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Hiatal hernia is a common condition in elderly patients, but the additional presence of prolapse of the pancreas is extremely rare. We herein report an 89-year-old woman who presented with liver function disorders and abdominal pain. Her laboratory tests revealed cholestasis, and imaging examinations showed stenosis of the common bile duct pulled toward the hernia sac. She was diagnosed with a common bile duct stricture due to pancreatic herniation and underwent laparoscopic surgery. Our review of the literature identified three types of pancreatic herniations: asymptomatic, bile duct complication, and acute pancreatitis. Pancreatic head herniation tends to induce bile duct complications.

We herein report a 44-year-old man suffering from systemic edema due to protein-losing enteropathy (PLE) with superior mesenteric vein (SMV) obstruction and development of collateral veins, which subsequently proved to be a chronic result of thrombosis and a complication of Crohn's disease (CD). PLE was supposedly induced by both intestinal erosion and thrombosis-related lymphangiectasia, which was histologically proven in his surgically-resected ileal stenosis. Elemental diet and anti-TNFα agent improved his hypoalbuminemia after surgery. The rarity of the simultaneous coexistence of SMV obstruction and PLE and the precedence of these complications over typical abdominal symptoms of CD made the clinical course complex.

Background There are currently no guidelines concerning the advisability and timing of tube removal following percutaneous transhepatic gallbladder drainage (PTGBD). The present study aimed to assess the feasibility and risks of early removal of the PTGBD tube under the scenario of subsiding inflammation, patent cystic and common bile ducts, and absence of intraperitoneal leakage. Methods Patient background and outcomes were assessed retrospectively in 701 cases of acute cholecystitis treated with PTGBD. The median times until tube removal and tube dislodgement and the cumulative rates of tube dislodgement were calculated. Results Tube removal was performed in 275 patients after a median time of 16 days (range: 6 to 213 d); biliary peritonitis was observed in 2 patients following tube removal. Tubes were removed in 8 and 35 patients within 7 and 10 days, respectively. Tube dislodgement was observed in 82 patients after a median time of 12 days (range: 1 to 125 d). Conclusion The present study suggests that drainage tube removal is safe and effective when performed after a short drainage period of 7 to 10 days if the criteria for the removal of the drainage tube were met.

Background: Isomaltodextrin (IMD) is a novel highly branched α-glucan and its function as a soluble dietary fiber is expected. Objective: The goal of this study was to evaluate the effects of IMD on postprandial glucose excursions in healthy people and to make the mechanism clear. Design: Twenty-nine subjects ingested a solution containing maltodextrin (MD) or sucrose with or without IMD. Fourteen subjects ingested a solution containing glucose with or without IMD. Blood glucose concentrations were then compared between the groups. Furthermore, in vitro digestion, inhibition of digestive enzymes, and glucose absorption tests were conducted. Results: IMD attenuated blood glucose elevation in the subjects with blood glucose excursions at the high end of normal following the ingestion of MD or sucrose or glucose alone. This effect of 5 g IMD was most clear. IMD was digested partially only by small intestinal mucosal enzymes, and maltase and isomaltase activities were weakly inhibited. Furthermore, IMD inhibited the transport of glucose from mucosal side to serosal side. Conclusions: IMD attenuated postprandial blood glucose, after the ingestion of MD or sucrose or glucose. As one of the mechanism, it was suggested that IMD inhibited the absorption of glucose on small intestinal mucosal membrane.

At present, the clinical trials of Boron Neutron Capture Therapy (BNCT) are being performed at research reactor facilities. However, an accelerator based BNCT has a merit that it can be built in a hospital. So, we just launched a development project for the BNCT based on an accelerator in order to establish and to spread the BNCT as an effective therapy in the near future. In the project, a compact proton linac installed in a hospital will be applied as a neutron source, and energy of the proton beam is planned to be less than about 10 MeV to reduce the radioactivity. The BNCT requires epithermal neutron beam with an intensity of around 1x109 (n/cm2/sec) to deliver the therapeutic dose to a deeper region in a body and to complete the irradiation within an hour. From this condition, the current of the proton beam required is estimated to be a few mA on average. Enormous heat deposition in the target is a big issue. We are aiming at total optimization of the accelerator based BNCT from the linac to the irradiation position. Here, the outline of the project is introduced and the moderator design is presented.

Production of a novel cyclomaltopentaose cyclized by an alpha-1,6-linkage, [ICG5; cyclo-{--6)-alpha-D-Glcp-(1--4)-alpha-D-Glcp-(1--4)-alpha-D-Glcp-(1--4)-alpha-D-Glcp-(1--4)-alpha-D-Glcp-(1--}], from starch was performed using isocyclomaltooligosaccharide glucanotransferase (IGTase) derived from Bacillus circulans AM7. The optimal conditions for ICG5-production from partially hydrolyzed starch were as follows: substrate concentration, 1.0% (w/v); pH, 5.5; temperature, 45 degrees C; reaction time, 24 h, IGTase, 1.0 unit/g-dry solid (DS); isoamylase, 2,500 units/g-DS. The yield of ICG5 reached 25.9% under optimal conditions. ICG5-production was achieved from partially hydrolyzed starch using a crude enzyme preparation containing IGTase. Finally, ICG5 was obtained in a yield of 17.9% (99.3% purity, 2,681 g-DS). A digestive test with a human salivary amylase, an artificial gastric juice, a pancreatic amylase, and small intestinal enzymes showed that ICG5 was an indigestible oligosaccharide.

A bacterial strain AM7, isolated from soil and identified as Bacillus circulans , produced two kinds of novel cyclic oligosaccharides. The cyclic oligosaccharides were produced from amylose using a culture supernatant of the strain as the enzyme preparation. The major product was a cyclomaltopentaose cyclized by an α-(1→6)-linkage, cyclo -{→6)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→}. The other minor product was cyclomaltohexaose cyclized by an α-(1→6)-linkage, cyclo -{→6)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→}. We propose the names isocyclomaltopentaose (ICG5) and isocyclomaltohexaose (ICG6) for these novel cyclic maltooligosaccharides having one α-(1→6)-linkage. ICG5 was digested by α-amylase derived from Aspergillus oryzae , cyclomaltodextrin glucanotransferase (CGTase) from Bacillus stearothermophilus , and maltogenic α-amylase. On the other hand, ICG6 was digested by CGTase from B. stearothermophilus and B. circulans , and maltogenic α-amylase. This is the first report of enzymatically produced cyclomaltopentaose and cyclomaltohexaose, which have an α-(1→6)-linkage in their molecules.

We investigated the acceptor specificity of a thermostable trehalose phosphorylase from Thermoanaerobacter brockii ATCC 35047 (TbTP) was examined using beta-D-glucose-1-phosphate (beta-G1P) as a glucosyl donor and oligosaccharides as the acceptor. Oligosaccharides with a reducing-end glucose residue as the C-6 substituent (e.g., isomaltose, gentiobiose, melibiose, isomaltotriose, and isopanose) were found to be successful acceptors. The transfer products of isomaltose, gentiobiose, and melibiose were isolated and characterized as 6-O-alpha-D-glucopyranosyl trehalose (alpha-GlcTre), 6-O-beta-D-glucopyranosyl trehalose (beta-GlcTre), and 6-O-alpha-D-galactopyranosyl trehalose (alpha-GalTre), respectively. To produce alpha-GalTre, a novel nonreducing trisaccharide, the reaction conditions of alpha-GalTre were examined using trehalose as a glucosyl donor. As a result, the yield of alpha-GalTre reached 40.5%.

Our previous study has shown that a soluble hesperidin derivative, glucosyl hesperidin (G-hesperidin), preferentially lowers serum triglyceride (TG) level in hypertriglyceridemic subjects through the improvement of very low-density lipoprotein (VLDL) metabolic abnormality. G-Hesperidin has also been found to decrease an elevated serum apolipoprotein B (apo B) level in the hypertriglyceridemic subjects, suggesting a possibility that this compound suppresses excess VLDL secretion in the liver. In the present study, to gain a better understanding of possible mechanisms by which G-hesperidin lowers serum TG, we examined whether this derivative affects apo B secretion from HepG2 human hepatoma cells, a model of hepatic VLDL secretion. As a result, G-hesperidin significantly reduced apo B secretion from the oleate-stimulated HepG2 cells. Furthermore, G-hesperidin significantly suppressed apo B secretion only in the oleate-stimulated cells and failed to act on the cells incubated without oleate. In the oleate-stimulated cells, G-hesperidin significantly decreased cellular cholesteryl ester (CE), although it had no effect on cellular TG or free cholesterol amounts. Moreover, the oleate-stimulated cells had a decrease in cellular apo B amounts by G-hesperidin exposure. These findings indicate that G-hesperidin down-regulates the assembly of apo B-containing lipoproteins via the reduction of CE synthesis augmented with oleate and results in suppressing excess apo B secretion from the cells. This effect is speculated to be associated with the improvement of VLDL metabolic abnormality in hypertriglyceridemic subjects and considered as a mechanism of lowering serum TG.

Malto-oligosyltrehalose synthase (EC 5.4.99.15, MTSase) catalyzes the conversion of α-1,4-glucan to glycosyltrehalose by forming an α,α-1,1-glucosidic linkage on the reducing side of the α-1,4-glucan. This enzyme also slightly hydrolyses the glucan and releases glucose from the reducing end of the glucan. We mutated the gene of MTSase from Sulfolobus acidocaldarius ATCC 33909 and expressed the mutated gene in E. coli. The mutants of Asp228, Glu255 or Asp443 corresponding to the catalytic residues of the α-amylase family enzymes showed no enzymatic activity. The transglycosylation activity of the mutants of Lys390 or Lys445 decreased, but the hydrolytic activity of the mutants increased in comparison to the wild-type enzyme. The substitution of Lys390 or Lys445 for a bulky residue, tryptophan, caused the loss of the transglycosylation activity of the enzyme, and provided a novel hydrolase reacting on the reducing side of the α-1,4-glucan.

A novel cyclic pentasaccharide (CPS) and a branched cyclic pentasaccharide (6G-CPS) consisting of d-glucopyranose were synthesized with 6-alpha-glucosyltransferase (6GT) and 3-alpha-isomaltosyltransferase (IMT) from Bacillus globisporus N75. The structure of CPS was cyclo-[--6)-alpha-D-Glcp-(1--3)-alpha-D-Glcp-(1--6)-alpha-D-Glcp-(1--3)-alpha-D-Glcp-(1--4)-alpha-D-Glcp-(1--]. The other, 6G-CPS, had the structure cyclo-[--6)-alpha-D-Glcp-(1--3)-alpha-D-Glcp-(1--6)-alpha-D-Glcp-(1--3)-[alpha-D-Glcp-(1--6)]-alpha-D-Glcp-(1--4)-alpha-D-Glcp-(1--]. The formation of CPS was presumed to occur after the following four successive reactions: a 6-glucosyltransfer reaction with 6GT, a 4-glucosyltransfer reaction with 6GT, a 3-isomaltosyltransfer reaction with IMT, and a cyclization reaction with IMT.

The glucosyl transfer reaction of kojibiose phosphorylase (KPase) from Thermoanaerobacter brockii ATCC35047 was examined using cyclo-{-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->} (CTS) as an acceptor. KPase produced four transfer products, saccharides 1-4. The structure of a major product, saccharide 4, was 2-O-alpha-d-glucopyranosyl-CTS, cyclo-{-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-[alpha-d-Glcp-(1-->2)]-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->}. The other transfer products, saccharides 1-3, were 2-O-alpha-kojibiosyl-, 2-O-alpha-kojitriosyl-, and 2-O-alpha-kojitetraosyl-CTS, respectively. These results showed that KPase transferred a glucose residue to the C-2 position at the ring glucose residue of CTS. This enzyme also catalyzed the chain-extending reaction of the side chain of 2-O-alpha-d-glycopyranosyl-CTS.

We investigated the interaction between trehalose and alkaline-earth metal ions. The nuclear relaxation times of carbon atoms of trehalose were shortened by addition of the alkaline-earth chloride salts, MgCl2, CaCl2, and SrCl2, indicating that trehalose formed metal-complexes with the alkaline-earth metal chlorides. From the data of the 1H-1H coupling constants of trehalose in the presence of the alkaline-earth chlorides, it appeared that trehalose formed complexes with MgCl2, and CaCl2 at the various complexing sites: Mg2+ was coordinated to O-4 and O-4' of trehalose, and Ca2+ to O-2 and O-3. We succeeded in the preparation of two types of crystals of the trehalose/CaCl2. One was a crystal consisting of trehalose, CaCl2, and water in a ratio of 1:1:1. The other was an anhydrous crystal containing trehalose and CaCl2 in a ratio of 1:2. Several applications of the complexing between trehalose and the metal ions for food processing are proposed.

Transglycosylation reactions to cyclic tetrasaccharide (CTS, cyclo[--6)-alpha-D-Glcp-(1--3)-alpha-D-Glcp-(1--6)-alpha-D-Glcp-(1--3)-alpha-D-Glcp-(1--]) and its derivatives were investigated. An enzyme, 6-alpha-glucosyltransferase, which is involved in CTS synthesis from starch, from Bacillus globisporus C11 produced 4-O-alpha-glucosyl-CTS (4G-CTS) from a mixture containing CTS and maltopentaose. Another enzyme, 3-alpha-isomaltosyltransferase, synthesized 3-O-alpha-isomaltosyl-CTS (3IM-CTS) from CTS and panose. Two novel branched CTSs, 3-O-alpha-isomaltosyl-4-O-alpha-glucosyl-CTS (3IM-4G-CTS) and 3-O-alpha-isomaltosyl-(4-O-alpha-glucosyl)-CTS [3IM-(4G)-CTS], were synthesized by the isomaltosyl transfer of IMT into 4G-CTS. IMT also produced a novel saccharide, 3-O-alpha-isomaltosyl-3-O-alpha-isomaltosyl-CTS (3IM-3IM-CTS) from 3IM-CTS. It was confirmed that the oligosaccharides, including 4G-CTS, 3IM-CTS, 3IM-4G-CTS, 3IM-(4G)-CTS and 3IM-3IM-CTS, remaining in the reaction mixture during the production of CTS from starch were the transfer products of 6GT and IMT into CTS.

Cyclomaltodextrin glucanotransferase (EC 2.4.1.19, abbreviated as CGTase) from Bacillus stearothermophilus produced a series of transfer products from a mixture of cyclomaltohexaose and D-fructose. After digestion with glucoamylase, the resultant product contained turanose and trehalulose in addition to D-glucose and D-fructose. These results showed that CGTase transferred the glucose residue to the C3- and C1-hydroxy groups of fructopyranose. The optimum conditions on the turanose production from α-CD and D-fructose using CGTase were examined. The reaction mixture containing 30% of α-CD and 30% of D-fructose and 300 U/g of α-CD of CGTase from B. stearothermophilus was incubated at 60°C and pH 5.5 for 24 h and then treated with glucoamylase. Turanose was produced in a yield of about 45%.

Cyclic tetrasaccharide (CTS; cyclo{→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→}) is a cyclic oligosaccharide four D-glucosyl residues linked in an alternating α-1,3- and α-1,6-fashion. CTS has been reported for enzymatic synthesis from alternan, an α-(1→3)-α-(1→6)-D-glucan synthesized from sucrose by Leuconostoc mesenteroides NRRL B-1355 alternansucrase, by endo-glucanase (alternanase) from Bacillus sp. NRRL B-21195. While searching for nonreducing oligosaccharides that can be produced from α-1,4-glucan as the substrate, we screened bacteria isolated from soil in our own way, and obtained Bacillus globisporus C11, which produces CTS from starch. Two kinds of glycosyltransferase, 6-α-glucosyltransferase (6GT) and α-isomaltosyltransferase (IMT), were purified from the culture supernatant of this strain. It was found that CTS is produced by the sequential action of both enzymes. The genes for IMT (CtsY) and 6GT (CtsZ) were linked together on the chromosome, forming ctsYZ. Both of the gene products showed similarities to α-glucosidases belonging to glycoside hydrolase family 31 and conserved two aspartic acids corresponding to the putative catalytic residues of these enzymes. CtsYZ and four open reading frames upstream of ctsYZ form a gene cluster, ctsUVWXYZ. The reaction conditions for CTS synthesis were examined using 6GT and IMT from B. globisporus C11. The optimum reaction conditions to obtain CTS from Pinedex #100 (partial hydrolyzate of starch, 1.3%-hydrolysis) were the following: substrate concentration, 3%; pH, 6-7; temperature, 30°C; enzyme dosages, 1 U/g-dry solid 6GT, 10 U/g-dry solid IMT. In these optimum conditions the CTS yields reached 62% at the reaction time of 48 h. A mass-production method of highly purified CTS crystals at a reasonable cost was established, and the functions and characteristics of CTS were studied.

本研究では,水/エタノール溶液系での不飽和脂肪酸自動酸化に及ぼすトレハロース添加の影響について,1)不飽和脂肪酸からのヒドロペルオキシド(HPOD)生成過程および,2)HPODの分解,アルデヒド生成過程を検討し,トレハロースの脂質酸化抑制作用を考察した.1) リノール酸(LA)からのHPOD生成はトレハロース添加濃度に応じて減少し,反応14日目のHPOD量は,トレハロース終濃度7.3mMでは2.57mg/gLA,14.6mMでは1.87mg/gLA,29.2mMでは1.28mg/gLAであった.一方,α-リノレン酸(LNA)からのHPOD生成量もLAと同様にトレハロース添加濃度に応じて減少し,トレハロース終濃度7.3mMでは7.80mg/gLNA,14.6mMでは6.30mg/gLNA,29.2mMでは4.50mg/gLNAであった.糖アルコールであるマルチトール添加系のHPOD生成量もトレハロース添加系と同様に低値であった.2) トレハロースおよびマルチトールによるHPOD分解およびHPODからの揮発性アルデヒド生成抑制作用は弱かった.以上の結果から,トレハロースは不飽和脂肪酸からHPODを生成する酸化初期反応を抑制することが示唆された.

We investigated the anti-tumor effect of pamidronate after obtaining a decrease of serum monoclonal immunoglobulin (Ig) level by conventional chemotherapy in patients with multiple myeloma (MM) in order to evaluate whether the drug is useful as maintenance therapy for MM. Eight patients with MM received 60 mg/d pamidronate every third week for 6-18 months without chemotherapeutic agents or corticosteroids after the treatment with melphalan and prednisolone, or vincristine, adriamycin and prednisolone. Serum Ig and beta2-microglobulin (b2MG) levels were maintained at the levels obtained after the termination of chemotherapy in six and four out of eight patients, respectively. Hemoglobin levels were maintained at, or increased to more than, the levels observed at the end of chemotherapy in six patients. Decreased plasma cells in the bone marrow after the chemotherapy were evident in five patients. Two patients were categorized as non-responders, because Ig and b2MG increased and anemia progressed after treatment with the drug. Despite the very small numbers, the results suggest that pamidronate may have anti-tumor activity and be useful for treatment after the conventional chemotherapy in some cases of MM.

Production of cyclo[--6)-alpha-D-Glcp-(1--3)-alpha-D-Glcp-(1--6)-alpha-D-Glcp-(1--3)-alpha-D-Glcp-(1--] (CTS, cyclic tetrasaccharide) from starch was attempted using 1,6-alpha-glucosyltransferase (6GT) and 1,3-alpha-isomaltosyltransferase (IMT) from Bacillus globisporus C11. The optimal conditions for production from partially hydrolyzed starch were as follows: substrate concentration, 3%; pH 6-7; temperature, 30 degrees C; 6GT, 1 unit/g-dry solid (DS); IMT, 10 units/g-DS. The production of CTS was demonstrated and 544 g of CTS hydrate crystal powders were obtained from 3500 g of partially hydrolyzed starch. Two major by-products were also isolated from the reaction mixture and identified as the branched derivatives of CTSs, 4-O-alpha-D-glucopyranosyl-CTS and 3-O-alpha-isomaltosyl-CTS.

Trehalose is a non-reducing disaccharide consisting of two glucose residues. Industrial mass-production of trehalose from starch has been achieved by using enzymatic reactions of maltooligo-syltrehalose synthase and maltooligosyltrehalose trehalohydrolase. This saccharide is available for various foods and has many useful functions; sweetening, low-damage to nutrients, and prevention of degeneration of starch and proteins. In this review, we present several functions of trehalose revealed by recent studies. Degradation of fatty acids and the formation of volatile aldehydes from fatty acids are remarkably suppressed by trehalose. This saccharide is preventive of the body odor (volatile aldehydes) of aged persons. In addition, trehalose has inhibitive effects on DNA-strand scission and protein modification by lipid oxidative products. Trehalose is effective in stabilizing vitamin C and SOD-like activity. Trehalose has shown ability to interact with minerals, including calcium chloride. Because of the interaction between trehalose and minerals, trehalose can be applied to improve of the property of mineral-containing products. From these results, it has been seen that trehalose has the benefits of lipid, minerals and vitamins other than starch and protein, including its function as a malti-functional saccharide. Trehalose has wide applications in foods, cosmetics and phamaceuticals.

The genes for isomaltosyltransferase (CtsY) and 6-glucosyltransferase (CtsZ), involved in synthesis of a cyclic tetrasaccharide from alpha-glucan, have been cloned from the genome of Bacillus globisporus C11. The amino-acid sequence deduced from the ctsY gene is composed of 1093 residues having a signal sequence of 29 residues in its N-terminus. The ctsZ gene encodes a protein consisting of 1284 residues with a signal sequence of 35 residues. Both of the gene products show similarities to alpha-glucosidases belonging to glycoside hydrolase family 31 and conserve two aspartic acids corresponding to the putative catalytic residues of these enzymes. The two genes are linked together, forming ctsYZ. The DNA sequence of 16,515 bp analyzed in this study contains four open reading frames (ORFs) upstream of ctsYZ and one ORF downstream. The first six ORFs, including ctsYZ, form a gene cluster, ctsUVWXYZ. The amino-acid sequences deduced from ctsUV are similar in to a sequence permease and a sugar-binding protein for the sugar transport system from Thermococcus sp. B1001. The third ctsW encodes a protein similar to CtsY, suggested to be another isomaltosyltransferase preferring panose to high-molecular-mass substrates.

The intact maltotetraose-forming exo-amylase from Pseudomonas stutzeri (G4-1), which has a raw starch binding domain, has been crystallized. The structure was identified (PDB entry 1GCY) by the molecular replacement method using the structure of its catalytic domain (G4-2). The result showed that the raw starch binding domain is in a disordered state, the corresponding electron densities being almost invisible. Superposition of these two enzyme forms showed evidence for the possible location of the raw starch binding domain (SBD). This crystal is a novel case, in that it forms a regular lattice incorporating flexibly bound SBD in the channel of crystal packing of the catalytic domains.

Arthrobacter ramosus S34, which produces trehalose from maltooligosaccharide, was isolated. A trehalose-producing operon, treYZ, was cloned from the genome. Expression experiments with treY and treZ confirmed that they coded malto-oligosyltrehalose synthase and malto-oligosyltrehalose trehalohydrolase, respectively. The amino acid sequence of TreY from A. ramosus S34 and that from Arthrobacter sp. Q36 did not show high identity, nor did those of TreZ.

Malto-oligosyltrehalose synthase (EC 5.4.99.15, MTSase) is a glycosyltransferase catalyzing the conversion of maltodextrins to malto-oligosyltrehaloses by forming α, α-1, l-glucosidic linkage. The enzyme slightly catalyzes the release of glucose from maltodextrins by hydrolysis of the α-1, 4 linkage of maltodextrins on the reducing end. The amino acid sequence of MTSase from Sulfolobus acidocaldarius ATCC 33909 showed significant homologies to those of not only other bacterial MTSases but also α-amylase family enzymes. The S. acidocaldarius MTSase lost the enzyme ac tivity by site-directed mutation of Asp228, G1u255, or Asp443 of the enzyme, which corresponded to the catalytic residues of α-amylase family enzymes. The mutation of Lys390 or Lys445 brought about a decrease of the transfer activity and an increase of the hydrolysis activity of the enzyme. The two lysine residues are conserved among MTSases but not α-amylase family enzymes. The tertialy structure analysis revealed that MTSase as well as α-amylase family enzymes contained a (β/α)8 barrel structure as the catalytic domain. The three carboxyl residues (Asp228, G1u255 and Asp443) proposed as the catalytic center of MTSase were found to be located in the active cleft and positioned similarly to those of a -amylase family enzymes. There were two loops and one he lix structures on one end of the active cleft, where a nearly closed space was formed. The lysine residues (Lys390 and Lys445) were found to be situated in the closed structure. It was suggested that several residues containing the two lysines in the closed structure were involved in catalysis of the α, α-1, 1 transfer activity of MTSase.

A gene located just upstream of the treYZ operon was isolated from Arthrobacter sp. strain Q36. The gene, designated treX, encoded an 823-amino acid protein. The amino acid sequence of the protein had 50% identity with the TreX protein (isoamylase) from Sulfolobus acidocaldarius ATCC 33909 which has a treZXY operon on the genome. We suggest that Arthrobacter treX is an isoamylase gene, and that it is a component of a treXYZ operon.

The three-dimensional structure of isoamylase from Pseudomonasamyloderamosa , which hydrolyzes α-1,6-glucosidic linkages of amylopectin and glycogen, has been determined by X-ray structure analysis. The enzyme has 750 amino acid residues and a molecular mass of 80 kDa, and it can be crystallized from ammonium sulfate solution. The structure was elucidated by the multiple isomorphous replacement method and refined at 2.2 A resolution, resulting in a final R -factor of 0.161 for significant reflections with a root-mean-square deviation from ideality in bond lengths of 0.009 A. The analysis revealed that in the N-terminal region, isoamylase has a novel extra domain that we call domain N, whose three-dimensional structure has not so far been reported. It has a (β/α) 8 -barrel-type supersecondary structure in the catalytic domain common to the α-amylase family enzymes, though the barrel is incomplete, with a deletion of an α-helix between the fifth and sixth β-strands. A long excursed region is present between the third β-strand and the third α-helix of the barrel but, in contrast to the so-called domain B that has been identified in the other enzymes of α-amylase family, it cannot be considered to be an independent domain, because this loop forms a globular cluster together with the loop between the fourth β-strand and the fourth α-helix. Isoamylase contains a bound calcium ion, but this is not in the same position as the conserved calcium ion that has been reported in other α-amylase family enzymes.

The three-dimensional structures of the catalytic residue Glu219-->Gln mutant of Pseudomonas stutzeri maltotetraose-forming exo-alpha-amylase, and its complex with carbohydrate obtained by cocrystallization with maltopentaose were determined. Two crystal forms were obtained for the complexed enzyme, and a bound maltotetraose was found in each. The structures were analyzed at 2.2 A and 1.9 A resolution, respectively for the uncomplexed and complexed mutant. These structures were compared with the wild-type enzyme structure. In the complexed crystals, the maltotetraose was firmly bound, extensively interacting with the amino acid environments in the active cleft. The non-reducing end glucose unit was hydrogen bonded to the side-chain of Asp160 and the main-chain nitrogen of Gly158, which seem to be predominantly required for the recognition of the non-reducing end of the substrate that determines the exo-wise degradation of this enzyme. The reducing end glucose unit of bound maltotetraose showed clear deformation, adopting a half-chair conformation with extensive hydrogen bonds to surrounding polypeptides. The C1-atom of this deformed glucose unit lies very close to Asp193OD1 with a distance of 2.6 A. The catalytic residue Asp294 is firmly hydrogen-bonded to the O2 and O3-hydroxyl groups of the deformed reducing end glucose unit. Upon binding of the carbohydrate, small but significant induced fits were observed in the regions of Asp294, Phe156, Ile157, and Asp160. Possible roles of the three catalytic residues are also discussed.

The three-dimensional structure of an exo-type alpha-amylase from Pseudomonas stutzeri which degrades starch from its non-reducing end to produce maltotetraose has been determined by X-ray structure analysis. The catalytic domain of this enzyme (G4-2), whose structure was determined, is a product of spontaneous limited proteolysis in culture broth. It has 429 amino acid residues and a molecular mass of 47,200, and crystallizes in ammonium sulfate solution at pH 7.5. The structure was elucidated by the multiple isomorphous replacement method and refined at 2.0 A resolution, resulting in a final R-factor of 0.178 for significant reflections with a root-mean-square deviation from ideality in bond distances of 0.013 A. The polypeptide chain of this molecule folds into three domains; the first with a (beta/alpha)8 barrel structure, the second with an excursed part from the first one, and the third with five-stranded antiparallel beta-sheets. The active cleft is formed on the C-terminal side of the beta-sheets in the (beta/alpha)8 barrel as in the known endo-type alpha-amylases. A histidine side-chain nitrogen ND1 is coordinated to one of the bound calcium ion. The recognition site of the non-reducing end of the amylose that determines exo-wise degradation is presumed to be at one end of this cleft where there is a disordered loop consisting of the 66th to 72nd residues, and a loop carrying an aspartic acid (Asp160). These structural features may be responsible for the binding of the non-reducing end of the substrate amylose.

Based on determination of the transferred products (maltooligosaccharides G n ), the plausible degradation modes of G 2 , G 3 , and G 4 were analyzed quantitatively, and it was made an attempt to evaluate the binding affinity A i on some subsites of cycloamylose glucanotransferase (CGT) from Bacillus stearothermophilus. This method can be employed instead of the technique using a radioisotope. The quantities of the substrates (G 2 -G 4 ) and products G n were measured by using the HPLC technique as a function fo time for the CGT-catalysed reaction.

The two genes encoding maltooligosyl trehalose synthase and maltooligosyl trehalose trehalohydrolase, which are related to biosynthesis of α,α-trehalose, were cloned from Rhizobium sp. M-11.Sequence analysis showed that the synthase gene composed of 2316 bp was connected with the hydrolase gene of 1788 bp by an overlap of one nucleotide. The deduced amino acid sequences of both enzymes have several regions common to amylolytic enzymes belonging to an “α-amylase family”.

We searched for a cyclic tetrasaccharide, cyclo-{→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→} (abbreviated as CTS) in various foods. GLC analysis revealed that sake lees and sake contained CTS in ranges of 61 to 378 μg/g and 0.23 to 7.05 μg/g, respectively. CTS isolated from sake lees, showed 1H- and 13C-NMR spectra completely identical to those of CTS enzymatically synthesized from starch. We examined CTS formation in the cells of Saccharomyces cerevisiae ATCC9763. Fed with additional sucrose on the 25th day of culture, cells (8.4 g) from a 39-day culture gave a significant amount of CTS (25.2 mg).

A novel enzyme, maltooligosyl trehalose trehalohydrolase from Arthrobacter sp. Q36 was purified from a cell-free extract to an electrophoretically pure state by successive column chromatography on Sepabeads FP-DA13, Butyl-Toyopearl 650M, DEAE-Toyopearl 650S, and Toyopearl HW-55S. The enzyme specifically catalyzed the hydrolysis of the alpha-1,4-glucosidic linkage that bound the maltooligosyl and trehalose moieties of maltooligosyl trehalose. The Km of the enzyme for maltosyl trehalose, maltotriosyl trehalose, maltotetraosyl trehalose, and maltopentaosyl trehalose was 5.5 mM, 4.6 mM, 7.0 mM, and 4.2 mM, respectively. The enzyme had a molecular mass of 62,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.1 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was threonine. The enzyme showed the highest activity at pH 6.5 and 45 degrees C, and was stable from pH 5.0 to 10.0 and up to 45 degrees C. The activity was inhibited by Hg2+, Cu2+, Fe2+, and Zn2+.

Egg white lysozyme was found to catalyze the transfer of N-acetylglucosamine to cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] (CTS). Structural analysis showed that the transfer product was 3-O-beta-N-acetylglucosaminyl CTS, cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[beta-GlcNAc-(1-->3)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. This branched saccharide is anticipated to be a model compound of the sugar chains of glycoproteins.

Cycloamylose glucanotransferase(cyclomalto-oligosaccharide glu-canotransferase, CGTase), obtained from Bacillus stearotheromophilus, was found to catalyze the synthesis of cyclomalto-oligosaccharides from maltotriose (G3) as a substrate. The synthetic reaction is one of the CGTase-catalysed reactions, so called “cyclisation”. A main saccharide produced by the cyclisation reaction under the experimental conditions employed was confirmed to be cyclomaltoheptaose CG7. The kinetic parameters, the Michaelis constant Km and the molar activity k0 were evaluated for the cyclisation reaction. Modification of tryptophan residues with N-bromosuccinimide (NBS) was found to effect on Km, but not on k0 of the cyclisation reaction, suggesting that the tryptophan residue (Trp97) carries an essential role in the binding (disproportionation process) of maltotriose. Tryptophanreste von Bakterien-Cycloamylose-Glucanotransferase: Einflus der Modifizierung mit N-Bromosuccinimid auf die enzymkatalysierte Synthese von Cyclomaltoheptaose aus Maltotriose. Es wurde gefunden, das aus Bacillus stearothermophilus gewonnene Cycloamylose-Glucanotransferase (Cyclomalto-oligosac-charid-glucanotransferase, CGTase) die Synthese von Cyclomalto-oligosacchariden aus Maltotriose (G3) als Substrat katalysiert. Die Synthese-Reaktion ist eine der CGTase-Reaktion, die sogenannte „Cyclisierung”. Ein durch die Cyclisierungsreaktion unter gegebenen experimentellen Bedingungen hauptsachliches Saccharid wurde als Cyclomaltoheptaose (CG7) bestatigt. Die kinetischen Parameter, die Michaelis-Konstante Km und die molare Aktivitat k0 fur die Cyclisierungsreaktion, wurden ermittelt. Es wurde gefunden, das die Tryptophanreste, die mit N-Bromosuccinimid (NBS) modifiziert worden sind, auf den Km, jedoch nicht auf k0 einwirken. Dies weist darauf hin, das der Tryptophanrest (Trp97) eine wesentliche Rolle bei der Bindung (Disproportionierungsprozes) von Maltotriose spielt.

Cycloamylose glucanotransferase (CGTase, EC 2.4.1.19) from Bacillus stearothermophilus was found to catalyze the production of cyclomalto-oligosaccharides from maltose (G2) as a substrate. This synthesis reaction, called “cyclisation” is one of the four CGTase-catalyzed reactions: disproportionation, coupling, hydrolysis, and cyclisation. On the reaction time-course observed by using the fluorescence method, a lag-phase was found prior to the production of cyclomalto-oligosaccharides. The lag-time may be referred to a reaction “disproportionation”. Based on the linear portion of the reaction curves, the kinetic parameters, the Michaelis constant Km and the molar activity k0, were evaluated for the G2 cyclisation. Modification of tryptophan residues with N-bromosuccinimide gives not so much effect on the Km and k0 values for cyclisation. However, an effect was found on the lag-time, suggesting that the Trp residue (Trp97) carries an essential role in the disproportionation process. Cycloamylose-glucanotransferase-katalysierte Cyclisierung fur ein Substrat Maltose. Modifizierung mit N-Bromosuccinimid an den Tryptophanresten. Es wurde gefunden, das Cycloamyloseglucanotransferase (CGTase, EC 2.4.1.19) aus Bacillus stearothermophilus die Produktion von Cyclomaltooligosacchariden aus Maltose (G2) als Substrat katalysiert. Diese „Cyclisierung” genannte Synthesereaktion ist eine der vier CGTase-katalysierten Reaktion: Disproportionierung, Kupplung, Hydrolyse und Cyclisierung. Bei dem Reaktions-Zeitverlauf wurde durch Anwendung der Fluoreszens-Methode eine Verzogerungsphase vor der Produktion von Cyclomalto-oligisacchariden gefunden. Die Verzogerungszeit durfte sich auf die Disproportionierungs-Reaktion beziehen. Basierend auf dem linearen Anteil der Reaktionskurven wurden die kinetischen Parameter, die Michaelis-Konstante Km und die molare Aktivitat k0 fur die G2-Cyclisierung bewertet. Die Modifizierung der Tryptophanreste mit N-Bromosuccinimid ergibt keinen besonderen Effekt auf die Km- und k0-Werte fur die Cyclisierung. Es wurde je-doch ein Einflus auf die Verzogerungszeit gefunden, was darauf hin-weist, das der Trp-Rest (Trp97) eine wesentliche Rolle im Disproportionierungsprozess spielt.

Four tryptophan residues in cyclomalto-oligosaccharide (cycloamylose, cyclodextrin) glucanotransferase (CGTase) from Bacillus stearothermophilus were modified with N -bromosuccinimide (NBS), one of which (“Trp 4 ”) was markedly less reactive than the others. The modification of Trp 4 by NBS corresponded with inactivation of the CGTase-catalysed hydrolysis of cyclomaltohexaose (CG 6 ). Trp 4 was protected against NBS by glucose and the maltosaccharides G 2 –G 4 , which indicates Trp 4 to be located at the substrate binding site of CGTase.

Field trials were carried out in calves using a live bovine respiratory syncytial (BRS) virus vaccine prepared from the attenuated BRS virus, strain rs-52. Two hundred seventy-five and 353 calves were vaccinated intranasally and intramuscularly, respectively. No undesirable postvaccinal reactions were observed in the vaccinated calves. Of the serum neutralizing (SN) antibody negative calves 89.7% (26/29) and 92.8% (90/97) developed SN antibody 1 month after intranasal and intramuscular vaccination, respectively. Most of the calves having SN antibody titers of 1:1 or 1:2 at the time of vaccination showed a significant increase in SN antibody titer. About 70% and 90% of the calves vaccinated intranasally and intramuscularly, respectively, maintained SN antibody for 6 months after vaccination. In a field trial, a natural BRS virus infection occurred about 5 months after the start of the trial. Ten of the 16 unvaccinated control calves showed respiratory symptoms due to BRS virus infection. On the contrary, all of the 68 vaccinated calves exhibited no symptoms at all, indicating efficacy of the vaccine.

本研究では, 歯周病患者での初診, 治療中からメインテナンスにかけてのプラークコントロールレベルの経時的な動態や病態との関連について検討を行った。対象者は, 初診からメインテナンスまで歯周治療が継続された35名 (平均年齢46.4歳) とした。被検項目としては, O'LearyのPlaque Control Record (PCR) とProbing Depth (PD) を用い, 以下の結果を得た。1) 対象者の初診時や初期治療中のプラークスコアの改善度とメインテナンス時のプラークスコアとは, 直接関連していなかった。2) 対象者のメインテナンス時ではプラークスコアが低い人ほどその変動が大きく表れることが示唆された。3) 対象者の初診時PCRあるいは初期治療によるPCRの改善と4mm以上のPD所有率とに関連は見られなかった

The bacterial strain PP710, isolated from soil and identified as Paenibacillus species, produced a low-digestibility alpha-glucan containing a large amylase-resistant portion. This alpha-glucan was obtained in high yields from maltodextrin (dextrose equivalent 3) by using the condensed culture supernatant of the strain as the enzyme preparation. The water-soluble dietary fiber content of the low-digestibility alpha-glucan was 80.2%, and showed resistance to a rat intestinal enzyme preparation. The alpha-glucan was found to be a novel highly branched alpha-glucan by acid hydrolysis, NMR analysis, gel permeation chromatography, methylation analysis, and enzymatic digestion.

During the studies on application of amylases, some a-amylases showed a remarkable transglycosylation action in a high concentration of starch. Using the action, the production of novel and useful saccharides was undertaken, and CGTase was selected. The enzyme catalyzes “cyclization” forming cyclodextrins, “disproportionation” and “coupling reaction, ” in which glycosyl residues are transferred from α-1, 4-glucan or cyclodextrins to an acceptor such as glucose or sucrose. Through the coupling reaction of CGTase we succeeded in the conjugation with glycosyl residue and various substances, and established their usages in food industry and related fields.

The template effects of alkali metal ions and organic ammonium ions on the rate of calixarene ring inversion were investigated. In the temperature-dependent 1 H NMR spectra in D 2 O, the coalescence temperature (T c ) of p-sulfonatocalix[4] arene (1 4 H) was enhanced up to 17-26 o C in the presence of alkali metal ions and up to 50-65°C in the presence of ammonium ions from 9°C in the absence of these ions. The temperature rise, which means the suppression of the ring-inversion rate, was explained as such that these ions have the template effects on the calixarene ring inversion

The 3-dimensional X-ray crystallographic structure of cyclodextrin glucanotransferase (CGT-ase) from B. stearothermophilus showed that the CGTase molecule fold into four globular domains, A, B, C and D. The N-terminal domains, A and B, are similar to those of Taka-amylase. The C and D domains, which are unique to this enzyme, both consist of antiparallel β-barrel structure. With a substrate binding analysis in the crystal, two binding sites have been found on the enzyme molecule, one in a cleft of the A-domain and the other in the D-domain. The first one is considered to be related to the enzyme activity in a same manner with a-amylase and the second to the raw starch binding activity of the CGTase.