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Breast cancer is the most frequent cancer threatening the lives of women between the ages of 30 and 64. The cancer antigen 15-3 assay (CA15-3) has been widely used for the detection of breast cancer recurrence; however, its sensitivity and specificity are inadequate. We previously found that the breast cancer cell line YMBS secretes mucin 1 possessing 3′-sulfated core1 (3Score1-MUC1) into the medium. Therefore, we here evaluated whether 3Score1-MUC1 is secreted into the blood streams of breast cancer patients, and whether it can serve as an improved breast cancer marker. We developed a lectin-sandwich immunoassay, called Gal4/MUC1, using a 3′-sulfated core1-specific galectin-4 and a MUC1 monoclonal antibody. Using the Gal4/MUC1 assay method, we found that 3Score1-MUC1 was profoundly expressed in the blood streams of patients with recurrent and/or metastatic breast cancer. The positive ratio of the Gal4/MUC1 assay was higher than that of the CA15-3 assay in both primary (n = 240) and relapsed (n = 43) patients, especially in the latter of which the positive ratio of Gal4/MUC1 was 86%. whereas that of CA15-3 was 47%. Furthermore, serum Gal4/MUC1 levels could more sensitively reflect the recurrence of primary breast cancer patients after surgery. Therefore, the Gal4/MUC1 assay should be an excellent alternative to the CA15-3 tumor marker for tracking the recurrence and metastasis of breast cancer.

Detailed structural analysis of high molecular weight human milk oligosaccharides (HMOs) is still a challenging task. Here we present a modular strategy for a flexible de novo structural characterization of this class of molecules. The protocol combines established techniques such as separation by two-dimensional high-performance liquid chromatography with different types of mass spectrometry, exoglycosidase digestion, and linkage analysis in an individual glycan-based manner. As a proof of principle, this approach was applied to two distinct HMO isomers representing a difucosylated octaose core and a trifucosylated decaose core. Obtained data revealed the presence of one terminal Lewis A and one internal Lewis X epitope in the case of the octaose and led to the identification of this molecule as a difucosylated iso-lacto-N-octaose. The trifucosylated, doubly branched lacto-N-neo-decaose was shown to represent a new type of HMO core structure in which the branched antenna is linked to carbon atom 3 of the innermost galactosyl residue. Hence, using this analytical protocol a novel HMO structure could be defined. Our results further demonstrate that a combination of different techniques may be required for de novo structural analysis of these molecules.

Human serum Krebs von den Lugen-6 (KL-6) antigen is a MUC1 glycoprotein (KL-6/MUC1) recognized by anti-KL-6 monoclonal antibody (KL-6/mAb) and has been utilized as a diagnostic marker for interstitial pneumonia. KL-6/mAb is thought to recognize the specific glycopeptides sequence of MUC1, but the precise glycan structure of the epitope is unclear. In this study, we determined the carbohydrate structures of KL-6/MUC1 to search the carbohydrate epitopes for KL-6/mAb. KL-6/MUC1 was purified from the culture medium of human breast cancer YMB-S cells by KL-6/mAb-affinity chromatography; the O-linked glycan structures were determined in combination with paper electrophoresis, several lectin column chromatographies, sialidase digestion and methanolysis. KL-6/MUC1 contained core 1 and extended core 1 glycans modified with one or two sialic acid/sulfate residues. Based on these structures, several synthetic glycans binding to anti-KL-6/mAb were compared with one another by surface plasmon resonance. Sequentially, related radiolabeled oligosaccharides were enzymatically synthesized and analyzed for binding to a KL-6/mAb-conjugated affinity column. 3'-sialylated, 6'-sulfated LNnT [Neu5Acα2-3(SO(3)(-)-6)Galβ1-4GlcNAcβ1-3Galβ1-4Glc], 3'-sialylated, 6-sulfated core 1 [Neu5Acα2-3Galβ1-3(SO(3)(-)-6)GalNAc] and disulfated core 1 SO(3)(-)-3Galβ1-3(SO(3)(-)-6)GalNAc exhibited substantial affinity for KL-6/mAb, and 3'-sulfated core 1 derivatives [SO(3)(-)-3Galβ1-3(±Neu5Acα2-6)GalNAc] and 3'-sialylated core 1 weakly interacted with KL-6/mAb. These results indicated that the possible carbohydrate epitopes of KL-6/mAb involve not only 3'-sialylated core 1 but also novel core 1 and extended core 1 with sulfate and sialic acid residues. Epitope expressing changes with suppression or over-expression of the Gal6ST (Gal 6-O-sulfotransferase) gene, suggesting that Gal6ST is involved in the biosynthesis of the unique epitopes of KL-6/mAb.

Galectin-8 has much higher affinity for 3'-O-sulfated or 3'-O-sialylated glycoconjugates and a Lewis X-containing glycan than for oligosaccharides terminating in Galβ1→3/4GlcNAc, and this specificity is mainly attributed to the N-terminal carbohydrate recognition domain (N-domain, CRD) (Ideo, H., Seko, A., Ishizuka, I., and Yamashita, K. (2003) Glycobiology 13, 713-723). In this study, we elucidated the crystal structures of the human galectin-8-N-domain (-8N) in the absence or presence of 4 ligands. The apo molecule forms a dimer, which is different from the canonical 2-fold symmetric dimer observed for galectin-1 and -2. In a galectin-8N-lactose complex, the lactose-recognizing amino acids are highly conserved among the galectins. However, Arg(45), Gln(47), Arg(59), and the long loop region between the S3 and S4 β-strands are unique to galectin-8N. These amino acids directly or indirectly interact with the sulfate or sialic acid moieties of 3'-sialyl- and 3'-sulfolactose complexed with galectin-8N. Furthermore, in the LNF-III-galectin-8N complex, van der Waals interactions occur between the α1-3-branched fucose and galactose and between galactose and Tyr(141), and these interactions increase the affinity toward galectin-8N. Based on the findings of these x-ray crystallographic analyses, a mutagenesis study using surface plasmon resonance showed that Arg(45), Gln(47), and Arg(59) of galectin-8N are indispensable and coordinately contribute to the strong binding of galectins-8N to sialylated and sulfated oligosaccharides. Arg(59) is the most critical amino acid for binding in the S3-S4 loop region.

GOTO cells, a neuroblastoma cell line retaining the ability to differentiate into neuronal or Schwann cells, were found to be rich in membrane rafts containing ganglioside GM2 and hypersensitive to lipid raft-disrupting methyl-beta-cyclodextrin (MbetaCD); the GM2-rich rafts and sensitivity to MbetaCD were markedly diminished upon their differentiation into Schwann cells. We first raised a monoclonal antibody that specifically binds to GOTO cells but not to differentiated Schwann cells and determined its target antigen as ganglioside GM2, which was shown to be highly concentrated in lipid rafts by its colocalization with flotillin, a marker protein of rafts. Disturbance of normal structure of the lipid raft by depleting its major constituent, cholesterol, with MbetaCD resulted in acute apoptotic cell death of GOTO cells, but little effects were seen on differentiated Schwann cells. Until this study, GM2-rich rafts are poorly characterized and MbetaCD hypersensitivity, which may have clinical implications, has not been reported.

While the CA 125 and SCC antigens are used as tumor markers for ovarian cancer and uterine cervical cancer, respectively, an effective marker for uterine corpus cancer has not been identified. We asked whether beta1,3-galactosyltransferase-4 and/or 5 (beta3Gal-T4/T5) could serve as novel tumor markers for detecting gynecological carcinomas, especially those of the uterine corpus.We obtained a monoclonal antibody and a polyclonal antiserum against beta3Gal-T5 and constructed a sandwich ELISA method. Western blot analysis and immunoprecipitation revealed that this ELISA recognizes both beta3Gal-T4 and beta3Gal-T5.We found beta3Gal-T4 and T5 enzymatic activity in ovarian cancer tissues, indicating that these enzymes are expressed at least in ovarian cancer. The cutoff value was determined by ROC analysis to be 5.4 ng/ml in the sera. The beta3Gal-T4/T5-positive rates for the sera from ovarian cancer and uterine cervical cancer patients were comparable with the CA 125- and SCC antigen-positive rates for these cancers, respectively. Significantly, the beta3Gal-T4/T5-positive rate was higher for uterine corpus cancer (64%) than the CA 125 (37%)- and CA 19-9 (24%)-positive rates. The stage I uterine corpus cancers had particularly high beta3Gal-T4/T5-positive rates (57%).beta3Gal-T4/T5 is a novel tumor marker for uterine corpus cancer and other gynecological cancers.

Enzymatic activities of some glycosyltransferases are markedly increased via complex formation with other transferases or cofactor proteins. We previously showed that β1,3-N-acetylglucosaminyltransferase-2 (β3Gn-T2) and β3Gn-T8 can form a heterodimer in vitro and that the complex exhibits much higher enzymatic activity than either enzyme alone (Seko, A., and Yamashita, K. (2005) Glycobiology 15, 943–951). Here we examined this activation and the biological significance of complex formation in differentiated HL-60 cells. β3Gn-T2 and -T8 were co-immunoprecipitated from the lysates of both-transfected COS-7 cells, indicating their association in vivo. We prepared inactive mutants of both enzymes by destroying the DXD motifs. The mixture of mutated β3Gn-T2 and intact β3Gn-T8 did not exhibit any activation, whereas the mixture of intact β3Gn-T2 and mutated β3Gn-T8 had increased activity, indicating the activation of β3Gn-T2 via complex formation. Next, we compared expression levels of β3Gn-T1-T8 in HL-60 cells and DMSO-treated differentiated HL-60 cells, which produce larger poly-N-acetyllactosamine chains. The expression level of β3Gn-T8 in the differentiated cells was 2.6-fold higher than in the untreated cells. Overexpression of β3Gn-T8, but not β3Gn-T2, induced an increase in poly-N-acetyllactosamine chains in HL-60 cells. These results raise a possibility that up-regulation of β3Gn-T8 in differentiated HL-60 cells increases poly-N-acetyllactosamine chains by activating intrinsic β3Gn-T2.

We evaluated the effects of the use of nonsteroidal anti-inflammatory drugs (NSAIDs) on endoscopic and histological findings in patients with rheumatoid arthritis (RA) before and after the eradication of Helicobacter pylori infection. Helicobacter pylori (H. pylori) eradication using lansoprazole 30 mg, amoxicillin 750 mg, and clarithromycin 200 mg twice daily for 1 week was conducted in 44 patients (mean age: 56.5 years) with RA. Using the updated Sydney system, endoscopic and histological findings of the greater curvature of the antrum, the greater curvature of the upper corpus, and the lesser curvature of the lower corpus were compared before and after eradication, for a mean follow-up period of 3.5 months. Overall, H. pylori eradication was successful in 32 patients (72.7%). Of these 32 patients, 23 were NSAID users. In the successful eradication group, (1) there was no significant change on endoscopic findings, including gastric erythema and erosion in all three regions irrespective of NSAIDs use; (2) of 17 active ulcers before eradication in NSAIDs users, all healed except for one duodenal ulcer that persisted, where one patient newly developed a gastric ulcer, one developed erosive duodenitis, and two developed reflux esophagitis, all in NSAID users; (3) neutrophil infiltration and chronic inflammation were significantly improved in all three regions after H. pylori eradication irrespective of use of NSAIDs, while atrophic change and intestinal metaplasia did not change. In the eradication failure group; (1) there was no significant change on endoscopic and histological findings in the three regions; (2) two of three ulcers present before eradication on NSAID users persisted even after eradication, and no new cases of gastric ulcer or erosive duodenitis occurred. In conclusion, over a mean follow-up period of 3.5 months, use of NSAIDs in Japanese patients with RA did not impair the healing process of gastric and duodenal ulcers nor did it affect the endoscopic and histological improvements associated with H. pylori eradication.

Breast cancer is the most frequent cancer threatening the lives of women between the ages of 30 and 64. The cancer antigen 15-3 assay (CA15-3) has been widely used for the detection of breast cancer recurrence; however, its sensitivity and specificity are inadequate. We previously found that the breast cancer cell line YMBS secretes mucin 1 possessing 3'-sulfated core1 (3Score1-MUC1) into the medium. Therefore, we here evaluated whether 3Score1-MUC1 is secreted into the blood streams of breast cancer patients, and whether it can serve as an improved breast cancer marker. We developed a lectin-sandwich immunoassay, called Gal4/MUC1, using a 3'-sulfated core1-specific galectin-4 and a MUC1 monoclonal antibody. Using the Gal4/MUC1 assay method, we found that 3Score1-MUC1 was profoundly expressed in the blood streams of patients with recurrent and/or metastatic breast cancer. The positive ratio of the Gal4/MUC1 assay was higher than that of the CA15-3 assay in both primary (n = 240) and relapsed (n = 43) patients, especially in the latter of which the positive ratio of Gal4/MUC1 was 86%. whereas that of CA15-3 was 47%. Furthermore, serum Gal4/MUC1 levels could more sensitively reflect the recurrence of primary breast cancer patients after surgery. Therefore, the Gal4/MUC1 assay should be an excellent alternative to the CA15-3 tumor marker for tracking the recurrence and metastasis of breast cancer.

A high-sensitivity immunoassay system with surface plasmon field-enhanced fluorescence spectrometry (SPFS) was constructed using a plastic sensor chip and then applied to the detection of total prostate-specific antigen (total PSA) and GalNAcβ1-4GlcNAc-linked prostate-specific antigen (LacdiNAc-PSA) in serum, to discriminate between prostate cancer (PC) and benign prostate hyperplasia (BPH). By using this automated SPFS immunoassay, the detection limit for total PSA in serum was as low as 0.04 pg/mL, and the dynamic range was estimated to be at least five digits. A two-step sandwich SPFS immunoassay for LacdiNAc-PSA was constructed using both the anti-PSA IgG antibody to capture PSA and Wisteria floribunda agglutinin (WFA) for the detection of LacdiNAc. The results of the LacdiNAc-PSA immunoassay with SPFS showed that the assay had a sensitivity of 20.0 pg/mL and permitted the specific distinction between PC and BPH within the PSA gray zone. These results suggested that high-sensitivity automated SPFS immunoassay systems might become a powerful tool for the diagnosis of PC and other diseases.

We characterized a novel member of the beta1,3-N-acetylglucosaminyltransferase (beta3Gn-T) gene family, beta3Gn-T8. A recombinant soluble form of beta3Gn-T8 was expressed in Pichia pastoris (P. pastoris), and its substrate specificity was compared with that of beta3Gn-T2. The two enzymes had similar substrate specificities and recognized tetraantennary N-glycans and 2,6-branched triantennary glycans in preference to 2,4-branched triantennary glycans, biantennary glycans, and lacto-N-neotetraose (LNnT), indicating their specificity for 2,6-branched structures such as [Galbeta1-->4GlcNAcbeta1-->2(Galbeta1-->4GlcNAcbeta1-->6)Manalpha1--> 6Man]. Interestingly, when soluble recombinant beta3Gn-T2 and beta3Gn-T8 were mixed, the Vmax/Km value of the mixture was 9.3- and 160-fold higher than those of individual beta3Gn-T2 and -T8, respectively. Sephacryl S-300 gel filtration of the enzymes revealed that apparent molecular weights of each beta3Gn-T2, beta3Gn-T8, and the mixture were 90-160, 45-65, and 110-210 kDa, respectively, suggesting that beta3Gn-T2 and -T8 can form a complex with enhanced enzymatic activity. This is the first report demonstrating that in vitro mixed glycosyltransferases show enhanced enzymatic activity through the formation of a heterocomplex. These results suggested that beta3Gn-T8 and beta3Gn-T2 are cooperatively involved in the elongation of specific branch structures of multiantennary N-glycans.

Interleukin (IL)-18 induces T cells and natural killer cells to produce not only interferon-gamma but also other cytokines by binding to the IL-18 receptor (IL-18R) alpha and beta subunits. However, little is known about how IL-18, IL-18Ralpha, and IL-18Rbeta form a high-affinity complex on the cell surface and transduce the signal. We found that IL-18 and IL-18Ralpha bind to glycosylphosphatidylinositol (GPI) glycan via the third mannose 6-phosphate diester and the second beta-GlcNAc-deleted mannose 6-phosphate of GPI glycan, respectively. To determine which GPI-anchored glycoprotein is involved in the complex of IL-18 and IL-18Ralpha, IL-18Ralpha of IL-18-stimulated KG-1 cells was immunoprecipitated together with CD48 by anti-IL-18Ralpha antibody. More than 90% of CD48 was detected as beta-GlcNAc-deleted GPI-anchored glycoprotein, and soluble recombinant human CD48 without GPI glycan bound to IL-18Ralpha, indicating that CD48 is associated with IL-18Ralpha via both the peptide portion and the GPI glycan. To investigate whether the carbohydrate recognition of IL-18 is involved in physiological activities, KG-1 cells were digested with phosphatidylinositol-specific phospholipase C before IL-18 stimulation. Phosphatidylinositol-specific phospholipase C treatment inhibited the phosphorylation of tyrosine kinases and the following IL-18-dependent interferon-gamma production. These observations suggest that the complex formation of IL-18.IL-18Ralpha. CD48 via both the peptide portion and GPI glycan triggers the binding to IL-18Rbeta, and the IL-18.IL-18Ralpha.CD48.IL-18Rbeta complex induces cellular signaling.

Tumor necrosis factor-alpha (TNF-alpha) binds to TNF-alpha receptors (TNFR) to produce a hexameric (TNF-alpha)(3)-(TNFR)(3) structure that stimulates apoptosis. We found by using ELISA that TNF-alpha binds to the glycosylphosphatidylinositol (GPI) anchor glycans of carcinoembryonic antigen, human placental alkaline phosphatase (hAP), and Tamm-Horsfall glycoprotein. These binding abilities were inhibited by 10(-6)M mannose-6-phosphate. Treatment of hAP with mild acid and phosphatase, which releases the N-acetylglucosamine (GlcNAc) beta1 --phosphate--6 residue from the GPI-anchor glycan of hAP, abrogated the binding of TNF-alpha to hAP. Thus, TNF-alpha binds to the GlcNAcbeta1--phosphate--6Man residue in GPI-anchor glycans. To investigate whether the carbohydrate-binding ability of TNF-alpha is related to its physiological functions, human lymphoma U937 cells were used. TNF-alpha stimulates U937 cell apoptosis in a dose-dependent manner and the presence of mannose-6-phosphate inhibited this. TNF-alpha-dependent tyrosine phosphorylation of several proteins in U937 cells was also diminished by mannose-6-phosphate. Phosphatidylinositol-specific phospholipase C-pretreatment also inhibited this tyrosine phosphorylation. These data suggest that TNF-alpha stimulates U937 cell apoptosis by forming a high-affinity nanomeric (TNF-alpha)(3)-(TNFR)(3)-(GPI-anchored glycan)(3) complex. The GPI-anchored glycoprotein involved remains to be identified.

Vesicular integral protein of 36 kDa (VIP36) is an intracellular lectin recognizing high-mannose type glycans and is highly expressed in salivary glands, especially the parotid gland, which secretes alpha-amylase in large quantities. Here immunoelectron microscopy demonstrated that VIP36 was primarily localized to secretory vesicles in the glandula parotis of the rat, where alpha-amylase also resided. A secretory vesicle fraction, prepared by Percoll density gradient centrifugation, contained both VIP36 and alpha-amylase. Moreover, alpha-amylase that was localized to these secretory vesicles contained high-mannose type glycans. In addition, VIP36 coprecipitated with alpha-amylase in an endo H treatment-sensitive manner. These results suggest that VIP36 is involved in the secretion of alpha-amylase in the rat parotid gland.

Glycosylphosphatidylinositol (GPI)-anchored proteins are ubiquitous in eukaryotes. The minimum conserved GPI core structure of all GPI-anchored glycans has been determined as EtN-PO4-6Manα1–2Manα1–6Manα1–4GlcN-myo-inositol-PO3H. Human placental alkaline phosphatase (AP) has been reported to be a GPI-anchored membrane protein. AP carries one N-glycan, (NeuAcα2→3)2Gal2GlcNAc2Man3GlcNAc(±Fuc)GlcNAc, and a GPI anchor, which contains an ethanolamine phosphate diester group, as a side chain. However, we found that both sialidase-treated soluble AP (sAP) and its GPI-anchored glycan bound to a Psathyrella velutina lectin (PVL)-Sepharose column, which binds β-GlcNAc residues. PVL binding of asialo-sAP and its GPI-anchored glycan was diminished by digestion with diplococcal β-N-acetylhexosaminidase or by mild acid treatment. After sequential digestion of asialo-sAP with β-N-acetylhexosaminidase and acid phosphatase, the elution patterns on chromatofocusing gels were changed in accordance with the negative charges of phosphate residues. Trypsin-digested sAP was analyzed by liquid chromatography/electrospray ionization mass spectrometry, and the structures of two glycopeptides with GPI-anchored glycans were confirmed as peptide-EtN-PO4-6Manα1→2(GlcNAcβ1-PO4→6)Manα1–6(±EtN-PO4→)Manα1→4GlcN, which may be produced by endo-α-glucosaminidase. In addition to AP, GPI-anchored carcinoembryonic antigen, cholinesterase, and Tamm-Horsfall glycoprotein also bound to a PVL-Sepharose column, suggesting that the β-N-acetylglucosaminyl phosphate diester residue is widely distributed in human GPI-anchored glycans. Furthermore, we found that the β-N-acetylglucosaminyl phosphate diester residue is important for GPI anchor recognition of aerolysin, a channel-forming toxin derived from Aeromonas hydrophila.

The Galbeta1-->4(SO(3)(-)-->6)GlcNAc moiety is present in various N-linked and O-linked glycans including keratan sulfate and 6-sulfosialyl-Lewis X, an L-selectin ligand. We previously found beta1,4-galactosyltransferase (beta4GalT) activity in human colonic mucosa, which prefers GlcNAc 6-O-sulfate (6SGN) as an acceptor to non-substituted GlcNAc (Seko, A., Hara-Kuge, S., Nagata, K., Yonezawa, S., and Yamashita, K. (1998) FEBS Lett. 440, 307-310). To identify the gene for this enzyme, we purified the enzyme from porcine colonic mucosa. The purified enzyme had the characteristic requirement of basic lipids for catalytic activity. Analysis of the partial amino acid sequence of the enzyme revealed that the purified beta4GalT has a similar sequence to human beta4GalT-IV. To confirm this result, we prepared cDNA for each of the seven beta4GalTs cloned to date and examined substrate specificities using the membrane fractions derived from beta4GalT-transfected COS-7 cells. When using several N-linked and O-linked glycans with or without 6SGN residues as acceptor substrates, only beta4GalT-IV efficiently recognized 6SGN, keratan sulfate-related oligosaccharides, and Galbeta1-->3(SO(3)(-)-->6GlcNAcbeta1-->6) GalNAcalpha1-O-pNP, a precursor for 6-sulfosialyl-Lewis X. These results suggested that beta4GalT-IV is a 6SGN-specific beta4GalT and may be involved in the biosynthesis of various glycoproteins carrying a 6-O-sulfated N-acetyllactosamine moiety.

The vesicular integral membrane protein VIP36 belongs to the family of animal lectins and may act as a cargo receptor trafficking certain glycoproteins in the secretory pathway. Immunoelectron microscopy of GH3 cells provided evidence that endogenous VIP36 is localized mainly in 70-100-nm-diameter uncoated transport vesicles between the exit site on the ER and the neighboring cis-Golgi cisterna. The thyrotrophin-releasing hormone (TRH) stimulation and treatment with actin filament-perturbing agents, cytochalasin D or B or latrunculin-B, caused marked aggregation of the VIP36-positive vesicles and the appearance of a VIP36-positive clustering structure located near the cis-Golgi cisterna. The size of this structure, which comprised conspicuous clusters of VIP36, depended on the TRH concentration. Confocal laser scanning microscopy confirmed the electron microscopically demonstrated distribution and redistribution of VIP36 in these cells. Furthermore, VIP36 colocalized with filamentous actin in the paranuclear Golgi area and its vicinity. This is the first study to show the ultrastructural distribution of VIP36 in the early secretory pathway in GH3 cells. It suggests that actin filaments are involved in glycoprotein transport between the ER and cis-Golgi cisterna by using the lectin VIP36.

Many reports show that N-glycans of glycoproteins play important roles in vectorial transport in MDCK cells. To assess whether structural differences in N-glycans exist between secretory glycoproteins and membrane glycoproteins, we studied the N-glycan structures of the glycoproteins isolated from MDCK cells. Polarized MDCK cells were metabolically labeled with [3H]glucosamine, and (3)H-labeled N-glycans of four glycoprotein fractions, secretory glycoproteins in apical and basolateral media, and apical and basolateral membrane glycoproteins, were released by glycopeptidase F. The structures of the free N-glycans were comparatively analyzed using various lectin column chromatographies and sequential glycosidase digestion. The four samples commonly contained high-mannose-type glycans and bi- and tri-antennary glycans with a bisected or non-bisected trimannosyl core. However, secretory glycoproteins in both media predominantly contained (sialyl)LacdiNAc sequences, +/-Sia alpha 2-->6GalNAc beta 1-->4GlcNAc beta 1-->R, which linked only to a non-bisected trimannosyl core. beta1-->4N-acetylgalactosaminyltransferase (beta 4GalNAc-T) activity in MDCK cells preferred non-bisected glycans to bisected ones in accordance with the proposed N-glycan structures. This secretory glycoprotein-predominant LacdiNAc sequence was also found in the case of human embryonic kidney 293 cells. These results suggest that the secretory glycoprotein-specific (sialyl)LacdiNAc sequence and the corresponding beta 4GalNAc-T are involved in transport of secretory glycoproteins.

Endothelins (ETs) participate directly and indirectly in angiogenesis via ET receptors. During early fetal angiogenesis, vascular endothelial growth factor (VEGF) and its receptors kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase-1 (Flt-1) are required for the development of the systemic vasculature. In late angiogenesis, stromal-cell-derived factor (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) act in an organ-specific manner to promote the formation and development of large blood vessels supplying the gastrointestinal tract. We studied the roles of these ligand receptors in angiogenesis during healing of gastric ulcers. We studied the following five groups, each consisting of ten cases of endoscopically confirmed gastric ulcer: active stage (GA), healing stage (GH) and scar stage (GS) of gastric ulcers located in the angulus; Helicobacter pylori (Hp)-positive gastritis (gast+); and Hp-negative gastritis (gast-). All cases in the ulcer groups were Hp-positive. The study materials consisted of frozen biopsy specimens of lesions arising in the angulus. ET-1 was measured by enzyme immunoassay. The other factors were assayed by reverse-transcription–PCR. The distributions of ET-1, ETA receptor (ETAR), SDF-1 and CXCR4 in the gastric mucosa were evaluated by enzyme immunoassay. ET-1 and ETAR reached peak levels during the GH (ET: P

Expression levels of sulfomucin in human colonic adenocarcinomas are lower than those in normal colonic mucosa; this should be in part caused by down-regulation of expression of sulfotransferases, but it remains unclear which Gal 3-O-sulfotransferase (Gal3ST) is responsible for the biosynthesis of sulfomucin. In this study, we first examined the substrate specificities of four Gal3STs cloned so far, and found that Galbeta1 3GlcNAcbeta1 3Galbeta1 4Glc (LNT) can be utilized only by Gal3ST-2 as an acceptor substrate. The substrate specificity of Gal3ST-2 is closely similar to those of Gal3ST activities present in human normal mucosa and adenocarcinomas, suggesting that Gal3ST-2 is the dominant Gal3ST in colon and colonic cancer. Secondly, using LNT as a substrate, we comparatively analyzed levels of Gal3ST-2 activities in non-mucinous adenocarcinoma, mucinous adenocarcinomas, and the adjacent normal mucosa. We found that levels of Gal3ST-2 activities in non-mucinous adenocarcinoma are significantly lower than those in the adjacent normal mucosa, while those in mucinous adenocarcinomas are not significantly different from those in the adjacent normal mucosa. Moreover, we showed by a competitive RT-PCR method that expression levels of transcript for Gal3ST-2 in non-mucinous adenocarcinoma are lower than those in normal mucosa. These results suggest that Gal3ST-2 is one of the enzymes responsible for biosynthesis of sulfomucin, and that expression levels of Gal3ST-2 are down-regulated in non-mucinous adenocarcinoma.

VIP36, an intracellular lectin that recognizes high mannose-type glycans (Hara-Kuge, S., Ohkura, T., Seko, A., and Yamashita, K. (1999) Glycobiology 9, 833-839), was shown to localize not only to the early secretory pathway but also to the plasma membrane of Madin-Darby canine kidney (MDCK) cells. In the plasma membrane, VIP36 exhibited an apical-predominant distribution, the apical/basolateral ratio being approximately 2. Like VIP36, plasma membrane glycoproteins recognized by VIP36 were found in the apical and basolateral membranes in the ratio of approximately 2 to 1. In addition, secretory glycoproteins recognized by VIP36 were secreted approximately 2-fold more efficiently from the apical membrane than from the basolateral membrane. Thus, the apical/basolateral ratio of the transport of VIP36-recognized glycoproteins was correlated with that of VIP36 in MDCK cells. Upon overproduction of VIP36 in MDCK cells, the apical/basolateral ratios of both VIP36 and VIP36-recognized glycoproteins were changed from approximately 2 to approximately 4, and the secretion of VIP36-recognized glycoproteins was greatly stimulated. In contrast to the overproduction of VIP36, that of a mutant version of VIP36, which has no lectin activity, was of no effect on the distribution of glycoproteins to apical and basolateral membranes and inhibited the secretion of VIP36-recognized glycoproteins. Furthermore, the overproduction of VIP36 greatly stimulated the secretion of a major apical secretory glycoprotein of MDCK cells, clusterin, which was found to carry at least one high mannose-type glycan and to be recognized by VIP36. In contrast to the secretion of clusterin, that of a non-glycosylated apical-secretion protein, galectin-3, was not stimulated through the overproduction of VIP36. These results indicated that VIP36 was involved in the transport and sorting of glycoproteins carrying high mannose-type glycan(s).

Galectin-4 is a member of galectin family and has two carbohydrate recognition domains. Although galectin-4 has been thought to function in cell adhesion, its precise carbohydrate binding specificity has not yet been clarified. We studied the carbohydrate binding specificity of galectin-4 comparatively with that of galectin-3, using surface plasmon resonance, galectin-3- or -4-Sepharose column chromatography and the inhibition assay of their binding to immobilized asialofetuin. Galectin-3 broadly recognized lactose, type 1, type 2, and core 1. The substitution at the C-2 and C-3 position of beta-galactose in these oligosaccharides with alpha-fucose, alpha-GalNAc, alpha-Neu5Ac, or sulfate increased the binding ability for galectin-3, whereas the substitution at the C-4 or C-6 position diminished the affinity. In contrast, galectin-4 had quite weak affinity to lactose, type 1, and type 2 (K(d) congruent with 8 x 10(-4) M). Galectin-4 showed weak binding ability to core 1 and C-2' or -3'-substituted lactose, type 1, and type 2 with alpha-fucose, alpha-GalNAc, or sulfate (K(d) : 5 x 10(-5) approximately 3 x 10(-4) M). Interestingly, the K(d) value, 3.4 x 10(-6) M, of SO(3)(-)-->3Galbeta1-->3GalNAc-O-Bn to galectin-4 at 25 degrees C was two orders of magnitude lower than that of core 1-O-Bn. 3'-Sialylated core 1 had very weak affinity to galectin-4, suggesting that 3'-O-sulfation of core 1 is critical for the recognition. These results suggest that galectin-4 has a unique carbohydrate binding specificity and interacts with O-linked sulfoglycans.

Serial lectin affinity chromatography is a convenient technique for characterizing glycan motifs (terminal glycan structures) of glycoproteins or released glycans. When these glycoconjugates are applied serially or in parallel to lectin-immobilized columns, information regarding the glycan motifs can be obtained. We demonstrate lectin affinity chromatographic methods for determining O-linked glycan structures of MUC1 purified from a breast cancer cell line, YMB-S, N-linked glycan structures of serum prostate-specific antigen from prostate cancer, and serum alkaline phosphatases from choriocarcinoma. These lectin-fractionated samples are analyzed quantitatively by measuring radioactivity, antigen contents are analyzed using enzyme-linked immunosorbent assay, and enzymatic activities are assessed.

Serial lectin affinity chromatography is a convenient technique for characterizing glycan motifs (terminal glycan structures) of glycoproteins or released glycans. When these glycoconjugates are applied serially or in parallel to lectin-immobilized columns, information regarding the glycan motifs can be obtained. We demonstrate lectin affinity chromatographic methods for determining O-linked glycan structures of MUC1 purified from a breast cancer cell line, YMB-S, N-linked glycan structures of serum prostate-specific antigen from prostate cancer, and serum alkaline phosphatases from choriocarcinoma. These lectin-fractionated samples are analyzed quantitatively by measuring radioactivity, antigen contents are analyzed using enzyme-linked immunosorbent assay, and enzymatic activities are assessed.

糖鎖認識能を持つサイトカインが存在することが報告されて、糖鎖認識によってサイトカインとその受容体との相互作用が修飾を受けている可能性が示唆されている。本報では、サイトカインの糖鎖認識能について概観し、さらにIL-2における糖鎖認識能の役割について述べる。CTLL-2細胞はIL-2依存的に増殖する。マンノースを5残基あるいは6残基持つ高マンノース型糖鎖によってCTLL-2細胞の増殖ならびにシグナル伝達が阻害されたことから、高マンノース型糖鎖がIL-2によるT細胞増殖のモジュレーターとして働くことが示された。また、IL-2受容体の各サブユニットを独立に発現させると、IL-2と弱い結合しか見られないことから、IL-2刺激によってどのようにIL-2とIL-2受容体α, β, γサブユニットとの高親和性複合体が形成されるのかは不明であった。しかし、我々はIL-2がIL-2受容体αサブユニット上のマンノースを5残基あるいは6残基持つ高マンノース型糖鎖と特定のペプチド鎖をデュアルに認識することを見出した。IL-2とIL-2受容体αサブユニット複合体形成がシグナル伝達に必要な高親和性複合体の形成の引き金をひくものと思われる。

The vectorial intracellular transport of N-glycan-linked glycoproteins is indispensable for biological functions. In order to sort these glycoproteins to the correct destination, animal intracellular lectins play important roles as sorting receptors. The roles of such lectins in the biosynthetic pathway from the endoplasmic reticulum (ER) to the cell surface are addressed in this review. Calnexin and calreticulin function via specific carbohydrates in quality control of newly synthesized glycoproteins in the ER, and ERGIC-53 seems to function in the transport of glycoproteins from ER to the Golgi complex. In addition to the well-understood role of mannose 6-phosphate receptor in lysosomal protein sorting, the vesicular integral protein of 36 kDa (VIP36) functions as a sorting receptor by recognizing high-mannose type glycans containing alpha1-->2Man residues for transport from Golgi to the cell surface in polarized epithelial cells.

The 36 kDa vesicular-integral membrane protein, VIP36, has been originally isolated from MDCK cells as a component of glycolipid-enriched detergent-insoluble complexes containing apical marker proteins, and its luminal domain shows homology to leguminous plant lectins and ERGIC-53. As the first step to identify the functional role of VIP36, the carbohydrate binding specificity of VIP36 was investigated using a fusion protein of glutathione- S -transferase and luminal domain of VIP36 (Vip36). It was found that VIP36 recognizes high-mannose type glycans containing alpha1-->2 Man residues and alpha-amino substituted asparagine. The binding of Vip36 to high-mannose type glycans was independent of Ca(2+)and theoptimal condition was pH 6.0 at 37 degrees C. The concentration at which half inhibition of the binding by Man(7-9).GlcNAc(2). N Ac. Asn occurred was 1.0 x 10(-9)M. The association constant between Man(7-9).GlcNAc(2)in porcine thyroglobulin and immobilized Vip36 was 2.1 x 10(8)M(-1)as determined by means of a biosensor based on surface plasmon resonance. These results indicate that VIP36 functions as an intracellular lectin recognizing glycoproteins which possess high-mannose type glycans, (Manalpha1-->2)(2-4).Man(5). GlcNAc(2).

A blood rheology study was conducted using Kikuchi's microchannel method in male Sprague Dawley rats with fatty liver induced by choline deficiency. Effects of eicosapentaenoic acid (EPA) on blood rheology were also evaluated. Male Sprague Dawley rats given normal feed served as the control. One group was given choline-deficient feed for 4 weeks (EPA (−) group), while another was given EPA (1000 mg/kg) once daily for 4 weeks together with choline-deficient feed (EPA (+) group). The microchannel passage time was determined using 100 mL of whole blood during observation under a microscope with a video camera. The passage time increased significantly in the EPA (−) group compared with the control group, while it was significantly shorter in the EPA (+) group compared with the EPA (−) group. Changes in platelet aggregation and leukocyte adhesion were less in the EPA (+) group than in the EPA (−) or the control groups. EPA supplementation appeared to correct blood flow changes induced by choline deficiency in rats.

Synchronized cultures of rat 3Y1 cells, prepared by the density-arrested method, were used to investigate the relationship of N-glycosylation to the cell cycle. Although total cellular proteins were synthesized independently of the cell cycle, the synthesis of membrane-bound proteins was found to be dependent on the cell cycle, the time of maximum synthesis being just before that of maximum DNA synthesis. The synthesis of lipid-linked oligosaccharides (LLO), which are utilized as intermediates in N-glycosylation, increased in the S phase to a level at least ten times higher than in the G1, G2, and M periods. Moreover, the activities of dehydrodolichyl diphosphate synthase and farnesyl diphosphate synthase, which synthesize the precursors of dolichol, also increased in the S phase concomitantly with LLO synthesis. However, since the cell cycle dependency curves of these two enzyme activities were somewhat broader than that of LLO synthesis, the rate-limiting enzyme in the regulation of LLO synthesis might be another one such as dehydrodolichol reductase. These results suggest that LLO synthesis is regulated by multiple synthetic enzymes which are activated in a cell cycle dependent manner.

著者は甲状腺疾患発見のため, 頸部の超音波検査時だけでなく腹部や乳腺などの超音波検査の際にも甲状腺を観察するよう努めている。甲状腺超音波検査を行なった632例について検討した。(1) 甲状腺超音波検査所見は結節性病変157例 (そのうち充実性腫瘤は52例), びまん性変化38例, 異常なし437例であった。(2) 結節性病変群には7例の甲状腺癌が含まれた。7例の年齢は41歳から92歳, 腫瘍径は8mmから28mm, 組織型は乳頭癌6例, 濾胞癌1例であった。腫瘍径25mm以上の3例では診察時に前頸部腫瘤を認めたが, 20mm以下の4例は腹部超音波検査の際に甲状腺を観察して発見したものであった。結節性病変に甲状腺機能異常を伴ったのは機能亢進2例と機能低下3例であった。(3) びまん性変化群には機能亢進5例 (亜急性甲状腺炎1例, バセドウ病4例), 機能低下2例 (慢性甲状腺炎) が含まれていた。そのうちバセドウ病の1例と機能低下2例では超音波検査前には甲状腺疾患を指摘されていなかった。甲状腺機能正常の31例の中にも10例の甲状腺自己抗体陽性例がみられた。(4) 超音波検査がきっかけとなって甲状腺機能異常が診断されたのは機能異常12例中5例で, この5例のうち4例が60歳以上の老年者であった。超音波検査は甲状腺癌だけでなく, 甲状腺機能異常の発見にも有用であった。甲状腺疾患は頻度が高く, 腹部などの超音波検査時に甲状腺の観察を追加することが有意義であった。

The major objective of the present study was to evaluate mechanisms by which teprenone, a gastric mucosal protecting agent, increases hepatic mucosal blood flow using male Sprague-Dawley rats. Hepatic and gastric blood flow was measured using a laser blood flow meter after administration of teprenone, dissolved in Tween 80, into the inferior vena cava. Teprenone itself increased hepatic and gastric blood flow. It also increased hepatic and gastric blood flow in rats with acute hepatic disorders due to carbon tetrachloride (CCL4) and improved histological changes, such as inflammatory cell infiltration and fatty changes in the liver. The fact that blood endothelin (ET) concentrations increased after administration of teprenone suggest that teprenone has great affinity for ET beta receptors and shows ET beta-receptor antagonist-like effects. Hepatic blood flow decreased after administration of N-nitro-L-arginine methyl ester, a nitric oxide (NO) synthetase inhibitor, suggesting that teprenone increase NO activity. Teprenone was thought to increase hepatic and gastric blood flow by different mechanisms, because it increased gastric mucosal prostaglandin E2 concentrations.

Trichosanthesjaponica agglutinin I (TJA-I) having a high affinity to the NeuAcα2-6Galβ1-4GIcNAc terminal sequence was used for an immunohistochemical study to analyze the expression of the NeuAcα2-6Gal structure in human colonic tissues. A TJA-I binding substance was detected in the mucosa of colonic carcinoma, but not in the normal and transitional mucosa. The TJA-I staining was correlated with the degree of histo logical differentiation of the carcinoma. TJA-I preferentially stained well-differentiated and moderately differentiated adenocarcinoma. In the carcinoma containing mucin lakes, the TJA-I staining reaction was either weak in intensity or negative. A comparative study using monoclonal antibody against sialosyl-Tn antigen (NeuAcα2-6-GaINAc) known to be expressed in colonic carcinoma tissues revealed that the areas exhibiting strong expressions of siaosyl-Tn antigen separated from the areas vividly reactive for the TJA-I staining. In addition, the expression of sialosyl-Tn antigen, unlike the TJA-I staining, was strong in the mucinous areas of the carcinoma. The results obtained in the present study suggest the possibility that a comparative staining method with TJA-I and monoclonal antibody against sialosyl-Tn antigen could be helpful to the diagnosis of human colonic carcinoma.

Investigation into the efficacy of bezafibrate against primary biliary cirrhosis, with histological references from cases receiving long term monotherapy

We previously reported that the carbohydrate-deficient glycoprotein (CDG) syndrome is an asparagine-N-linked sugar chain transfer deficiency [Yamashita et al. (1993) J. Biol. Chem. 268, 5783-5789]. In order to confirm this hypothesis, we applied electrospray ionization-mass spectrometric analysis to transferrin isoforms purified from patients with the CDG syndrome. Transferrin isoforms containing 4, 2, and 0 sialic acid residues, S4, S2, and S0, were separated by Mono Q anion exchange column chromatography from serum of a patient with the CDG syndrome. The molecular masses of S4, S2, and S0 were determined to be 79,570 +/- 5, 77,364 +/- 6, and 75,157 +/- 6 Da by electrospray ionization mass spectrometry (ESI/MS). The differences between S4 and S2, and between S2 and S0 were both in accordance with the molecular mass of a disialylated biantennary sugar chain [Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->6(Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc] (2,206 Da), showing that S0 is nonglycosylated, and that S4 and S2 carry 2 and 1 mol of asparagine-N-linked sugar chains, respectively. The nonglycosylated asparagine site of S2 was elucidated to be random by high performance liquid chromatography-ESI/MS of a tryptic peptide of reduced and pyridylethylated S2.ESI/MS analysis of transferrin purified through one step from serum is applicable for a definite diagnosis of the CDG syndrome.

The structure of over 93% of the sugar chains of serum transferrin purified from three patients with carbohydrate-deficient glycoprotein (CDG) syndrome was Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->6 (Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->3)Man beta 1-->4GlcNac beta 1-->4GlcNAc, similar to that in a healthy control. On chromatofocusing, CDG syndrome transferrin was separated into three major isoforms, S4, S2, and S0, containing 4, 2, and 0 sialic acids/molecule at pH 5.12 (5.16), 5.42, and 5.80, respectively. On 7.5% SDS-polyacrylamide gel electrophoresis, the molecular masses of transferrin isoforms S4, S2, and S0 were 80, 77, and 74 kDa, respectively. Transferrin isoforms S4 and S2 were linked to 2 and 1 mol of sialylated biantennary sugar chain/transferrin molecule, on the other hand, isoform S0 was not linked to any asparagine-N-linked oligosaccharide. Accordingly, CDG syndrome can be concluded to be an asparagine-N-linked oligosaccharide transfer deficiency, although the primary deficient enzyme has not yet been determined.

A prostate-specific antigen (PSA) is widely used as a diagnostic marker for prostate cancer (PC) because of its high specificity. However, elevated serum PSA does not occur only in PC but also in benign prostatic hyperplasia (BPH). Since the structural changes of N-glycans during carcinogenesis are common phenomena, we investigated whether PC-specific N-glycans are linked to PSA. We first analyzed the carbohydrate structures of PSA derived from seminal fluid, serum of BPH and PC patients, and PC cell line, namely, LNCaP using eight lectin-immobilized columns and then with enzyme-linked immunosorbent assay (ELISA). The fraction of serum PSA from PC patients bound to both Fucα1-2Gal and βGalNAc binding Trichosanthes japonica agglutinin-II (TJA-II) column, while that from BPH patients did not exhibit this binding ability, thereby implying that there is elevated expression of α1,2-fucosylation and β-N-acetylgalactosaminylation of PSA during carcinogenesis. We then performed a real-time polymerase chain reaction (PCR) and confirmed that these structural changes

A prominent lectin in the root tubers of Trichosanthes japonica was purified by affinity chromatography on a porcine stomach mucin-Sepharose column and termed TJA-II. The molecular mass of the native lectin was determined to be 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and TJA-II was separated into two different subunits of 33 and 29 kDa in the presence of 2-mercaptoethanol. The respective subunits contained mannose, N-acetylglucosamine, fucose, and xylose. It was determined by equilibrium dialysis to have two equal binding sites per molecule, the association constant toward tritium-labeled Fuc alpha 1-->2Gal beta 1-->3GlcNAc beta 1-->3Gal beta 1-->4GlcOT being K alpha = 3.05 x 10(5) M-1. The precise carbohydrate binding specificity of immobilized TJA-II was studied using various tritium-labeled oligosaccharides. A series of oligosaccharides possessing Fuc alpha 1-->2Gal beta 1--> or GalNAc beta 1--> groups at their nonreducing terminals showed stronger binding ability than ones with Gal beta 1-->GlcNAc (Glc) groups, indicating that TJA-II fundamentally recognizes a beta-galactosyl residue and the binding strength increases on substitution of the hydroxyl group at the C-2 position with a fucosyl or acetylamino group. This lectin column is useful for fractionating oligosaccharides or glycoproteins containing blood group type 1H, type 2H, and Sd antigenic determinants.

Two lectins were purified from tuberous roots of Trichosanthes japonica. The major lectin, which was named TJA-II, interacted with Fuc alpha 1-->2Gal beta/GalNAc beta 1-->groups, and the other one, which passed through a porcine stomach mucin-Sepharose 4B column, was purified by sequential chromatography on a human alpha 1-antitrypsin-Sepharose 4B column and named TJA-I. The molecular mass of TJA-I was determined to be 70 kDa by sodium dodecyl sulfate gel electrophoresis. TJA-I is a heterodimer of 38-kDa (36-kDa) and 32-kDa (30-kDa) subunits with disulfide linkage(s), and the difference between 38 and 36 kDa, and between 32 and 30 kDa, is due to secondary degradation of the carboxyl-terminal side. It was determined by equilibrium dialysis that TJA-I has four equal binding sites per molecule, and the association constant toward tritium-labeled Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4GlcOT is Ka = 8.0 x 10(5) M-1. The precise carbohydrate binding specificity was studied using hemagglutinating inhibition assay and immobilized TJA-I. A series of oligosaccharides possessing a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc or HSO3(-)-->6Gal beta 1-->4GlcNAc group showed tremendously stronger binding ability than oligosaccharides with a Gal beta 1-->4GlcNAc group, indicating that TJA-I basically recognizes an N-acetyllactosamine residue and that the binding strength increases on substitution of the beta-galactosyl residue at the C-6 position with a sialic acid or sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)

Male Sprague-Dawley rats were fed a 0.1% ethionine-added choline-deficient diet for 8 weeks to induce liver cirrhosis. At the same time 100 mg/kg/day teprenone was administered orally in order to evaluate its effects on the liver and gastric mucosal blood flow. Blood flow increased not only in gastric mucosa but also in liver tissues in the teprenone group. Serum transaminase levels and histopathologic findings of the liver also improved. These findings suggest that teprenone alleviates hepatocellular injuries. This effect may be partly attributable to cytoprotective effects of the catenoid isoprenoid moiety of teprenone on liver cells.

In our previous study, we determined changes in hepatic blood flow using a Laser Doppler blood flow meter after i.v. injection of endothelin-1 (ET-1) or endothelin-3 (ET-3) at 2 nmol/kg in rats and found that ET-3 caused greater decreases in blood flow than ET-1. In the present study, we determined how the arachidonic acid cascade, mainly thromboxane A2 (TXA2), is related to ET-1 and ET-3 using indomethacin (INDO), which inhibits the biosynthesis of prostaglandin (PG), and OKY-046, a selective inhibitor of TXA2 synthesis. In the first series of experiments, ET-1 and ET-3 were administered after inhibiting the biosynthesis of PG by s.c. injection of 2 mg/kg of INDO. While INDO failed to inhibit the slight decrease in hepatic blood flow induced by ET-1, it significantly inhibited the marked decrease in hepatic blood flow elicited by ET-3. In the next series of experiments, ET-1 and ET-3 were administered after administration of 20 mg/kg of OKY-046. OKY-046 showed no effects in animals treated with ET-1, as in those pre-treated with INDO, while it significantly inhibited the decreases in hepatic blood flow induced by ET-3. These findings suggest that ET-1 decreases hepatic blood flow due to its direct effects although to a lesser extent than ET-3, while ET-3 does so due not only to its direct effects but also to TXA2-mediated effects. It is therefore likely that in addition to ET family peptides, PG-mediated mechanisms are involved in the regulation of hepatic microcirculation by ETs.

Changes in gastric mucosal and hepatic tissue blood flow were simultaneously determined using a laser-Doppler blood flow meter in rats given i.v. injection of endothelin-1 (ET-1) and endothelin-3 (ET-3), both at 2 nmol/kg. Gastric mucosal blood flow decreased significantly after administration of ET-1 compared to after administration of ET-3. Decreases in blood flow due to ET-1 were reversed by pre-treatment with 10 mg/kg of BQ-123 (sodium salt), an ETA receptor antagonist, to levels comparable to those induced by ET- 3, but BQ-123 had no effects on decreases in blood flow due to ET-3. On the other hand, decreases in hepatic tissue blood flow by ET-3 were significant compared to those by ET-1. Decreases in hepatic tissue blood flow due to ET-1 were slightly inhibited by pre-treatment with 10 mg/kg of BQ-123, but it had no effect at all on decreases due to ET-3. These findings indicate that decreases in gastric mucosal blood flow are mainly caused by ET-1 via ETA receptors inhibited by BQ-123, while decreases in hepatic tissue blood flow are caused mainly by ET-3 via non-ETA receptors not inhibited by BQ-123. The fact that ET-3 decreases blood flow more significantly than ET-1 suggests the involvement of ET-3 selective receptors (ETc). The findings obtained in the present study indicate that complex mechanisms are involved in the regulation of tissue blood flow by ET, with different receptor subtypes and ET family peptides being involved according to the type of tissue.

Bezafibrate in the treatment of primary biliary cirrhosis: comparison with ursodeoxycholic acid

A prostate-specific antigen (PSA) is widely used as a diagnostic marker for prostate cancer (PC) because of its high specificity. However, elevated serum PSA does not occur only in PC but also in benign prostatic hyperplasia (BPH). Since the structural changes of N-glycans during carcinogenesis are common phenomena, we investigated whether PC-specific N-glycans are linked to PSA. We first analyzed the carbohydrate structures of PSA derived from seminal fluid, serum of BPH and PC patients, and PC cell line, namely, LNCaP using eight lectin-immobilized columns and then with enzyme-linked immunosorbent assay (ELISA). The fraction of serum PSA from PC patients bound to both Fucalpha1-2Gal and betaGalNAc binding Trichosanthes japonica agglutinin-II (TJA-II) column, while that from BPH patients did not exhibit this binding ability, thereby implying that there is elevated expression of alpha1,2-fucosylation and beta-N-acetylgalactosaminylation of PSA during carcinogenesis. We then performed a real-time polymerase chain reaction (PCR) and confirmed that these structural changes were responsible for the elevated expression of fucosyltransferase I (FUT1) and beta-N-acetylgalactosaminyltransferase 4(B4GALNT4). Second, we measured TJA-II-bound PSA contents and the binding ratios of TJA-II column chromatography in serum PSA samples from 40 patients of both PC and BPH. The results indicated that both TJA-II-bound PSA content and TJA-II binding ratios (%) could be used to discriminate between PC and BPH with more than 95% probability, and TJA-II-bound PSA can be regarded as a potential marker of PC.

Galectins are a family of beta-galactoside-binding proteins that are widely found among animal species and that regulate diverse biological phenomena. To study the biological function of glycolipid-binding galectins, we purified recombinant Caenorhabditis elegans galectins (LEC-1-11) and studied their binding to C. elegans glycolipids. We found that LEC-8 binds to glycolipids in C. elegans through carbohydrate recognition. It has been reported that Cry5B-producing Bacillus thuringiensis strains can infect C. elegans and that the C. elegans Cry5B receptor molecules are glycolipids. We found that Cry5B and LEC-8 bound to C. elegans glycolipid-coated plates in a dose-dependent manner and that Cry5B binding to glycolipids was inhibited by the addition of LEC-8. LEC-8 is usually expressed strongly in the pharyngeal-intestinal valve and intestinal-rectal valve and is expressed weakly in intestine. However, when C. elegans were fed Escherichia coli expressing Cry5B, intestinal LEC-8::EGFP protein levels increased markedly. In contrast, LEC-8::EGFP expression triggered by Cry5B was reduced in toxin-resistant C. elegans mutants, which had mutations in genes involved in biosynthesis of glycolipids. Moreover, the LEC-8-deficient mutant was more susceptible to Cry5B than wild-type worms. These results suggest that the glycolipid-binding lectin LEC-8 contributes to host defense against bacterial infection by competitive binding to target glycolipid molecules.