Since the past, some genetic backgrounds have been pointed out as one of a cause of inflammatory bowel disease (colitis ulcerative, Crohn's disease). There genetic backgrounds include HLA typing and genotype of polymorphism site of cytokine genes. It is for a purpose of this study to make clear genetic factors of the susceptibility of inflammatory bowel disease in Japanese. We examined the association between inflammatory bowel disease and TNF gene, TNFRSF1A gene, TNFRSF1B gene, and IL18 gene. Furthermore, we studied it on CARD15/NOD2 gene. We found a significant difference in carrier frequency for haplotype AT (1466 A, 1493 T) of the TNFRSF1B gene between CD patients and the controls. The significance proved to be greater in CD patients with both internal and external fistula, and in those who were poor responders (n=22) to our treatments, which consisted of nutritional therapy, medical therapy and surgical therapy. In patients with Crohn's disease, the frequency of TCC-allele carriers was significantly higher than in healthy controls. And the magnitude of the association was more remarkable in females. The TCC-allele at codon 35 of IL18 may increase the risk for Crohn's disease, especially in females. It was reported that CARD15/NOD2 gene variants related to Crohn's disease in Europe and America in 2001. However, the same variants was not detected in Crohn's disease patient in Japan. Now we have been detecting CARD15/NOD2 genetic new variants. We have to make clear how CARD15/NOD2 gene variants is related to the onset of inflammatory bowel disease in Japanese.

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The pyridylamination method was originally described in 1978 as a means of analyzing glycan structures with high-sensitivity. Subsequently, the method has been applied to structure analyses of glycans including glycosidase digestion, 2D-mapping by various kinds of HPLC's, partial acetolysis, Smith degradation, methylation analysis, nuclear magnetic resonance, mass spectrometry, and affinity assay for lectins. Glycans on glycoconjugates are liberated by hydrazinolysis followed by N-acetylation. Reducing ends of the released glycans are tagged with 2-aminopyridine by reductive amination. Pyridylamino (PA-) derivatives of glycans with fluorescence and a positive charge have the following advantages: 1. high sensitivity in detection; 2. excellent separation in reversed phase HPLC; 3. high chemical stability under the conditions for structure elucidation; 4. applicable to many authentic methods for glycan analysis.

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Mucin 2 (MUC2) is the major intestinal mucin. O-glycans are attached to MUC2 in a potentially diverse arrangement, which is crucial for their interaction with endogenous and exogenous lectins. In the present report, a PTTTPITTTTK peptide corresponding to a portion of the MUC2 tandem repeat domain was synthesized, and incubated with UDP-N-acetyl-D-galactosamine (UDP-GalNAc) and detergent-soluble microsomes, prepared from the human colon carcinoma cell line LS174T. The products were fractionated by reverse-phase HPLC and characterized by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. Oligopeptides with GalNAc residues derived from PTTTPITTTTK, containing consecutive threonine residues, were found to be glycosylated with 1-7 GalNAc residues per single peptide. The sequences of all glycopeptides were determined. The results indicated that the predominant sites of the first through to the sixth GalNAc incorporation were Thr-3, Thr-6, Thr-5, Thr-2, Thr-4 and Thr-1, respectively. An exception was the presence of a glycopeptide with three GalNAc residues at Thr-1, Thr-4 and Thr-5. When the putative biosynthetic intermediates, PTTT^*PITTTTK and PT^*TTPITTTTK, were used as acceptors, all the products detected and analyzed were the same as those obtained when the unsubstituted peptide and microsome fractions of LS174T cells were incubated. Thus, the preferential order and maximum number of GalNAc incorporation into threonine residues of MUC2 core peptides are strictly regulated, when the microsome fraction of LS174T cells is used as a source of N-acetyl-D-galactosaminyltransferases.

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Recent progress of glycobiology has revealed that specific sugar chains on glycoproteins are concerned with cell adhesion, birth, differentiation and cancer metastasis. However, remodeling of cell surface proteins by glycosyltransferase genes (glycogenes), transgenic mice and even knock-out mice could not demonstrate real functions of oligosaccharide structures for each phenotype of glycosyltransferase transfectants, because many glycoproteins were glycosylated by glyco-genes. We should remember that glycoproteins modified with gene arrangement were not randomized but specific for each glycosyltransferase. In this study, we identified intracellular target proteins for glyco-genes using 2-dimensional electrophoresis followed by lectin blots. Used materials were transgenic mice of N-acetylglucosaminyltransferase III (GnT-III) and α1-6 fucosyltransferase (α1-6FucT) transfected Hep3B cells. GnT-III is involved in the synthesis of branching formation on N-glycans and α1-6FucT is involved in the attachment of fucose in the innermost GlcNAc on N-glycans. Treatment with diethylnitrosamine (DEN) of GnT-III transgenic and control mice brought tumor formation in the liver. The numbers of hepatic tumors were significantly smaller in GnT-III transgenic mice than in control mice. One of the target molecules for GnT-III in the serum of GnT-III transgenic mice was identified as haptoglobin. When α1-6FucT transfectants of Hep3B cells were injected into the spleen of athymic mice, tumor formation in the liver was dramatically suppressed as compared to their control cells. One of the causative molecules for this suppression of intrahepatic metastasis was identified as α5β1 integrin which is strongly α1-6 fucosylated. These approaches provide a new insight into glycobiology area and further functional glycomics would be required in the 21st century.

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Carbohydrates expressed on cell surface not only alter during carcinogenesis but also play important roles in cancer invasion and metastasis. Clinical significances of glycolipid and glycoprotein expression in urological malignancies were reported. In bladder tumor, large amounts of ganglioside GM 3 accumulate in superficial, compared with invasive bladder tumors and exogenous GM 3 inhibits the invasive potential of bladder tumor cells. A murine bladder carcinoma cell line (MBT-2) was transfected with a GM 3 synthase cDNA, because this line does not naturally express GM 3. Stable transfectants that over-expressed GM 3 were characterized by a reduced potential for cell proliferation, motility, invasion, xenograft tumor growth. N-acetylglucosaminyltransferase-V (GnT-V) may play important roles in early carcinogenesis of bladder tumor. Core 2-GnT (C 2-GnT) was up-regulated according to invasiveness of bladder tumor. In testicular tumor, Gb 3 was accumulated in seminoma and non-seminomatous germ cell tumors. Gb 3 may be a new marker of testicular tumors, especially seminomas, for which useful markers are so far lacking. Differentiation between seminoma and malignant lymphoma is sometimes difficult by histopathological findings but it is considered to be greatly facilitated by examination of the pattern of glycolipid expression. Only the presence or absence of galactosylgloboside (Gb 5), but of no other glycolipids or gangliosides, clearly correlated with metastatic potential in patients with seminoma. Immunohistochemistry using anti-C 2-GnT monoclonal antibody revealed high positive rate in embryonal carcinoma and choriocarcinoma, indicating possible involvement of C 2-GnT in hematogenous metastasis of testicular tumors. For prostate cancer, we analyzed lectin-binding activity of serum prostate specific antigen (PSA) to clarify cancerous change of carbohydrates expressed on PSA. Maackia amurensis (MAA) bound fraction of free PSA showed distinct difference between prostate cancer and benign prostate hypertrophy.

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Mucous glycoproteins secreted from the gastroduodenal mucosa and pancreatic duct showing gastric metaplasia characteristically contain GlcNAcα1→4Galβ→R structures in terminal ends of O-glycans, and this unique carbohydrate is frequently expressed in gastric cancer cells. We have isolated a cDNA encoding human α1, 4-N-acetylglucosaminyltransferase (α4GnT) responsible for the biosynthesis of GlcNAcα1 →4Galβ→R by expression cloning and characterized the substrate specificity and tissue localization of this enzyme. Using an antibody specific for α4GnT, we then demonstrated that this enzyme is frequently expressed in gastric cancer cells but not in peripheral blood cells. Thus, the expression level of α4GnT mRNA in the mononuclear cell fraction of peripheral blood was quantitatively measured using real-time RT-PCR to detect the circulating tumor cells. The transcripts of α4GnT were detected in 26 patients (63.4%) of gastric cancer patients but not in any of 23 healthy volunteers examined. In chronic active gastritis, 3 patients (33.3%) were positive for this assay, but the relative amounts of α4GnT mRNA to GAPDH mRNA in the chronic gastritis patients were 30-fold lower than those of the gastric cancer patients. Surprisingly, the transcripts of α4GnT were detectable even in early gastric cancer patients, when the cancer cells were limited to the submucosal layer. In addition, the expression level of the α4GnT was increased in association with the tumor progression. These combined results indicate that the quantitative analysis of α4GnT mRNA in the peripheral blood based on the real-time RT-PCR is useful for the detection of gastric cancer.

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さらなるゲノム解析の高速化のために, 革新的なゲノム解析方法である1分子ゲノム解析を目指して, そのために必要とされるDNA分子のマニピュレーション技術の開発を行った. 今回紹介した方法は, アガロースゲル中のDNA分子に10Hz前後の周波数の交流電場を印加すると, DNA分子が電場方向に伸張するという現象を利用したものである. この伸張現象を, 高分子物理学的な見地から説明するとともに, これを利用することにより, 最終的には100万塩基対を超える染色体DNAをも伸張させることに成功した. この方法は, 従来法に比べて非常に簡便でより高速に染色体DNAを伸張させることができるため, 1分子ゲノム解析への可能性が示唆された.

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Carbohydrates are widely distributed in nature and have multilateral functions. Recently glycobiology has evoked from the special interest in diverse functions of carbohydrates covalently bound to proteins and lipids. It has been revealed that the carbohydrate chains in such glycoconjugatges on cell surface are deeply associated with various cell events including division, differentiation, morphogenosis, fertilization, inflammation and metastasis. Although they are not formed by direct translation as in genomes and proteomes, they are important because many kinds of enzymes relating to their biosynthesis are under the control of genomes. Therefore, the carbohydrates in glycoconjugates are attractive as postscriptomes, and the method development for carbohydrate analysis is an urgent theme in glycobiology. Since a number of structurally resembling species of carbohdrates coexist in biological samples, their analysis requires the most efficient methods capable of high resolution and sensitive detection. For this reason capillary electrophoresis (CE) has become the most important tool for carbohydrate analysis. CE is, however, basically a method for ions, hence carbohydrates, which are generally neutral, are not good objects of CE. In addition, carbohydrates are normally difficult to sensitively detect, because they lack in chromophore/fluorophore. Chemical/enzymatic derivatization is the most appropriate strategy to solve these problems simultaneously. In this paper a number of methods for derivatization of carbohydrates for CE are compared and criticized, and the 1-phenyl-3-methyl-5-pyrazolone (PMP) method and the methylglycamine-4-nitro-2, 1, 3-benzodiazole tagging method (MG-NBD method) are selected as the most suitable methods for routine and ultramicro analyses of carbohydrates, respectively. A number of examples for their application are presented. A technique of automation of such analysis by CE with in-capillary derivatization is also presented. Miniaturization of the PMP method to microchip electrophoresis (ME) has realized rapid analysis of carbohydrates in few ten seconds. The versatile functions of carbohydrates stem from their binding with particular proteins, and therefore the study of carbohydrate-protein binding is also important. CE can be performed in free solution, not necessitating addition of any supporting materials. Owing to this single-phase property of CE one can observe interaction occurring in a capillary as the migration time change of either of these reactants in running buffer containing the counterpart. By mathematical treatment of such migration time change, we could develop a series of methods for micro-ultramicro estimation of association constant. On the other hand we could succeed in finding out a number of proteins in biological samples, which can specifically bind to glycans and glycoconjugates.

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Matrix metalloproteinase-3 (MMP-3) is expressed in a variety of cancer tissues. Recently, it was shown that the MMP-3 gene has a promoter polymorphism due to a variation in the length of a polymorphic track of adenosines located at position -1171, resulting in one allele having 5 adenosines (5A) and the other allele having 6 adenosines (6A). In this study, we detected this polymorphism by using MALDI-TOF mass spectrometry based on mini-sequencing assay. Among 3 mini-sequencing assays so far reported, VSET assay was most suitable for detecting MMP-3 promoter 5A/6A polymorphism. Relationship between the MMP-3 genotype and endometrial cancer was then examined for 190 samples including 122 healthy women and 68 patients with endometrial cancer. There was no significant association between them, but frequency of the 5A genotype was significantly increased in the patients with stages III and IV compared to those with stages I and II.

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The author has been focused on research and development of the following methodologies using lipoprotein fractionation by electrophoresis since 1970.
1. Development and application of thin layer agarose gel film and polyethylene tele-phthalate (PET) backing with hydrophilic treated surface for tough adhesion to agarose and polyacrylamide gels (1973)
2. Electrophoresis chamber with electronic cooling system based on Peltier effect (1980) and fine fractionation of HDL by α-cyclodextrin inclusion agarose isoelectric focusing electrophoresis (1982)
3. Enzymatic formazan staining for fractionation of cholesterol (Chol), triglycerides (TG), phospholipids (PL) or total lipids (TL=Chol+TG+PL)
a) Chol fraction (1981)
Cholesterol Esterase-Cholesterol Dehydrogenase-NAD-Diaphorase-NTB reaction
b) TG fraction (1983)
Lipoprotein Lipase-Glycerol Kinase-(Glycerol-3-phosphate Dehydrogenase)-NAD-Diaphorase-NTB reaction
c) PL fraction (1983)
Phospholipase D-Choline Oxidase-FAD-(1-m-PMS)-NTB reaction
d) TL (Chol+TG+PL) fraction (1983)
Complex reaction by combined reagent for the above three reactions
4. Development of lipid profile (Chol, TG, PL and TL fractionations) into 3-Dimensional (3-D) skyscraper analysis and bird's-eye overview of lipid metabolic abnormalities (1985)
5. Specific staining for precise fractionation of Chol in VLDL, LDL, HDL and degenerate LDL after polyacrylamide gel electrophoresis (1991)
With great expectation of wide propagation in the field of lipid research, the above technologies were transferred to a sophisticated specialist in separation analysis by electrophoresis, Helena Laboratories (Japan) for their commercialization. Consequently, the commercial products with automatic rapid electrophoresis (REP) procedure for Chol and TG based on Peltier effect and enzymatic formazan staining have come out into one of the most valuable laboratory diagnostic tools (1997).
Recently, it is said that lipotoxicity and adipotoxicity due to visceral obesity are background factors of multiple risk factor syndrome (MRFS) and primary preventiv emeasures is therefore most urgent, critical subject for the health and welfare administration. Accordingly, aggressive approach to investigate new, exciting laboratory tests and methodologies are keenly interested in detection of MRFS at the stage of preliminary group in order to prevent from advancing toward onset. For example, individual laboratory test result of HbAlc, HDL-Chol or LDL-Chol is too small to utilize as signal for MRFS even if disease state is in borderline type. Contrarily, ratio of increasing or decreasing components with progress of disorders such as LDL-Chol↑/HDL-Chol↓ and HDL-Chol↓/HbAlc↑ becomes surprisingly important information with wide dynamic range, which may be reliable index to MRFS. Under the circumstances, 21st century subject is how to analyze and reflect medical information on majority of adult withoutdisease so far, i.e., “population with unbalance” of eating and exercise lifestyle in visual pattern by 3-D skyscraper or 2-D analysis.

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In vitro における血清リポ蛋白の安定性について報告する. 37℃加温による血清外観の経日変化は, free では5日目, 10日目で濁りが減少し, その後, 経日的に濁りは増す. 一方, V. EやEDTAでは15日目まで変化なく, 20日目で増加した. PAGEによるリポ蛋白の経日変化については, LDL亜分画の出現, IDLの出現, LDLの陽極への移動, HDLの減少が認められた. 脂質項目の経日変化は, V. EとEDTAで安定した値が得られた. EDTAは良好なリポ蛋白変性防止効果が認められ, 血清中の金属類が酸化変性に大きく関与していることが示唆された. また同時に, in vitro において血清リポ蛋白は, 酸化変性以外にも変化することも確認された.

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Small dense LDL is oxidized easily compared with LDL of the large particle diameter. Small dense LDL gave strongly rise to cause arteriosclerosis. Usually, human serum is ultra-centrifuged to measure the size of LDL particle, and the obtained LDL fraction is analyzed by gradient gel electrophoresis (GGE). The GGE method is very difficult in clinical laboratory. However, the method of using Lipophor can be done very handily. The separation analysis of the lipoprotein by the electrophoretic method which uses PAG will be expected of a clinical application as a risk factor of various diseases including arteriosclerosis with degenerated LDL which makes small dense LDL and oxidized LDL. A relative mobility for the LDL fraction showed the tendency to long in patient's serum of high concentration of triglyceride and Apo B. In each case with hyperlipidemia, diabetes, cardiac infarction, arteriosclerosis, cerebral infarction, and angina, abnormal rate of RM value admitted high to be tended intentionally compared with the healthy person group. The RM value especially tended to high for IIb and IV type of hyperlipidemia. As for the relation of mobility (RM) by the electrophoretic method compared with LDL-Chol/LDL-apoB which had been used as an index of small dense LDL for the LDL fraction, the relation of the reverse-correlation was admitted. Therefore, the method by the electrophoretic method of small dense LDL is very clinical usefull.

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コレステロール分画・トリグリセライド分画同時解析法を用いて, 虚血性心疾患, 動脈硬化を起こしやすい糖尿病・腎疾患, 脂質異常を高頻度に認める肝疾患・悪性腫瘍・膠原病の計6疾患群の脂質分画と変性LDLを疾患別に比較検討した. 変性LDLの出現率は, 腎疾患で最も高率で, 高脂血症のみならず正脂血症や低脂血症でも高率であった. 脂質項目の平均値を, 高脂血症と正脂血症に分けて変性LDL陽性例と陰性例を比較すると疾患による特徴を認めた. 変性LDL陽性例では, 高TG・低HDL-C・IDLの出現は高率であったが, 高LDL-Cの出現では陰性例と差を認めなかった. CHDでは, 低HDL-Cが高率で, 陽性例では93%出現した. 糖尿病では, TCおよびTGが高値で, IDLの出現が高率であった. 高脂血症表現型は, 変性LDL陽性例でII b型・IV型が多くII a型は少なかった. 以上の結果, 脂質分画と変性LDLの出現は疾患により異なり, 本法により脂質分画の詳細を把握することは臨床的に有用であった.

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PAG電気泳動のLDL分画像から変性LDLを捉え, 加齢に伴う変化や糖尿病や動脈硬化性疾患との関係について検討した. LDLの分画像の形状を, S, A, N, Dの4型に分類し, IDLやレムナントを含むN型とD型を重視した. また, LDL分画像のピークおよびLDL分画の最も速い移動部位の相対移動度から small LDLと fast β-Lpの有無を捉え, この3点を変性LDLの所見とした. LDL分画像で捉えるものとRLP-Cとは異質であり, 酸化LDLはN型・D型と fast β-Lp の異常分画に存在した. N型・D型と fast β-Lp の増加には加齢に伴う変化が観察され, 特に40歳代と50歳代との間に大きな有意差を認めた. 加齢に伴う同様の変化はT-CやLDL-Cにおいてもみられるが, その変化よりも顕著であった. 若年者 (中学2年生) のLDL分画像は, N型とD型が少ないのに対し, fast β-Lp は50歳代と同等以上の高い陽性率を示し, 酸化LDLも fast β-Lp と同様の傾向を示した. 若年者の酸化LDLは主として fast β-Lp に存在し, 成人で増加するN型・D型に存在する酸化LDLとは異質であろうと考えられた. 糖尿病のLDL分画像は, 蛋白尿 (+) 群では, N型とD型が多く, 健常者に対しても, 蛋白尿 (-) 糖尿病群に対しても有意差を認めた. 50歳代健常者と比べ, 動脈硬化性疾患群にはN型・D型と small LDLが有意に多く, fast β-Lp 陽性も増加していた.

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