Biochemical characterization of plasma-derived tissue factor pathway inhibitor: post-translational modification of free, full-length form with particular reference to the sugar chain.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a physiological protease inhibitor that inhibits the initial reactions of the extrinsic blood coagulation pathway. Most TFPI in human plasma is associated with lipoproteins; however, the most functionally active form is thought to be the free, full-length form (f-pTFPI). Cell culture derived TFPI and recombinant TFPI (rTFPI) exhibit variations in their respective anticoagulant activity, which may be caused by post-translational modifications, such as the frequent differences in sugar chain structures among recombinant proteins. Sugar chain structures in rTFPI expressed in Chinese hamster ovary (CHO) cells have been reported previously, but those of plasma TFPI have not been. OBJECTIVES: To purify f-pTFPI and analyze the sugar chain structures. RESULTS AND CONCLUSION: f-pTFPI was purified to homogeneity from blood plasma using a combination of anion-exchange, heparin affinity, immunoaffinity, and reversed-phase chromatographies, resulting in a yield of 76%. f-pTFPI showed a partially phosphorylated glycoprotein comprising a total of 276 amino acids by peptide mapping. The sugar chain structures were analyzed by two-dimensional sugar mapping combined with exoglycosidase digestion of the pyridylamino sugar chains and the following results were obtained. (Sialyl) Galbeta1-3GalNAc was linked to Thr(175), partially to Thr(14) and Ser(174); sialyl complex-type sugar chains to Asn(117) and Asn(167), whereas Asn(228) was not glycosylated. Neuraminidase-resistant acidic sugar chains including sulfated sugar chains were not observed significantly. The protease inhibitory activities of f-pTFPI towards activated factor (F) X and tissue factor-activated FVII complex were identical to those of full-length rTFPI expressed in CHO cells.

Overexpression of the Arabidopsis thaliana EDS5 gene enhances resistance to viruses.

The Arabidopsis thaliana ENHANCED DISEASE SUSCEPTIBILITY 5 gene (EDS5) is required for salicylic acid (SA) synthesis in pathogen-challenged plants. SA and EDS5 have an important role in the Arabidopsis RCY1 gene-conferred resistance against the yellow strain of Cucumber mosaic virus [CMV(Y)], a Bromoviridae, and HRT-conferred resistance against the Tombusviridae, Turnip crinkle virus (TCV). EDS5 expression and SA accumulation are induced in response to CMV(Y) inoculation in the RCY1-bearing ecotype C24. To further discern the involvement of EDS5 in Arabidopsis defence against viruses, we overexpressed the EDS5 transcript from the constitutively expressed Cauliflower mosaic virus 35S gene promoter in ecotype C24. In comparison to the non-transgenic control, the basal level of salicylic acid (SA) was twofold higher in the 35S:EDS5 plant. Furthermore, viral spread and the size of the hypersensitive response associated necrotic local lesions (NLL) were more highly restricted in CMV(Y)-inoculated 35S:EDS5 than in the non-transgenic plant. The heightened restriction of CMV(Y) spread was paralleled by more rapid induction of the pathogenesis-related gene, PR-1, in the CMV(Y)-inoculated 35S:EDS5 plant. The 35S:EDS5 plant also had heightened resistance to the virulent CMV strain, CMV(B2), and TCV. These results suggest that, in addition to R gene-mediated gene-for-gene resistance, EDS5 is also important for basal resistance to viruses. However, while expression of the Pseudomonas putida nahG gene, which encodes the SA-degrading salicylate hydroxylase, completely suppressed 35S:EDS5-conferred resistance against CMV(Y) and TCV, it only partially compromised resistance against CMV(B2), indicating that SA-dependent and -independent mechanisms are associated with 35S:EDS5-conferred resistance against viruses.

PKA activation in concert with ARIS and asterosap induces the acrosome reaction in starfish.

The acrosome reaction (AR) is a fundamental event for fertilization, which is induced in concert with acrosome reaction-inducing substance (ARIS) and asterosap, both of which are components of starfish egg jelly (EJ). During the AR, a spermatozoon undergoes a series of physiological changes, such as in intracellular cGMP concentration ([cGMP]i), pHi and intracellular Ca2+ concentration ([Ca2+]i). Affinity purification of cGMP-binding protein resulted in the isolation of a regulatory subunit of the cAMP-dependent protein kinase A (PKA), suggesting the involvement of a cAMP-dependent pathway in the AR. By using a cAMP enzyme immunoassay, [cAMP]i was found to increase in starfish spermatozoa when stimulated with ARIS and asterosap. ARIS could also increase the [cAMP]i in the presence of high pH seawater. Pretreatment of spermatozoa with two specific and cell-permeable PKA inhibitors, H89 and KT5720, prevented the induction of the AR in a concentration-dependent manner. These results suggest that PKA activity participates in the induction of the AR with ARIS and asterosap. To investigate this, we have cloned a gene that encodes a regulatory subunit of PKA that had been identified in starfish spermatozoa.

Activation of defense-related gene expression and systemic acquired resistance in cucumber mosaic virus-infected tobacco plants expressing the mammalian 2'5'oligoadenylate system.

Tobacco plants expressing the mammalian 2'5'oligoadenylate system (2-5A system) exhibit resistance to cucumber mosaic virus (CMV). Here, to characterize the molecular aspect of the resistance to CMV in 2-5A system-expressing tobaccos, the activation of defense-related genes and systemic acquired resistance (SAR) as the markers for the hypersensitive resistance (HR), were elucidated. Northern hybridization analysis indicated that the expression of four pathogenesis-related (PR) protein genes and five HR-related genes were induced in CMV-infected tobaccos expressing 2-5A system. Furthermore, the induction of SAR against Pseudomonas syringae pv. tabaci as second challenge, was observed on CMV-inoculated tobaccos expressing 2-5A system. These results suggested that the resistance to CMV in tobacco expressing 2-5A system is associated with the establishment of an HR-like response.

Up-regulation of COX-2 by inhibition of COX-1 in the rat: a key to NSAID-induced gastric injury.

BACKGROUND: A recent study demonstrated that inhibition of both cyclooxygenase (COX)-1 and COX-2 is required for the development of nonsteroidal anti-inflammatory drug (NSAID)-induced gastric lesions. However, the role of COX-1 or COX-2 inhibition in the pathogenisis of these lesions remains unclear. AIM: To examine the gastric ulcerogenic properties of selective COX-1 and COX-2 inhibitors in rats and to investigate further the relationship of COX inhibition to various events involved in the process of NSAID-induced gastric lesions. METHODS: Animals were given various COX inhibitors p.o., either alone or in combination, and killed 8 h later. Under the treatment, gastric damage, prostaglandin (PG) E2 content, mucosal permeability, myeloperoxidase (MPO) activity as well as gastric motility were examined. RESULTS: The nonselective COX inhibitor indomethacin inhibited PGE2 production, enhanced gastric motility, and provoked severe lesions in the stomach, with an increase in mucosal permeability and MPO activity. In contrast, the selective COX-2 inhibitor rofecoxib did not induce any damage in the stomach and had no effect on mucosal PGE2 content. Similarly, the selective COX-1 inhibitor SC-560 also caused no gastric damage, despite inhibiting PGE2 production. The combined administration of SC-560 and rofecoxib, however, provoked gross damage in the gastric mucosa, in a dose-dependent manner for each drug. SC-560, but not rofecoxib, caused marked gastric hypermotility and an increase in mucosal permeability, although an increase in MPO activity was observed only when rofecoxib was coadministered. The normal gastric mucosa expressed COX-1 mRNA and not COX-2 mRNA, but COX-2 mRNA was expressed in the stomach after administration of SC-560 as well as indomethacin but not rofecoxib. CONCLUSION: These results suggest that the gastric ulcerogenic properties of NSAIDs are not accounted for solely by COX-1 inhibition, but require the inhibition of both COX-1 and COX-2. The inhibition of COX-1 up- regulates COX-2 expression, and COX-2/PGs may, in turn, counteract the deleterious affects of gastric hypermotility due to COX-1 inhibition.

Inhibition of both COX-1 and COX-2 is required for development of gastric damage in response to nonsteroidal antiinflammatory drugs.

We examined the gastric ulcerogenic property of selective COX-1 and/or COX-2 inhibitors in rats, and investigated whether COX-1 inhibition is by itself sufficient for induction of gastric damage. Animals fasted for 18 h were given various COX inhibitors p.o., either alone or in combination, and they were killed 8 h later. The nonselective COX inhibitors such as indomethacin, naproxen and dicrofenac inhibited PG production, increased gastric motility, and provoked severe gastric lesions. In contrast, the selective COX-2 inhibitor rofecoxib did not induce any damage in the stomach, with no effect on the mucosal PGE(2) contents and gastric motility. Likewise, the selective COX-1 inhibitor SC-560 also did not cause gastric damage, despite causing a significant decrease in PGE(2) contents. The combined administration of SC-560 and rofecoxib, however, provoked gross damage in the gastric mucosa, in a dose-dependent manner. SC-560 also caused a marked gastric hypermotility, whereas rofecoxib had no effect on basal gastric motor activity. On the other hand, the COX-2 mRNA was expressed in the stomach after administration of SC-560, while the normal gastric mucosa expressed only COX-1 mRNA but not COX-2 mRNA. These results suggest that the gastric ulcerogenic property of conventional NSAIDs is not accounted for solely by COX-1 inhibition and requires the inhibition of both COX-1 and COX-2. The inhibition of COX-1 up-regulates the COX-2 expression, and this may counteract the deleterious influences, such as gastric hypermotility and the subsequent events, due to a PG deficiency caused by COX-1 inhibition.

Protection by aspirin of indomethacin-induced small intestinal damage in rats: mediation by salicylic acid.

Most of non-steroidal anti-inflammatory drugs (NSAIDs) except aspirin (ASA) produce intestinal damage in rats. In the present study, we re-examined the intestinal toxic effect of ASA in rats, in comparison with various NSAIDs, and investigated why ASA does not cause damage in the small intestine, in relation to its metabolite salicylic acid (SA). Various NSAIDs (indomethacin; 10 mg/kg; flurbiprofen; 20 mg/kg; naproxen; 40 mg/kg; dicrofenac; 40 mg/kg; ASA; 20-200 mg/kg) were administered s.c., and the small intestinal mucosa was examined macroscopically 24 h later. All NSAIDs tested, except ASA, caused hemorrhagic lesions in the small intestine, with a decrease of mucosal PGE(2) contents. ASA did not provoke any damage, despite inhibiting (prostaglandin) PG production, and prevented the occurrence of intestinal lesions induced by indomethacin, in a dose-related manner. This protective action of ASA was mimicked by the equimolar doses of SA (17.8-178 mg/kg). Indomethacin caused intestinal hypermotility, in preceding to the occurrence of lesion, and this event was followed by increases of enterobacterial translocation in the mucosa. Both ASA and SA prevented both the intestinal hypermotility and the bacterial translocation seen after indomethacin treatment. In addition, the protective effect of SA was not significantly influenced by either the adenosine deaminase or the adenosine receptor antagonists. Following administration of ASA, the blood SA levels reached a peak within 30 min and remained elevated for more than 7 h. These results suggest that SA has a cytoprotective action against indomethacin-induced small intestinal lesions, and this action may be associated with inhibition of the intestinal hypermotility and the bacterial translocation, but not mediated by endogenous adenosine. Failure of ASA to induce intestinal damage may be explained, at least partly, by a protective action of SA, the metabolite of ASA.

Structural analysis of the sugar chains of human urinary thrombomodulin.

The sugar chains of human urinary thrombomodulin were studied. N- and O-linked sugar chains were simultaneously liberated by hydrazinolysis followed by N-acetylation and were tagged with 2-aminopyridine. Then the structures of the N- and O-linked pyridylamino (PA-) sugar chains were analyzed by two-dimensional sugar mapping combined with exoglycosidase digestion. The major N-linked sugar chains of human urinary thrombomodulin were found to be monosialo- and disialofucosylbiantennary chains, while the major O-linked sugar chain was +/-Siaalpha2-3Galbeta1-3(+/-Siaalpha2-6)GalNAc. Thrombomodulin also contained the reported structure SO4-3GlcAbeta1-3Galbeta1-3(+/-Siaalpha2-6)Galbeta1-4Xyl [H. Wakabayashi, S. Natsuka, T. Mega, N. Otsuki, M. Isaji, M. Naotsuka, S. Koyama, T. Kanamori, K. Sakai, and S. Hase (1999) J. Biol. Chem. 274, 5436-5442]. In addition to these sugar chains, a single Glc was linked to Ser 287.

Intestinal protection by lafutidine, a histamine H(2)-receptor antagonist, against indomethacin-induced damage in rats--role of endogenous nitric oxide.

BACKGROUND: We previously reported that lafutidine ((I)-2-(furfurylsulfinyl)-N-[4-[4-(piperidinomethyl)-2-pyridyl]oxy-(Z)-2-butenyl] acetamide), a novel histamine H(2)-receptor antagonist, protects the small intestine against indomethacin-induced damage, mediated by capsaicin-sensitive afferent neurons (CSN). MATERIAL AND METHODS: In the present study, we investigated whether or not the protective action of lafutidine against indomethacin-induced intestinal damage is mediated by endogenous nitric oxide (NO). Male SD rats were given indomethacin (10 mg/kg, s.c), killed 24 hr later, and the small intestinal mucosa was examined. Lafutidine (10 mg/kg) and capsaicin (10 mg/kg) was given p.o. twice 0.5 hr before and 9 hr after indomethacin. The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME: 10 mg/kg) or the selective iNOS inhibitor aminoguanidine (10 mg/kg) was given s.c. 1 hr before lafutidine, while L-arginine (200 mg/kg) was given i.p. 10 min before L-NAME. RESULTS: Indomethacin produced severe lesions in the small intestine, accompanied by increases in enterobacterial translocation in the mucosa. Both lafutidine and capsaicin significantly reduced the severity of these lesions, together with suppression of bacterial translocation. The protective action of lafutidine as well as capsaicin was almost totally abolished by L-NAME but not aminoguanidine, in a L-arginine-sensitive manner. Both lafutidine and capsaicin significantly increased intestinal mucus secretion, and these effects were also attenuated by prior administration of L-NAME. The exogenous NO donor NOR-3 prevented indomethacin-induced intestinal lesions at the dose that stimulated the mucus secretion and inhibited the bacterial translocation. CONCLUSIONS: These results suggest that lafutidine protects the small intestine against indomethacin-induced damage, the action being dependent on CSN and mediated by endogenous NO produced by cNOS. The protective action of lafutidine may be attributable to suppression of the bacterial translocation following indomethacin, probably due to stimulation of intestinal mucus secretion.

A fibronectin-binding protein from rice bran with cell adhesion activity for animal tumor cells.

A rice bran 57-kDa protein was isolated by affinity chromatography with fibronectin immobilized on agarose. This fibronectin-binding protein designated as RB-57 had an amino-terminal amino acid sequence identical with that of a putative mature form of rice hydroxyproline-rich glycoprotein. A distinct feature of the amino acid composition of RB-57 was the high contents of hydroxyproline and proline representing about 45% of the total amino acids. The sugar analysis indicated that arabinose represented 46.8% of the total carbohydrates. RB-57 showed cell adhesion activity for murine Lewis lung carcinoma cells. The result suggests that RB-57 may play a role in plant cell adhesion, although cell adhesion-promoting activity for plant cells remains to be tested.

Intravenous chlorhexidine gluconate causing acute respiratory distress syndrome.

CASE REPORT: A 67-year-old man undergoing a colectomy for colon cancer was unintentionally administered 0.8 mg of chlorhexidine gluconate intravenously and subsequently developed acute respiratory distress syndrome. The operation was discontinued immediately. Respiratory failure progressed despite three cycles of plasma exchange beginning on day 1. Extracorporeal membrane oxygenation for 72 h beginning on day 3 was associated with dramatic improvement. The patient showed complete recovery of intellectual function and subsequently underwent a colectomy with lymph node dissection for colon cancer. CONCLUSION: For acute respiratory distress syndrome secondary to chlorhexidine gluconate intoxication, consideration should be given to the treatment of initial respiratory distress and subsequent pneumonia. The benefit of extracorporeal membrane oxygenation and plasma exchange may merit further investigation.

Molecular cloning and characterization of a cDNA for a novel ethylene receptor, NT-ERS1, of tobacco (Nicotiana tabacum L.).

The cDNA encoding a novel member (NT-ERS1) of ethylene receptor family of tobacco (Nicotiana tabacum L.) was obtained by a combination of RT-PCR and 5'-/3'-RACE cloning. The cDNA was 2,092 nucleotides long and had an open reading frame of 1,911 bp encoding 637 amino acids. The deduced polypeptide lacked a response regulator domain, indicating that the ethylene receptor belongs to an ERS-group. The amino acid sequence was similar to respective members of the tobacco ethylene receptor family: 67.8% to NT-ETR1, 39.1% to NTHK1 and 31.1% to NTHK2. Comparison of amino acid sequence suggested that NT-ERS1 is the counterpart of Nr in the ethylene receptor family of tomato, which belongs to Solanaceae as does tobacco. Northern blot analysis showed that mRNA of NT-ERS1 was present in leaf, shoot and root tissues, and accumulated in leaves treated with exogenous ethylene. A mutated NT-ERS1 cDNA transgene, obtained by introducing one nucleotide substitution into NT-ETR1 cDNA, conferred ethylene insensitivity in tobacco plants, indicating that the translation product of the cDNA actually functioned in the plants.

Micrococcus luteus teichuronic acids activate human and murine monocytic cells in a CD14- and toll-like receptor 4-dependent manner.

Teichuronic acid (TUA), a component of the cell walls of the gram-positive organism Micrococcus luteus (formerly Micrococcus lysodeikticus), induced inflammatory cytokines in C3H/HeN mice but not in lipopolysaccharide (LPS)-resistant C3H/HeJ mice that have a defect in the Toll-like receptor 4 (TLR4) gene, both in vivo and in vitro, similarly to LPS (T. Monodane, Y. Kawabata, S. Yang, S. Hase, and H. Takada, J. Med. Microbiol. 50:4-12, 2001). In this study, we found that purified TUA (p-TUA) induced tumor necrosis factor alpha (TNF-alpha) in murine monocytic J774.1 cells but not in mutant LR-9 cells expressing membrane CD14 at a lower level than the parent J774.1 cells. The TNF-alpha-inducing activity of p-TUA in J774.1 cells was completely inhibited by anti-mouse CD14 monoclonal antibody (MAb). p-TUA also induced interleukin-8 (IL-8) in human monocytic THP-1 cells differentiated to macrophage-like cells expressing CD14. Anti-human CD14 MAb, anti-human TLR4 MAb, and synthetic lipid A precursor IV(A), an LPS antagonist, almost completely inhibited the IL-8-inducing ability of p-TUA, as well as LPS, in the differentiated THP-1 cells. Reduced p-TUA did not exhibit any activities in J774.1 or THP-1 cells. These findings strongly suggested that M. luteus TUA activates murine and human monocytic cells in a CD14- and TLR4-dependent manner, similar to LPS.

Isolation and characterization of an N-linked oligosaccharide that is significantly increased in sera from patients with non-small cell lung cancer.

The structures of N-linked oligosaccharides present in human sera from 12 healthy volunteers and from 14 patients with non-small cell lung cancer (NSCLC) were analyzed by our recently developed partially automated systematic method. Thirty different structures of oligosaccharides were deduced, and these accounted for 84.1% of the total N-linked oligosaccharides present in human sera. All of the quantified oligosaccharide levels in healthy human sera were within twice the standard deviation. The amount of a triantennary trigalactosylated structure with one outer arm fucosylation (A3G3Fo) was found to be markedly increased in NSCLC patients in comparison to that in healthy volunteers (p < 0.01). No significant positive correlation with other clinical data was found. Serum A3G3Fo levels can thus be a novel marker for the diagnosis of NSCLC.

Induction of necrosis factor-alpha and interleukin-6 in mice in vivo and in murine peritoneal macrophages and human whole blood cells in vitro by Micrococcus luteus teichuronic acids.

Earlier studies showed that Micrococcus luteus cells and cell walls induced anaphylactoid reactions leading to death, in some instances within 1 h, in C3H/HeN mice primed with muramyl dipeptide (MDP). They also induced serum cytokines in the surviving mice. The present study investigated the structural components responsible for these activities. Teichuronic acids, a component of M. luteus cell walls, induced tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in MDP-primed C3H/HeN mice. Peptidoglycans had little effect on the cytokine-inducing activities. Reducing teichuronic acids, i.e., teichuronic acids whose carboxyl groups had been reduced, lost their cytokine-inducing activities. Neither peptidoglycans nor teichuronic acids induced anaphylactoid reactions in the MDP-primed mice. Purified teichuronic acids also induced TNF-alpha and IL-6 production in C3H/HeN murine peritoneal macrophages and human whole-blood cells in the culture, but reduced teichuronic acids did not. The purified teichuronic acids induced no TNF-alpha and only low levels of IL-6 in MDP-primed C3H/HeJ mice, and neither cytokine in peritoneal macrophage cultures from C3H/HeJ mice with a single point of mutation in Toll-like receptor 4 (TLR4) gene. These findings suggest that induction of cytokines by teichuronic acids is mainly TLR4-dependent.

Lack of small intestinal ulcerogenecity of nitric oxide-releasing indomethacin, NCX-530, in rats.

AIM: To evaluate the intestinal ulcerogenic property of nitric oxide-releasing indomethacin (NCX-530) in the rat, in comparison with indomethacin. METHODS: Animals were given indomethacin or NCX-530 subcutaneously and killed 24 h later for macroscopic examination of the small intestine. RESULTS: A single administration of indomethacin (10 mg/kg) provoked damage, mainly in the jejunum and ileum, accompanied by an increase in myeloperoxidase and inducible nitric oxide synthase activities as well as bacterial translocation. NCX-530 at an equimolar dose (14.2 mg/kg) caused no gross damage in the small intestine, nor any significant change in inducible nitric oxide synthase and myeloperoxidase activities or bacterial translocation. NOR-3, the nitric oxide donor (6.0 mg/kg), when administered subcutaneously together with indomethacin, significantly prevented the occurrence of intestinal lesions and other mucosal changes. Indomethacin reduced mucus and fluid secretions in the small intestine, while both NCX-530 and NOR-3 enhanced these secretions. NCX-530 reduced the mucosal prostaglandin E2 contents and exhibited an anti-inflammatory action against carrageenan-induced paw oedema, with equal effectiveness to indomethacin. CONCLUSION: NCX-530 does not cause intestinal damage, despite inhibiting cyclooxygenase activity. The reduced intestinal toxicity of NCX-530 may be attributable to inhibition of enterobacterial translocation, partly by increasing the mucus and fluid secretions mediated by nitric oxide released from this compound.

Structural analysis of N-linked sugar chains of human blood clotting factor IX.

The structures of N-glycans of human blood clotting factor IX were studied. N-Glycans liberated by hydrazinolysis were N-acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine. The pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. Each PA-sugar chain was analyzed by two-dimensional sugar mapping combined with glycosidase digestion. The major structures of the N-linked sugar chains of human factor IX were found to be sialotetraantennary and sialotriantennary chains with or without fucose residues. These highly sialylated sugar chains are located on the activation peptide of the protein.

Processing pathway deduced from the structures of N-glycans in Carica papaya.

A processing The processing pathway of N-glycans in Carica papaya was deduced from the structures of N-glycans. The N-glycans were liberated by hydrazinolysis followed by N-acetylation. Their reducing-end sugar residues were tagged with 2-aminopyridine and the pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. Eleven PA-sugar chains were found, and their structures were analyzed by two-dimensional sugar mapping combined with partial acid hydrolysis and exoglycosidase digestion. The structures of the N-glycans were of the highmannose types with xylose and fucose; however, among them two new N-glycans, Manalpha1-6(Manalpha1-3)Manalpha1-6(Xylbeta1-2)+ ++Manbeta1-4GlcNAcbeta1- 4(Fucalpha1-3)GlcNAc and Manalpha1-3Manalpha1-6(Xylbeta1-2)Manbeta1-4G lcNAcbeta1-4(Fucalpha1-3 )GlcNAc, were found. Judging from these structures together with Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) (Xylbeta1-2)Manbeta1- 4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc reported previously [Shimazaki, A., Makino, Y., Omichi, K., Odani, S., and Hase, S. (1999) J. Biochem. 125, 560- 565], a processing pathway for N-glycans in C. papaya is inferred in which the activity of Golgi alpha-mannosidase II is incomplete.

Health beliefs and perceptions of women presenting or not presenting for mammographic screening in a rural health setting.

Breast cancer continues to be a major health concern among Australian women. Recently, free mammography screening has been offered as a joint State and Commonwealth initiative to enable early detection. This program has particular significance in rural areas where access to health-care facilities, particularly those of a specialist nature, is limited. Attendance for screening is critical to the success of this type of program. Several lines of evidence suggest that health beliefs play a major role in compliance with recommended health behaviour. The present study investigated the role of five health beliefs: response efficacy, seriousness, concern, susceptibility and barriers to the likelihood of attending the North Coast Breast Screening Program in northern NSW. A questionnaire that measured these health beliefs was completed by 127 women who attended breast screening and 185 women who knew about the service but had not attended. Compared with those who had not undergone mammography, those presenting for screening were more health conscious, more likely to have had a mammography previously, more aware that mammography reduced the risk of developing severe breast cancer and less concerned about having a mammogram. A number of barriers to having a mammogram were also identified. The present study raises a number of concerns regarding the level of knowledge about breast self-examination and mammography as important preventive health measures among women in a rural setting.

beta1-4Galactosyltransferase activity of mouse brain as revealed by analysis of brain-specific complex-type N-linked sugar chains.

We previously reported two brain-specific agalactobiantennary N-linked sugar chains with bisecting GlcNAc and alpha1-6Fuc residues, (GlcNAcbeta1-2)(0)(or)(1)Manalpha1-3(GlcNAcbeta1-2M analpha1-6)(GlcNA cbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)Glc NAc [Shimizu, H., Ochiai, K., Ikenaka, K., Mikoshiba, K., and Hase, S. (1993) J. Biochem. 114, 334-338]. Here, the reason for the absence of Gal on the sugar chains was analyzed through the detection of other complex type sugar chains. Analysis of N-linked sugar chains revealed the absence of Sia-Gal and Gal on the GlcNAc residues of brain-specific agalactobiantennary N-linked sugar chains. We therefore investigated the substrate specificity of galactosyltransferase activities in brain using pyridylamino derivatives of agalactobiantennary sugar chains with structural variations in the bisecting GlcNAc and alpha1-6Fuc residues as acceptor substrates. While the beta1-4galactosyltransferases in liver and kidney could utilize all four oligosaccharides as substrates, the beta1-4galactosyltransferase(s) in brain could not utilize the agalactobiantennary sugar chain with both bisecting GlcNAc and Fuc residues, but could utilize the other three acceptors. Similar results were obtained using glycopeptides with agalactobiantennary sugar chains and bisecting GlcNAc and alpha1-6Fuc residues as substrates. The beta1-4galactosyltransferase activity of adult mouse brain thus appears to be responsible for producing the brain-specific sugar chains and to be different from beta1-4galactosyltransferase-I. The agalactobiantennary sugar chain with bisecting GlcNAc and alpha1-6Fuc residues acts as an inhibitor against "brain type" beta1-4galactosyltransferase with a K(i) value of 0.29 mM.