Biomedical subjects

S Kaneko

Publications and source records attributed to S Kaneko.

At least 217 records · Page 12Linked to original sources

PubMed1999

Effects of zonisamide on K+ and Ca2+ evoked release of monoamine as well as K+ evoked intracellular Ca2+ mobilization in rat hippocampus.

To clarify the effects of zonisamide (ZNS) on neurotransmission and intracellular Ca2+ mobilization, both Ca2+ and K+ evoked hippocampal releases of dopamine (DA) and serotonin (5-HT) were determined by in vivo microdialysis, and K+ (25 and 50 mM) evoked elevation of intracellular Ca2+ level was determined by fluorescence microscopy in vitro. Therapeutic concentrations of ZNS had different effects on Ca2+ and K+ evoked release of monoamine. ZNS stimulated Ca2+ evoked monoamine release, while ZNS inhibited K+ evoked monoamine release. ZNS inhibited K+ evoked elevation of hippocampal intracellular Ca2+ levels in a concentration dependent manner. These results suggest that ZNS inhibits the depolarization induced by neuronal excitation, whereas ZNS might enhance the N-type Ca2+ channel activity or N-type Ca2+ channel related exocytosis mechanisms.

Animals↗

PubMed1999

Intrauterine growth in the offspring of epileptic women: a prospective multicenter study.

The aim of the present study was to evaluate the risk of intrauterine growth delay in the offspring of epileptic mothers and to quantify the risks of intrauterine exposure to antiepileptic drugs (AEDs). Data concerning 870 newborns, prospectively collected in Canada, Japan and Italy, using the same study design, were pooled and analyzed. The overall proportion of newborns whose body weight (7.8%) or head circumference (11.1%) at birth were below the 10th percentile was not increased. However, logistic regression analysis showed that the risk of small head circumference was significantly higher in Italian than in Japanese (RR 4.2; 95% CI: 2.2-8.0) or Canadian children (RR 2.6; 95% CI: 1.1-6.5), and in children exposed to polytherapy (RR 2.7; 95% CI: 1.2-6.3), phenobarbital (PB) (RR 3.6; 95% CI: 1.4-9.4) and primidone (PRM) (RR 4.5; 95% CI: 1.5-13.8). Country was also the only factor affecting low body weight, with Italian children having a higher risk than Japanese (RR 5.2; 95% CI: 2.6-10.4) or Canadian (RR 8.8; 95% CI: 2.0-38.1) children. Due to the small categories, the influence of AED doses and plasma concentrations was studied for each individual AED, without adjustment for the other potential confounding factors. A clear dose-dependent effect was found for PB and PRM in terms of both small head circumference and low body weight, and a concentration-dependent effect for PB in terms of small head circumferences. The size of the difference between the Italian and the other two populations, which is only partially explained by differences in therapeutic regimens, suggests that genetic, environmental and ethnic factors also need to be taken into account when considering possible explanations.

Anticonvulsants↗

PubMed1999

Fur mites induce dermatitis associated with IgE hyperproduction in an inbred strain of mice, NC/Kuj.

An inbred strain of mice, NC, has been introduced as an animal model for atopic dermatitis because the mice develop dermatitis associated with severe scratch preceded by elevated serum IgE level when kept in conventional conditions. Although hypersensitivity to some environmental factors is suggested to cause dermatitis, the precise factor remains unclear. As the mice maintained under conventional conditions were often infected with fur mites, we investigated whether an infection of fur mites induces skin lesions in NC. Infection with the fur mites induced NC to develop skin lesions associated with highly elevated serum IgE, whereas no obvious skin lesions were observed in BALB/c and C57BL/6, and the elevation of serum IgE level was minimal in these two strains of mice. The role of the fur mites in the manifestation of skin lesions and IgE hyperproduction was confirmed by eliminating the fur mites by treatment with ivermectin. In addition, the existence of specific IgE antibody to Myocoptes musculinus antigen in the sera of mite-infested NC was detected by the antigen-induced histamine release from bone marrow-derived cultured mast cells after sensitization with the serum. These results suggest that continuous exposure to fur mite antigen is a potential factor in the development of dermatitis in NC. We provide a new model system of antigen-induced dermatitis for investigating the role of IgE in eliciting dermatitis.

Animals↗

PubMed1999

Can urinary eicosanoids be a potential predictive marker of clinical response to thromboxane A2 receptor antagonist in asthmatic patients?

Thromboxane (TX) A2 is an important bronchoconstrictor in the pathogenesis of asthma. Seratrodast, known as AA-2414, is a new oral TXA2 receptor antagonist which is currently prescribed in asthma therapy in Japan. However its clinical effects have been very different in individual subjects. To assess whether the clinical efficacy of TXA2 antagonist is predictable on the basis of urinary arachidonic acid metabolites in urine of patients with asthma, an open and multicentre trial was conducted. Fifty adult asthmatic subjects (women/men = 28/22) were enrolled [resting mean forced expiratory volume in 1 sec (FEV1)% was 82%; range, 50-96%]. Urinary levels of 11-dehydro-TXB2, leukotriene (LT) E4, 2,3-dinor-6-keto-prostaglandin F1alpha and creatinine in 3-h urine collected in the morning at the start of seratrodast (80 mg day(-1), once a day at evening for 4 weeks) were measured. Responders were defined by improvements of asthma symptoms score and peak expiratory flow rate (PEFR). Of the 50 subjects, 45 completed this study. Eighteen patients were responders and the other 27 were nonresponders. There were no significant differences between the two groups in patients' characteristics, baseline lung functions, treatments and baseline urinary eicosanoids. The 11-dehydro-TXB2/LTE4 ratio of responders was significantly higher (P = 0.0091) than that of non-responders (mean +/- SE, 7.49+/-0.71 vs. 5.09+/-0.67). Eleven patients out of 18 responders agreed to continue this drug for 6 months, the 11-dehydro-TXB2/LTE4 ratio decreased during this period, but not significantly. Our data demonstrated that responders and non-responders to TXA2 receptor antagonist existed in patients with asthma, and it suggests that the ratio of urinary eicosanoids might be a possible predictor of the effects of TXA2 receptor antagonist.

6-Ketoprostaglandin F1 alpha↗

PubMed1999

Enzymatic synthesis of no-carrier-added 6-[18F]fluoro-L-dopa with beta-tyrosinase.

An enzymatic synthesis of nca 6-[18F]fluoro-L-dopa has been developed. The process consists of a chemical synthesis of 4-[18F]fluorocatechol and its enzymatic reaction with beta-tyrosinase. The 4-[18F]fluorocatechol was prepared by nucleophilic aromatic substitution of the NO2 group on 6-nitroveratoraldehyde with [K/222]+18F-, followed by decarbonylation with tris(triphenylphosphine) rhodium(I) chloride and hydrolysis with hydroiodic acid. By C18 Sep-Pak purification, 4-[18F]fluorocatechol was obtained in ethanol with a radiochemical yield of 9.2%. An enzymatic reaction of 4-[18F]fluorocatechol, ammonium and pyruvate catalyzed by beta-tyrosinase in an ethanolic Tris-HCl buffer (pH 9.0) containing ascorbate gave within 5 min 6-[18F]fluoro-L-dopa with an approximate radiochemical yield of 60% without any isomers. The deproteinized reaction mixture was applied to a preparative reverse phase column, and the radiochemically and enantiomerically pure 6-[18F]fluoro-L-dopa was obtained with a radiochemical yield of 2.0% based on [18F]F- (decay-corrected). The synthesis time was 150 min from the EOB and the specific activity was > 200 GBq/micromol.

Chromatography, High Pressure Liquid↗

PubMed1999

Effects of a bolus injection of adenosine triphosphate on atrioventricular conduction and hemodynamics in patients undergoing coronary artery bypass grafting.

OBJECTIVE: To investigate the effects of adenosine triphosphate (ATP) on atrioventricular (A-V) conduction and hemodynamics in patients undergoing coronary artery surgery. DESIGN: A prospective study. SETTING: University teaching hospital. PARTICIPANTS: Patients scheduled for elective coronary artery bypass graft surgery. INTERVENTIONS: Anesthesia was induced with the same dose of intravenous fentanyl (300 microg) and midazolam (10 mg) for each patient. After the baseline measurement, a bolus dose of 10 mg of ATP (0.19+/-0.03 mg/kg, mean +/- SD) was rapidly injected through a central venous catheter. Right ventricular pacing was conducted at a rate of 40 beats/min during the development of complete A-V block. MEASUREMENTS AND MAIN RESULTS: ATP induced an A-V block in 10 of 12 patients (2:1 block in two patients and complete A-V block in eight patients). The onset of the A-V block was rapid (17.1+/-3.4 seconds) and the duration was short (12.1+/-7.0 seconds). Radial arterial pressure (AP) decreased with the appearance of the A-V block (p < 0.01). Heart rate (HR) returned to the baseline value quickly after the restoration of the A-V block, but recovery of radial AP was delayed. HR increased 1 minute after the ATP injection (p < 0.01). Both HR and radial AP returned to the baseline values 2 minutes after the ATP injection. ST segments on both leads II and V5 of electrocardiograms decreased more than 0.10 mV 1 minute after the ATP injection in one patient. CONCLUSION: A bolus injection of 10 mg of ATP successfully induced a transient A-V block in 83% of the 12 patients. The countermeasures, including ventricular pacing, are indispensable to cope with the subsequent hypotension that may lead to myocardial ischemia, especially in patients with ischemic heart disease.

Adenosine Triphosphate↗

PubMed1999

Leukotriene (LT)-receptor antagonist is more effective in asthmatic patients with a low baseline ratio of urinary LTE4 to 2,3-dinor-6-keto-prostaglandin (PG)F1alpha.

BACKGROUND: To test the hypothesis that urinary levels of arachidonic acid metabolites may be a predicting factor of the effects of pranlukast, a selective leukotriene (LT) antagonist, on chronic adult asthma, we investigated the relationship between its clinical efficacy and urinary eicosanoid levels. METHODS: An open, multicenter trial was conducted involving 38 stable moderate and severe asthmatic patients (mean percent predicted FEV1 was 71%). All patients received pranlukast (225 mg twice daily) for 4 weeks after a 2-week run-in period. Urinary levels of LTE4, 11-dehydro-thromboxane (TX) B2, 2,3-dinor-6-keto-prostaglandin (PG) F1alpha, and creatinine were measured in 3-h urine collected on day 1 of the treatment. The responder was defined by an improvement of asthma symptom scores and peak expiratory flow rate (PEFR). RESULTS: One patient was excluded because of an adverse effect, nausea. Thirteen out of 37 subjects were responders and 24 were nonresponders. There were no significant differences in patients' backgrounds and urinary arachidonate levels between the two groups. The urinary LTE4 to 2,3-dinor-6-keto-PGF1alpha ratio in the responder was significantly lower (P=0.01) than that in the nonresponder. In all patients, a significant inverse correlation was revealed between the baseline urinary LTE4/2,3-dinor-6-keto-PGF1alpha ratio and the improvement of PEFR in the morning (r=-0.43, P=0.007). CONCLUSIONS: These data suggested that the urinary ratio of LTE4 to 2,3-dinor-6-keto-PGF1alpha might be one of the predictive markers of the clinical efficacy of this LT-receptor antagonist in asthmatic subjects.

Adult↗

PubMed1999

The quantitative and qualitative defect of CD4+ CD45RO+ memory-type T cells are involved in the abnormality of TH1 immunity in atopic dermatitis patients.

Peripheral blood mononuclear cells (PBMCs) obtained from atopic dermatitis (AD) patients produced low levels of IFN-gamma in response to Dermatophagoides farinae antigen (Der f Ag) plus IL-2 or OKT3 MoAb in contrast with PBMCs obtained from healthy donors. The reduced IFN-gamma production in AD patients' T cells appeared to be derived from the defect of CD4+ T cells but not CD8+ T cells. Indeed, from the cytoplasmic staining analysis of cytokines, it was demonstrated that the frequency of IFN-gamma producing CD4+ T cells (TH1 cells) in AD patients was markedly lower than that of healthy donors. From the phenotypic analysis using flow cytometry, it was also found that the number of CD4+ CD45RO+ memory type T cells was significantly reduced in AD patients compared with that of healthy donors. In addition to quantitative defect of memory type CD4+ T cells, functional defect of CD4+ CD45RO+ memory type T cells was also demonstrated in AD patients. Enriched CD4+ CD45RO+ T cells obtained from AD patients, who exhibited greatly reduced delayed-type hypersensitivity (DTH) response in tuberculin test, showed no significant TH1 immunity in terms of IFN-gamma production by stimulation with OKT3 MoAb or purified protein derivative (PPD). Thus, the immunological abnormality of TH1 immunity in AD patients appeared to be induced in concomitant with both the quantitative and qualitative defect of memory type CD4+ T cells.

Antibodies, Monoclonal↗

PubMed1999

Competitive interaction of seven in absentia homolog-1A and Ca2+/calmodulin with the cytoplasmic tail of group 1 metabotropic glutamate receptors.

BACKGROUND: Group 1 metabotropic glutamate receptors (mGluR1 and mGluR5) are coupled to inositol trisphosphate/Ca2+ signaling via G proteins and play an important role in excitatory synaptic transmission. To explore the regulation of group 1 mGluR function, we applied the yeast two-hybrid system using the intracellular carboxy-terminal domain of group 1 mGluRs (group 1 ct-mGluRs) and attempted to identify novel protein-protein interactions of group 1 mGluRs. RESULTS: The two-hybrid screening revealed a specific interaction between group 1 ct-mGluRs and Siah-1A, the mammalian homolog of Drosophila seven in absentia which is involved in photoreceptor cell differentiation via the ubiquitin/proteasome-dependent mechanism. This interaction occurs within a homologous 27-28 amino acid stretch within group 1 ct-mGluRs and requires the latter two-thirds of Siah-1A. Following coexpression in COS-7 cells, myc-tagged Siah-1A was coimmunoprecipitated with the flag-tagged ct-mGluR1 by anti-flag antibody. Furthermore, in vitro binding revealed that Siah-1A and Ca2+/calmodulin (CaM) binding sites overlap, such that Siah-1A binding is competitively inhibited by CaM in a Ca2+-dependent manner. CONCLUSIONS: The results demonstrate a direct interaction between group 1 mGluRs and Siah-1A and suggest a novel modulatory mechanism mediated by a competitive interaction between Ca2+/CaM and Siah-1A.

Amino Acid Sequence↗

PubMed1999

Shift of hepatitis E virus RNA from hepatocytes to biliary epithelial cells during acute infection of rhesus monkey.

Hepatitis E virus (HEV) has been considered to be the major cause of enterically transmitted non-A, non-B hepatitis in developing countries. However, little is known about viral replication and localization in the liver. The aim of this study was to examine the distribution of HEV-infected cells in experimentally infected animals. Seven captured wild rhesus monkeys were inoculated intravenously with faecal extract derived from a Myanmar strain of HEV. Animals were killed at different time-points of clinical illness: during early infection, during prehepatitis with viral-like particles in bile, during acute hepatitis and during convalescence. Intrahepatic localization of HEV was analysed using non-isotopic thymine dimer in situ hybridization (NITDISH). Both plus and minus strands of HEV RNA were found in hepatocytes during the early infection period. Staining in the submembranous cytoplasmic region of hepatocytes was observed. In the prehepatitis period, both plus and minus strand HEV RNAs appeared in the canalicular side of isolated bile epithelial cells. Subsequently, HEV RNA became universally distributed in the cytoplasm of medium-size bile epithelial cells. After recovery, HEV RNA disappeared.

Acute Disease↗

PubMed1999

Factors responsible for the prolongation of school refusal.

Two hundred and one school refusers (< or =18 years old), excluding schizophrenia, were treated at the Department of Neuropsychiatry, Hirosaki University Hospital between April 1975 and March 1995. Of 56 cases of school refusal, 31 did not improve for more than 2 years (group P), and 25 cases had improved in the degree of school refusal and social impairment (group B). The remaining cases were excluded from the analysis for several reasons. There was no significant difference between groups B and P in age at the first psychiatric evaluation as well as the age of onset of school refusal. The duration from school absence to the first evaluation of group P was significantly longer than that of group B. The duration of school refusal significantly and positively correlated with the duration from school absence to the first evaluation. Introversion and nervousness prolonged the duration of school refusal. The non-presence of volition for school attendance, and a low frequency of school attendance during the 1 month prior to the first evaluation influenced the prolongation of school refusal. The 'duration from school absence to the first evaluation', the 'patient's character', the 'non-presence of volition for school attendance' and the 'frequency of school attendance' influenced the prolongation of school refusal. The introduction of treatment within 10 months of the onset of school refusal is an important factor in preventing the prolongation of the school refusal.

Absenteeism↗

PubMed1999

Differential effects of adenosine receptor subtypes on release and reuptake of hippocampal serotonin.

To clarify the effects of adenosine receptor subtypes (A1, A2 and A3) on hippocampal serotonin (5-HT) release and 5-HT reuptake activity, hippocampal extracellular 5-HT levels were determined in vivo by microdialysis in freely moving rats. Selective 5-HT reuptake inhibitor (SSRI) fluoxetine and DU24565 increased extracellular 5-HT levels. Adenosine and A1 receptor agonist, 2-chloro-N6-cyclopentyl-adenosine (CCPA), decreased extracellular 5-HT levels, whereas non-selective antagonist, caffeine, and A1 antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT) increased them. When 5-HT reuptake activity was inhibited by DU24565 and fluoxetine, the effects of CPT and CCPA on 5-HT level were enhanced. A2A receptor agonist, CGS21680, A2 receptor agonist, PD125944, A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), and A3 receptor agonist, N6-2-(4-aminophenyl)ethyladenosine (APNEA) did not affect 5-HT levels; however, when A1 receptor was blocked by CPT, 5-HT levels were increased by adenosine, CGS21680 and PD125944, and decreased by DMPX and APNEA. Under conditions of A1 receptor blockade, pretreatment with DU24565 or fluoxetine, enhanced the stimulatory effects of CGS21680 and PD125944 as well as inhibitory effects of DMPX on 5-HT level, whereas the inhibitory effect of APNEA was abolished. These results indicate that the stimulatory effects of A2 receptor and inhibitory effects of A3 receptor on hippocampal extracellular 5-HT levels are masked or abolished by the inhibitory effects of A1 receptor. In addition, hippocampal serotonergic transmission might be modulated by hippocampal presynaptic adenosine receptor subtypes, and hippocampal 5-HT reuptake activity might be modulated by hippocampal A3 receptor.

Adenosine↗

PubMed1999

Depletion of intracellular glutathione increases susceptibility to nitric oxide in mesencephalic dopaminergic neurons.

Using primary neuronal cultures, we investigated the effects of GSH depletion on the cytotoxic effects of glutamate and NO in dopaminergic neurons. Intracellular GSH was depleted by 24-h exposure to L-buthionine-[S,R]-sulfoximine (BSO), an irreversible inhibitor of GSH synthase. BSO exposure caused concentration-dependent reduction of the viability of both dopaminergic and nondopaminergic neurons. In contrast, 24-h exposure of cultures to glutamate or NOC18, an NO-releasing agent, significantly reduced the viability of nondopaminergic neurons without affecting that of dopaminergic neurons. Pretreatment with N-acetyl-L-cysteine for 24 h ameliorated the NOC18-induced toxicity in nondopaminergic neurons. In dopaminergic neurons, sublethal concentrations of BSO reduced intracellular GSH content and markedly potentiated glutamate- and NOC18-induced toxicity. These results suggested that glutamate toxicity was enhanced in dopaminergic neurons by suppression of defense mechanisms against NO toxicity under conditions of GSH depletion. Under such conditions, free iron plays an important role because BSO-enhanced NO toxicity was ameliorated by the iron-chelating agent, deferoxamine. These results suggest that GSH plays an important role in the expression of NO-mediated glutamate cytotoxicity in dopaminergic neurons. Free iron may be related to enhanced NO cytotoxicity under GSH depletion.

Animals↗

PubMed1999

Transcriptional regulation of the CDK inhibitor p16INK4a gene by a novel pRb-associated repressor, RBAR1.

p16, also known as INK4a, CDKN2, or MTS1, plays an important role in the control of the cell cycle progression, and retinoblastoma protein (pRb) is suggested to be involved in the transcriptional regulation of p16. However, it is not fully understood how pRb regulates transcription of the p16 gene. Nuclear proteins prepared only from the pRb-nonfunctional human tumor cells were found to bind to the 5'-flanking sequence of the p16 gene in the presence of Zn2+ by electrophoretic mobility shift assay (EMSA). EMSA using mutagenized 5'-flanking sequences as competitors suggested that the sequence at position -97 to -87 relative to first ATG of the p16 gene was critical for protein binding. Transient reporter assay indicated that the sequence identified by EMSA acted as a silencer element in the pRb-nonfunctional tumor cells, showing the presence of a transcriptional repressor associated with functional pRb (RBAR1).

Cyclin-Dependent Kinase Inhibitor p16↗

PubMed1999

Localization of a gene for benign adult familial myoclonic epilepsy to chromosome 8q23.3-q24.1.

Benign adult familial myoclonic epilepsy is an autosomal dominant idiopathic epileptic syndrome characterized by adult-onset tremulous finger movement, myoclonus, epileptic seizures, and nonprogressive course. It was recently recognized in Japanese families. In this study, we report that the gene locus is assigned to the distal long arm of chromosome 8, by linkage analysis in a large Japanese kindred with a maximum two-point LOD score of 4.31 for D8S555 at recombination fraction of 0 (maximum multipoint LOD score of 5.42 for the interval between D8S555 and D8S1779). Analyses of recombinations place the locus within an 8-cM interval, between D8S1784 and D8S1694, in which three markers, D8S1830, D8S555, and D8S1779, show no recombination with the phenotypes. Although three other epilepsy-related loci on chromosome 8q have been recognized-one on chromosome 8q13-21 (familial febrile convulsion) and two others on chromosome 8q24 (KCNQ3 and childhood absence epilepsy)-the locus assigned here is distinct from these three epilepsy-related loci. This study establishes the presence of a new epilepsy-related locus on 8q23.3-q24.11.

Adolescent↗

PubMed1999

Upregulation of type I interferon receptor by IFN-gamma.

Type I interferon (IFN) receptor has a multichain structure composed of at least two distinct subunits, IFNAR-1 and IFNAR-2. In the present study, we demonstrated that IFN-gamma induced the expression of mRNA for IFNAR-1 and IFNAR-2 in a human hepatoma cell line, HepG2 cells. The induction was dose and time dependent. Because of this result, we examined the effect of combined treatment with type I IFN and IFN-gamma. The intracellular 2-5A-synthetase activity induced by combined treatment was significantly higher than that by type I IFN alone. This study suggests that combined treatment with type I IFN and IFN-gamma may be more effective than that of type I IFN alone and that the upregulation of type I IFN receptor may be one of the reasons. Our findings may have some relevance to the clinical use of IFN.

2',5'-Oligoadenylate Synthetase↗

PubMed1999

Epidemiological analysis of site relationships of synchronous and metachronous multiple primary cancers in the National Cancer Center, Japan, 1962-1996.

BACKGROUND: Multiple primary cancer (MPC) has been recognized as a problem commonly encountered in routine medical practice. A study of MPC is necessary not only to provide insights into the etiology of cancer, but also to provide information for effective medical care by clinical oncologists. METHODS: A cohort of 49,751 cancer patients who were admitted to the National Cancer Center Hospital between 1962 and 1996 was used to study the site relationship of MPC. Logistic and Poisson regression analyses using an internal reference group within the cohort were applied for the calculation of the prevalence odds ratio (POR) for site relationships of synchronous MPC and the incidence rate ratio (IRR) for those of metachronous MPC. RESULTS: Three site combinations with elevated risks for both synchronous and metachronous MPCs, eight with elevated risk for synchronous MPC, five with elevated risk for metachronous MPC and six with decreased risk for synchronous MPC were identified with statistical significance. Among them, the increased risk of metachronous stomach cancer following lymphoma and myeoloma (POR = 1.0 and 1.1, P > 0.05; IRR = 2.5, P < 0.05) and the inverse site-correlation of synchronous MPC between [trachea, bronchus and lung] and other sites of the upper aerodigestive tract [lip, oral cavity and pharynx] (POR = 0.5 and 0.3, P < 0.05) and esophagus (POR = 0.7 and 0.3, P < 0.05) have not been reported previously. CONCLUSIONS: Our results suggest that interventions for lymphoma and myeloma might affect the development of subsequent stomach cancer and additional etiological factors other than tobacco smoking are associated with the development of cancer in the upper aerodigestive tract.

Cohort Studies↗

PubMed1999

Chitosanase-catalyzed hydrolysis of 4-methylumbelliferyl beta-chitotrioside.

4-Methylumbelliferyl beta-chitotrioside [(GlcN)(3)-UMB] was prepared from 4-methylumbelliferyl tri-N-acetyl-beta-chitotrioside [(GlcNAc)(3)-UMB] using chitin deacetylase from Colletotrichum lindemuthianum, and hydrolyzed by chitosanase from Streptomyces sp. N174. The enzymatic deacetylation of (GlcNAc)(3)-UMB was confirmed by (1)H-NMR spectroscopy and mass spectrometry. When the (GlcN)(3)-UMB obtained was incubated with chitosanase, the fluorescence intensity at 450 nm obtained by excitation at 360 nm was found to increase with proportion to the reaction time. The rate of increase in the fluorescence intensity was proportional to the enzyme concentration. This indicates that chitosanase hydrolyzes the glycosidic linkage between a GlcN residue and UMB moiety releasing the fluorescent UMB molecule. Since (GlcN)(3) itself cannot be hydrolyzed by the chitosanase, (GlcN)(3)-UMB is considered to be a useful low molecular weight substrate for the assay of chitosanase. The k(cat) and K(m) values obtained for the substrate (GlcN)(3)-UMB were determined to be 8.1 x 10(-5) s(-1) and 201 microM, respectively. From TLC analysis of the reaction products, the chitosanase was found to hydrolyze not only the linkages between a GlcN residue and UMB moiety, but also the linkages between GlcN residues. Nevertheless, the high sensitivity of the fluorescence detection of the UMB molecule would enable a more accurate determination of kinetic constants for chitosanases.

Amidohydrolases↗