6Galβ1pMP, and GlcNAcα1,4GlcAβ1pMP substrates, this enzyme may represent a specific glycosidase required for degrading α-GlcNAc-capped O-glycans of the class III mucin secreted from the stomach and duodenum. Deletion of the C-terminal region containing several
carbohydrate-binding module 32 (CBM32) domains significantly reduced the activity for porcine gastric mucin; however, activity
against GlcNAcα1,4Galβ1pMP was markedly enhanced. Dot blot and ELISA analyses revealed that the deletion construct containing the C-terminal CBM-C2
to CBM-C6 domains binds strongly to porcine gastric mucin. Consequently, tandem CBM32 domains located near the C terminus
of AgnC should function by increasing the affinity for branched or clustered α-GlcNAc-containing glycans. The agnC gene-disrupted strain showed significantly reduced growth on the class III mucin-containing medium compared with the wild
type strain, suggesting that AgnC might have an important role in dominant growth in intestines.
Journal of Biological Chemistry 02/2011; 286(8):6479-6489. · 4.77 Impact Factor
