The crystal structure of rutin, 3-[(6-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyl)oxy]-2-(3, 4-dihydroxy-phenyl)-5, 7-dihydroxy-4H-1-benzopyran-4-one, has been determined by the X-ray diffraction method. The crystals are orthorhombic with a=8.748 (3), b=23.567 (10), c=36.052 (12) Å and the space group is C222₁. The final R value is 0.102 for 1789 observed reflections after full-matrix least-squares refinement. The dihedral angle between the benzopy-ran and the phenyl rings is 19.0 (7)°, and the quercetin and the rutinose portions are linked together with the torsion angles of 114.1 (18)°and 143.1 (15)°, around the C(3)-O(3) and O(3)-C(1') bonds, respectively. There are two kinds of intramolecular hydrogen bonds between the quercetin and glucose moieties. The structure-function relationship of the flavonoids involving rutin as inhibitors of aldose reductase is also discussed.

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5-Methyl-7, 8, 9, 10-tetrahydro-5H-azepino[1, 2-α]benzimidazol-7-one (6) was synthesized by treating 1-(3-carboxypropyl)-2, 3-dimethylbenzimidazolium iodide (11) with 1, 1'-carbonyldiimidazole in the presence of triethylamine. Refluxing 2-(5-chloro-2-oxopentyl)-1-methylbenzimidazole (4) in ethyl acetate did not afford 6 but gave (Z)-1-methyl-2-(2, 3, 4, 5-tetrahydro-2-furylidenemethyl)benzimidazole (5), which was rearranged to the corresponding (E)-isomer (7) by heating in ethyl acetate in the presence of basic alumina. The structures of 5, 6 and 7 were determined by X-ray crystallography.

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Several 5-deaza-10-oxaflavin (2H-chromeno[2, 3-d]pyrimidine-2, 4(3H)dione) derivatives (1) were prepared in satisfactory yields through new, practical routes. These 5-deaza-10-oxaflavins showed a strong redox property in the oxidation of some alcohols to the corresponding carbonyl compounds under slightly acidic conditions, while they were reduced to the stable 1, 5-dihydro derivatives. Participation of a direct hydride transfer from the alcohol was demonstrated by an experiment using deuterium-labeled alcohol.

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For the purpose of development of functional biomimetic coenzyme models, we prepared some new types of chiral 5-deazaflavin derivatives. Syntheses of 5-deazaflavin derivatives possessing a chiral substituent at the C(6) position, (13), and a chiral tertiary asymmetric carbon center at C(5) (15a, b, 17a, b, and 18a, b) were achieved in rather satisfactory yield. Preliminary experiments on asymmetric reduction of ethyl benzoylformate with 15a, b were carried out in order to investigate the efficiency of the models thus obtained, resulting in moderate or low asymmetric induction.

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The methylation of cyclomaltohexaose (α-CD) and cyclomalto-octaose (γ-CD) gave two major components in each case. These methylated compounds were isolated by high-performance liquid chromatography and characterized by carbon-13 unclear magnetic resonance spectroscopy, fast-atom bombardment mass spectrometry, and fragmentation analysis. The two major products from α-CD were hexakis(2, 6-di-O-methyl)cyclomaltohexaose and pentakis(2, 6-di-O-methyl)-mono(2, 3, 6-tri-O-methyl)cyclomaltohexaose and those from γ-CD were octakis(2, 6-di-O methyl)cyclomalto-octaose and heptakis(2, 6-di-O-methyl)-mono(2, 3, 6-tri-O-methyl)cyclomalto-octaose.

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(+)-Indolmycenic ester 4, a key intermediate for the synthesis of (-)-indolmycin (1), was prepared from (2S, 3R)-epoxybutyrate 5 via (2S, 3S)-2-hydroxy-3-chlorobutyrate 6 or (2R, 3R)-2-mesyloxy-3-hydroxybutyrate 12, which were obtained by lipase-catalyzed kinefic resolution of the corresponding 2-acetates.

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The Lewis acid-catalyzed cyclocondensation of methylthio (or phenylthio)-silyloxydienes (2), easily derived from stable sulfur ylides (1), with several aromatic aldehydes (3) was investigated. Namely, in the case of boron trifluoride normal reaction products, 2, 3-dihydro-4-pyrones (4), were obtained in good yields. Novel reaction products, thiolanium ylides (6 and 7), were obtained in the presence of titanium(IV) chloride. The structures of 6g was established by X-ray crystallography.

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Two chiral spirocyclic dioxinones (S-5 and R-5) useful in teh so-called de Mayo reaction as alternatives to β-diketones (enones) have been synthesized; they not only act as formylacetates (inactive by themselves as enones) but also display remarkable diastereofacial selectivities in photoaddition to alkenes. The sythesis of these dioxinones is described, together with a practicl one-pot synthesis of enantiomerically pure (1S, 4R)-(+)-6-formyl-2-oxabicyclo[3.3.0]oct-6-en-3-one, the key intermediated of prostaglandin synthesis, from the (6S)-spirocyclic dioxinone (S-5).

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The derivatives of ethyl 2-arylamino-4-oxo-4, 5-dihydrofuran-3-carbodylates (1) undergo dealkoxycarbonylation when they are refluxed in dimethylformamide. A mechanism is proposed.

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Two sesterterpenoids, cheilanthenetriol (1) and cheilanthenediol (2), were isolated from the leaves of Aleuritopteris Khunii together with hop-22(29)-ene, adiantone, hydroxyhopane, a mixture of sterols, 3, 5-dihydroxy-7, 4'-dimethoxyflavone, 5-hudroxy-3, 7, 4'-trimethoxyflavone and 4-methoxybenzoic acid. Cheilanthenetriol was found to be identical with authentic samples of cheilanthatriol and cheilarinosin isolated from Indian ferns. The new sesterterpenoid, cheilanthenediol, wes established as (17Z)-13α, 19-dihydroxycheilanth-17-ene.

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Two new triterpenoid hydrocarbons were isolated from the rhizomes of Davallia mariesii MOORE, together with neohop-13(18)-ene, hop-21-ene, hop-17(21)-ene, hydroxyhopane, cyclolaudenol and cyclobalanol. On the other hand, fern-9(11)-ene, ferna-7, 9(11)-diene, fern-7-ene, hop-22(29)-ene, hydroxyhopane and dryocrassol were isolated from the leaves of the same fern. The structures of two new compounds, hop-16-ene (1) and isohop-22(29)-ene (2), were elucidated on the basis of spectral data and chemical correlations with known compound.

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A versatile chiral synthon, (1R, 6S)-6-methoxycarbonyl-3-cyclohexene-1-carboxylic acid, was obtained by an enantioselective ydrolysis of the corresponding meso diester with pig liver esterase. This enzymatic hydrolysis can easily be carried out on a multi-hundred gram scale. The chiral monoester thus obtained can be further converted into all stereoisomers of 1-amino-2-alkoxycarbonyl-4-cyclohexene derivatives in an enantio- and stereocontrolled manner. These derivatives are considered as potential key intermediates for synthesizing a variety of biologically interesting compounds such as aminocyclitol and carbapenem antibiotics.

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Substitution of a p-toluenesulfonyloxy group in the sugar portion of uracilnucleosides by an alkyl group was investigated by using Gilman reagents (R₂CuLi). In the cases of 5'-O-tosyl derivatives of 2', 3'-O-isopropylideneuridine, moderate vields of 5'-alkylated products were obtained. In contrast to this, the reactions of the corresponding 2'-deoxyuridine derivatives gave higher yields of products. A similar substitution reaction at the 3'-position of 2'-deoxyuridine derivatives was also examined.

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(±)-Oxylubimin (4), the most highly oxygenated spirovetivane phytoalexin, was synthesized stereoselectively from a key intermediate, (2RS, 5RS, 9SR, 10RS)-9-hydroxy-6, 10-dimethyl-2-pivaloyloxyspiro[4.5]dec-6-en-8-one (3). Its methoxymethyl ether (8) was reduced with NaBH₄ and CeCl₃·7H₂O to give predominantly the desired alcohol (9a) (9a/9b=6.4). The alcohol (9a) was successfully transformed into oxylubimin (4) via several steps, involving introduction of a bis(ethoxycarbonyl)methyl group at the C₂, catalytic hydrogenation of the C₆-C₇ double bond, and pyridinium chlorochromate oxidation of the hydroxymethyl group at C₆.

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Photocycloaddition reactions of 4-ethoxycarbonyl-5-aryl-1H-pyrrole-2, 3-dione (1) with cyclopentadiene, indene, and cyclopentene gave the dihydropyridones 8, 11, and 12 as major products, respectively. Formation of them can be rationalized by assuming the formation of a 2s + 2a adduct predicted by the stereoselection rule and subsequent skeletal rearrangement. The stereochemical results seem to support the hypothesis that the donor-acceptor interaction as theoretically deduced by Epiotis plays an important role in determining the stereochemical pathway of photocycloaddition.

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Photocycloaddition reaction of 4-ethoxycarbonyl-5-phenyl-1H-pyrrole-2, 3-dione (1) with cyclohexadiene and dihydropyran gave the cis-fused cyclobutane 5b and the trans-fused cyclobutane 6b as major adducts, respectively. The structures including stereochemistry were established by X-ray crystallographic analysis.The results for these systems markedly contrast with those for the photocycloaddition of 1 to five-membered cycloolefins, cyclopentadiene and dihydrofuran, which yield the adducts with skeletal rearrangements. The difference of product depending on the ring size can be related to the difference in the stereochemical pathways. The distance between donor and aceeptor is suggested to be an important factor determining the polarity of the donor-acceptor pair and, therefore, the stereochemistry of the photocycloaddition.

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Grains of Quinoa, Chenopodium quinoa have been used as a staple food in the Andes, South America. In a continuing study on saponin constituents of this plant, seven oleanane saponins were isolated from brans of the grains. Two of them were identified as known saponins of oleanolic acid, chikusetsusaponin IVa (10) from rhizomes of Panax spp. and quinoside D (12) from seeds of this plant, respectively. The other five compounds (7, 8, 9, 11 and 13) are new saponins, being designated as quinoa-saponins-6-10. The structures of these saponins were elucidated as follows : 7, 8 and 9, 3-O-[β-glucopyranosyl(1→2)-β-glucopyranosyl(1→3)-α-arabinopyranoside]-28-O-β-glucopyranoside of 30-O-methyl spergulagenate, oleanolic acid and phytolaccagenic acid, respectively; 11, hederagenin 3-O-β-glucuronide-28-O-β-glucopyranoside; and 13; hederagenin 3-O-β-xylopyranosyl(1→3)-β-glucuronide-28-O-β-glucopyranoside.

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Three neutral 3, 28-O-bisdesmosidic glycosides of quillaic acid, named bubiosides D, E and F, were isolated from the tuber of Thladiantha dubia BUNGE (Cucurbitaceae). Their structures were elucidated on the basis of chemical and spectral evidence.All dubiosides have a common prosapogenin structure, 3-O-[O-β-D-glucopyranosyl-(1→3)-[O-β-D-galactopyranosyl-(1→2)]-β-D-glucopyranosyl]-quillaic acid, and differ only in the structures of the estr-linked sugar moieties. Dubioside D is a 28-[O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl] ester, dubioside E, a 28-[O-β-D-xylopyranosyl-(1→4)-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl] ester and dubioside F, a 28-[O-β-D-xylopyranosyl-(1→3)-O-β-D-xylopyranosyl-(1→4)-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl] ester of the prosapogenin.

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α-Human atrial natriuretic peptide (α-hANP) was synthesized by assembling six peptide fragments in solution followed by deprotection with HF and subsequent air-oxidation. The trimethylbenzyl group was employed as an S-protecting group of cysteine. The HF-dimethylselenide-m-cresol system was employed as a final deprotecting reagent and, at the same time, as a reducing reagent of Met(O). Syntheric α-hANP elicited potent diuretic and natriuretic activity in rats.

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An alternative method for the synthesis of 2'-deoxy-6, 2'-methanouridine (1), -cytidine (3) and -4-thiouridine (13) is described which involves a condensation of an isopropyl β-D-2-pentuloside (5) and a carbanion formed from 6-trimethylsilylmethylpyrimidine (6), followed by intramolecular glycosylation.

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Treatment of (E)-3-(2-hydroxypropylidene)-4-methyl-1-phenylazetidin-2-one (11) with 10% Pd/C gave (E)-(12), (Z)-3-(2-oxopropylidene)-4-methyl-1-phenylazetidin-2-one (13), 3, 4-cis-(14a) and 3, 4-trans-3-(2-oxopropyl)-4-methyl-1-phenylazetidin-2-one (14b). Among them, 12 and 13 were found to show potent inhibitory activities against rabbit platelet-rich plasma aggregation induced by adenosine diphosphate or collagen. Ring-expanded homologous derivatives and an acyclic analogue-of 12 were also synthesized and tested for the biological activities. The azetidin-2-one skeleton bearing a 2-oxoalkylidene moiety at the 3 position was found to be essential for the platelet aggregation inhibitory activities of these compounds.

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Oxidation of 3-isobutyryl-2-isopropylpyrazolo[1, 5-α]pyridine (IBPP) was carried out with various chemical model systems for cytochrome P-450 in comparison with the liver microsomal system of rats or humans. α-Hydroxylation of side chains and ring hydroxylation at the 6 and 7 positions were the main reactions in both systems. A pattern analysis of products using two dimensional thin layer chromatography was employed to compare the functions of the chemical model systems with those of microsomal systems. The reaction profile of IBPP by the catalyst/Pt-colloid/H₂, O₂ system was most similar to that of human or rat microsomal system. The utility of these chemical models is discussed from the viewpoint of drug metabolism.

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A series of [MeTyr¹, MeArg⁷]-Dynorphin A (Dyn)(1-8)-OH analogues, modified at position 8 with various amino acids, is described. Their biological activities were determined in the three bioassays [guinea pig ileum (GPI), mouse vas deferens (MVD), and rabbit vas deferens (RVD)] and in the mouse tail-pinch test after subcutaneous administration. None of the analogues tested displayed more potent κ-opioid activity in the RVD than [MeTyr¹, MeArg⁷, D-Leu⁸]-Dyn(1-8)-NHEt (1), which is a potent analgesic peptide with similar opioid receptor selectivity to that of Dyn.However, [MeTyr¹, MeArg⁷, Melle⁸]-Dyn(1-8)-OH (11) showed about a twofold more potent analgesic effect than 1. Based on the obtained results it is conceivable that in the case of Dyn(1-8)-OH analogues both a lipophilic L-amino acid in position 8 and an unchanged 7-8 amide bond are essential to maintain potent κ-opioid activity.

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Sine azido dervatives of 1, 5-benzothiazepine Ca antagonist available for photoaffinity labeling are required for further studies of voltage-sensitive Ca channels, we synthesized 3-(p-azidobenzoyloxydeacetyl)- and 3-(4-azidobutyryloxydeacetyl)-diltiazem, and studied their pharmacological properties. Both azido compounds showed similar relaxing actions to diltiazem in K⁺ -depolarized dog arteries. They also showed a similar increasing action to diltiazem, but less potent, on the coronary and vertebral blood flow in the anesthetized dog. Moreover, their negative inotropic effects in the guinea pig papillary muscle were similar to or slightly more potent than that of diltiazem under physiological conditions, but were less potent when studied in K⁺ depolarizing solution. A radioligand binding study in rat skeletal muscle microsomes revealed that the azido derivatives had similar properties to diltiazem, but the nonspecific binding of 3-(p-azidobenzoyloxydeacetyl)-diltiazem was too high to allow estimation of its K_D and B_max values. In conclusion, we synthesized azido derivatives of diltiazem which were considered to share a common binding site on the voltage-dependent Ca channel with diltiazem in skeletal muscle microsomes and in vascular smooth muscle.

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Eleven triterpenoidal saponins having oleanolic acid or hederagenin as the aglycone and two flavonoidal glycosides were isolated from the leaves of Aralia elata SEEM. (Araliaceae). The structures of four new saponins (1, 2, 3, and 4) were established to be 3-O-α-L-rhamnopyranosyl(1→2)-α-L-arabinopyranosyl-hederagenin 28-O-β-D-xylopyranosyl-(1→6)-β-D-glucopyranosyl ester, 3-O-α-L-rhamnopyranosyl(1→2)-α-L-arabinopyranosyl-oleanolic acid 28-O-β-D-xylopyranosyl(1→6)-β-D-glucopyranosyl ester, 3-O-β-D-glucopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)-α-L-arabinopyranosyl-hederagenin 28-O-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl ester, and 3-O-β-D-glucopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)-α-L arabinopyranosyl-hederagenin, respectively, on the basis of spectroscopic and chemical evidence.

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Four new polyacylated sucrose derivatives were found in the bark of Prunus padus. All the compounds were shown to be derivatives of 3-O-p-coumaroyl-β-D-fructofuranosyl α-D-glucopyranoside, and their full structures were elucidated on the basis of spectroscopic and chemical evidence.

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The effects of Shosaikoto, one of the Kampo medicines used for therapy for chronic hepatitis, on liver functions were studied in mice. Oral administration of Shosaikoto for 5 d enhanced the well-known induction of tyrosine aminotransferase (TAT) activity by dexamethasone. Furtehr, TAT activity in mice treated with Shosaikoto was induced effectively by a smaller dose of dexamethasone, as compared with that in control mice. However, Shosaikoto itself did not induce TAT activity in the liver on oral administration or in cultured hepatocytes by direct addition. Moreover, Shosaikoto did not affect the induction of TAT activity by butyryl-adenosine 3', 5'-cyclic monophosphate. The amplifying effect of Shosaikoto seemed to be specific for induction by dexamethasone. These data suggest that Shosaikoto makes the liver sensitive to glucocorticoid by some unknown mechanism.

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Six more anisatin-like sesquiterpene lactones were isolated from the pericarps of Illicium majus. The structures of these sompounds (4-9) were characterized as majucins having a 2-oxy group. It is noteworthy that one of the six compounds has a (10S^*)-hydroxyl group, whereas usual anisatin- and majucin-type compounds have a (10R^*)-hydroxyl group. All these structures were determined on the basis of spectral data, compard with those of majucin and neomajucin. These compounds did not exhibit convulsive toxicity to mice, although the (10S^*)-hydroxy compound was not examined.

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Two diacyl rhamnopyranosylcatalpols were isolated from the leaves of Premna japonica. The structures of the compounds were determined to be 6-O-α-L-(2"-O-isoferuloyl, 4"-O-acetyl)rhamnopyranosylcatalpol and 6-O-α-L-(3"-O-isoferuloyl, 4"-O-acetyl)rhamnopyranosylcatalpol. The locations of acyl moieties were determined by long-range proton selective decoupling experiments and by partial alkaline hydrolysis.

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The effect of ginger root (Zingiberis Rhizoma) on gastrointestinal motility was examined based on its ability to enhance charcoal meal transport in mice. Oral administrations of the acetone extract of ginger (which contains volatile oils and bitter substances) at 75 mg/kg, [6]-shogaol at 2.5 mg/kg, or a [6]-, [8]- or [10]-gingerol at 5 mg/kg enhanced the transport of a charcoal meal. The effects of these substances were similar to or slightly weaker than those of metoclopramide and donperidone.

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The roots of Caraganas chamlagu LAMARCK (Leguminosae) are used as an anti-neuralgic, anti-rheumatic, anti-arthritic, etc. in the folk medicine of Korea. An ether extract was fractionated with monitoring of the anti-inflammatory activity, and teh active principle was elucidated as (+)-α-viniferin on the basis of spectroscopic data, including two-dimensional nuclear magnetic resonance and circular dichroism spectra.

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Cultivation of Bupleurum falcatum L. and determination of saikosaponins by high-performance liquid chromatography (HPLC) were studied. The yueld of dry roots decreased, while the weight of each root increased, as the cultivation period became longer in one-year-old plants. The yield and the weight of each root were shown to be higher in two-year-old plants than in one-year-old plants. The number of plants that survived, however, was greatly decreased by two-year cultivaion. By clipping the aerial part of the plant at the height of 50 or 70 cm, the weight of each root was increased, while the yield of the root was decreased. Mulching of the field with transparent or black vinyl sheet increased the weight of root while silver sheet was ineffective. The content of saikosaponin a or d (SA or SD) was higher in one-year- than in two-year-old plant. The correlation between the root weight and the content of SA or SD was poor. However, a close correlation was found between the contents of SA and SD in the same root.

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Three new diterpenoids, rabdoternins A (1), B (2) and C (3) have been isolated from the bried leaves of Rabdosia ternifolia (D. DON) HARA. Their structures have been elucidated on the basis of spectroscopic and chemical evidence.

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High-performance frontal analysis (HPFA) was applied to simultaneous determinations of the free and total carbamazepine (CBZ) concentrations in human plasma. When 1.5 ml of human plasma containing CBZ at a clinical therapeutic level (free fraction, about 30%) was directly injected into an internal-surface reversed-phase silica column, the CBZ was separated from the plasma bland and was eluted as a zonal peak with a plateau height corresponding to the free CBZ concentration in protein binding equilibrium. Slow and continuous introduction of the plasma sample and the use of titanium filters permitted us to inject the sample repeatedly while avoiding a rapid increase in column pressure. The free and total CBZ concentrations were determined simultaneously from the peak height at the plateau region and the area of the CBZ peak, respectively. The within-run and day-to-day reproducibilities were satisfactory (C.V.≤1.63%, n=5).

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To establish the optimal condition for preparing mouse antiserum specific to a drug, the optimal age and sex of mice for the immune response were studied by measuring the mouse serum levels of total immunoglobulin G (IgG) and Specific antibody to viomycin, as well as the changes in weight of mice immunized with a viomycin immunogen. It was observed that age was a more important factor than sex, and strongly affected productions of both total and specific IgGs of mice. The mice aged 8 weeks yielded the highest levels of both total IgG and the specific antibody. In the study on the influence of booster schedule, the number of boosters given had a larger influence on the immune response than the interval between priming and boosters. The greater the number of booster shots given, the less was the production of total and specific antibodies. The decrease in the weight of mice after immunization was also studied in more detail; it was found that it only occurred in the first week after priming but not after a booster injection. The mice aged eight weeks showed the largest weight loss.

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Ribonucleosides and ribonucleotides produce fluorescence (excitation maxima, ca. 340 nm; emission maxima, ca. 470 nm) when heated in an acidic solution with meso-1, 2-diarylethylenediamines which have phenyl groups substituted with electron-donating groups at the 4-positions, after periodate oxidation. Of the 1, 2-diarylethylenediamines tested, meso-1, 2-bis(4-methoxyphenyl)ethylenediamine was the most sensitive and permitted the fluorometric determination of ribonucleosides and ribonucleotides at concentrations as low as 60-500 pmol/ml. The compound also produces fluorescence with reducing sugars.

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A sensitive and specific enzyme immunoassay (EIA) for somatostatin (SS)-like immunoreactivity (SS-LI) was developed with the use of β-D-galactosidase labeled antigen. The minimum amount of SS-like immunoreactive substance (SS-IS) detectable by this method was 1.0 fmol/well (25 pmol/I). The level of SS-IS in bovine foremilk was about 20 pmol/I, and the level was unchanged after delivery. On the other hand, the levels of gastrin releasing peptide (GRP)-IS and vasoactive intestinal polypeptide (VIP)-IS in bovine foremilk were very high, but fell during 1 week after delivery to about 10% of those in foremilk.

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Two hepatoma cell lines designated Kagura-1 and Kagura-2 were established from rat hepatocellular carcinomas induced by aflatoxin B₁, and have been propagated for over two years. Both cell lines grew as monolayered sheets with a population doubling time of about 20 h. Chromosome counts of Kagura-1 cells ranged from 34 to 45 with a modal number of 40, while that of Kagura-2 cells ranged widely from 40 to 130 with a modal number of 65. Subcutaneous inoculation of cultured cells of both these lines into unde mice resulted in tumor formation. The histopathological appearances of the induced tumors were similar to those of the original tumors. Kagura-1 and Kagura-2 cell lines express at least two tumor markers, glutathione-S-transferase P and γ-glutamyl transpeptidase; the level of c-myc messenger ribonucleic acid was also highly elevated.

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Streptomyces subtilisin inhibitor (SSI) is a protein characterized by both its potent inhibitory activity toward subtilisin and its structure, composed of two homologous subunits. It binds two molecules of subtilisin to form a tetrameric complex.Intermolecularly cross-linked subtilisin is expected to form a polymeric complex with SSI. This could provide a useful model of protein-protein associatin. Therefore, Preparation of fluorescence-labeled and cross-linked subtilisin was carried out.

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In the preceding paper, the preparation of fluorescent cross-linked subtilisin was described. In this paper we present the catalytic and binding properties of the modified enzyme. Kinetic analysis showed that the cross-linked dimeric subtilisin retained both catalytic activity and binding affinity toward synthetic substrates. These kinetic characteristics of the modified enzyme were nearly identical to those of the native enzyme. The modified enzyme also exhibited a specific interaction with Streptomyces subtilisin inhibitor (SSI) with 1 to 1 stoichiometry. The formation of a polymeric complex, which is the expected product with cross-linked subtilisin, was demonstrated.

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The β-adrenoceptor-cyclic adenosine monophosphate (AMP) dependent glycogenolytic cascade was examined in normal rat hepatocytes and rat ascites hepatoma AH130 cells. The cyclic AMP content in AH130 cells was half of that in normal hepatocytes, and the cyclic AMP levels in both kinds of cells were clearly increased by isoproterenol (IPN). Cyclic AMP-dependent protein kinase activity was higher in AH130 cells than in normal hepatocytes. Phosphorylase kinase activities in 10000 × g supernatant of normal hepatocytes and AH130 cells were also increased in the presence of cyclic AMP. Phosphorylase a activities in the supernatant of both kinds of cells gradually decreased during incubation with 40 mM glucose at 37 °C, and the enzyme activity of normal hepatocytes was completely restored by the addition of Mg²⁺-adenosine triphosphate (ATP), but in the case of the hepatoma cells the recovery was small. The decreased phosphorylase a activity in the hepatoma cells was increased by additional glycogen but did not exceed the level before the incubation. In the case of normal hepatocytes it was not affected by glycogen. This indicates that glycogen contained in the cells influences the activation of phosphorylase; the glycogen content in AH130 cells was far less than in normal hepatocytes. On the other hand, when intact cells were incubated with a high concentration of glucose, phosphorylase a activity in the homogenate of normal hepatocytes was decreased and could be restored by IPN and dibutyryl cyclic AMP, but the enzyme activity in the homogenate of AH130 cells was very low and hardly changed after the incubation and treatment with these agents. Phosphorylase phophatase activity was lower in AH130 cells than in normal hepatocytes. Only phosphorylase b converted from the a form by the phosphatase may be able to act as the substrate of phosphorylase kinase. The present study indicates that the key enzymes in the glycogenolytic cascade of AH130 cells functionally act as well as normal hepatocytes, but glycogen phosphorylase is unrsponsive to stimulation through cyclic AMP. A part of the lack of response of the phosphorylase in AH130 cells may be due to its low ability to interconvert between the active form and the inactive form in the tumor cells owing to their low glycogen content and low phosphatase activity.

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The effects of pentagalloylglucose (1, 2, 3, 4, 6-penta-O-galloyl-β-D-glucose) on the aerobic electron transport system of Escherichia coli were studied. The activity of nicotineamide adenine dinucleotide (NADH) reductase was inhibited by pentagalloylglucose, but the activities of succinate dehydrogenase, D-lactate dehydrogenase, and ubiquinol-1 (Q₁H₂) oxidase were not susceptible to the inhibitor. Because the presence of two kinds of NADH dehydrogenase in respiratory chain of "Escherichia coli has been reported, we examined the effect of galloylglucose independently on both NADH dehydrogenases.Pentagalloylglucose is potent and specific inhibitor of both NADH dehydrogenases. One of the NADH dehydrogenases (NADH dh II) is more sensitive to the inhibitor than the other (NADH dh I).

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Changes of biological activites manifested by (1→6)-branched (1→3)-β-D-glucans of various molecular weights obtained by heat treatment of the corresponding intact β-glucan at 150°C (HD-LE) were examined. The activities assessed in this study were as follows : an antitumor activity, activation of alternative complement pathway, glucose consumption by macrophages, macrophage-mediated lysosomal enzyme activity in culture supernatant and cell lysate, interleukin-1 (IL-1) activity, and adjuvant activity. HD-LE could be classified into three groups : 1) HD-LE 0 h (MW 800000) which activated all of the biological activities tested, 2) HD-LE 0.5 and 3 h (MW 250000 and 21000) which lacked or exhibited low levels of activities such as activation of alternative complement pathway and lysosomal enzyme secretion, 3) HD-LE 6 h (MW 6400) which only activated glucose consumption and synthesis of lysosomal enzyme. These results suggest that an antitumor glucan is not always a multiple enhancer of host defense mechanisms and that a large molecular weight is required to augment multiple immunological activities.

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Three polysaccharides, named ukonan A, ukonan B and ukonan C, were isolated from the rhizomes of Curcuma longa L. They were homogeneous on electrophoresis and gel chromatography, and showed remarkable reticuloendothelial system-potentiating activity in a carbon clearnce test. They are composed of L-arabinose : D-xylose : D-galactose : D-glucose : L-rhamnose : D-galacturonic acid in the molar ratios of 12 : 4 : 12 : 1 : 4 : 10 (ukonan A), 12 : 4 : 12 : 1 : 2 : 4 (ukonan B) and 8 : 3 : 6 : 14 : 2 : 3 (ukonan C), in addition to small amounts of peptide moiety. Methylation analysis, carbon-13 unclear magnetic resonance and periodate oxidation studies indicated the structural features of ukonan B, the major one in terms of the activity. It has acidic arabino-galactan type structural units.

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Human splenin (hSP) was synthesized by assembling eight peptide fragments followed by deprotection with 1 M trifluoromethanesulfonic acid-thioanisole (molar ratios, 1 : 1) in trifluoroacetic acid in the presence of m-cresol and dimethylselenium. Finally, the depretected peptide was insubated with dithiothreitol to reduce sulfoxide on the methionine side chain. Incubation of peripheral lymphocytes isolated from uremic patients with the synthetic hSP showed an enhancing effect on the reduced B-lymphocytes, but had no restoring effect on the impaired blastogenic response of T-lymphocytes.

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The hydrolysis of nafamostat mesilate (NM) in aqueous solution was found to be accelerated by sodium bisulfite (SBS) using high-performance liquid chromatographic assay. The hydrolysis of NM catalyzed by SBS was pseudo-first-order and was considered to be a reaction between nafamostat cation (N⁺) and sulfite ion. The effects of SBS concentration and temperature on the hydrolysis of NM in buffer solution were examined. From the findings obtained, we estimated the compatible pH range of the intravenous admixture (mixed infusion) of NM after a constant storage time at a constant SBS concentration and temperature employing a simulated pH-profile for the mixed infusion. In order to evaluate the compatibility of the prescribed mixed infusion, the method of pH estimation for the mixed infusion was also investigated using the pH titration curve of each preparation.

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Rectal absorption of morphine from various kinds of suppository bases was investigated. The extent of bioavailability of morphine by rectal administration varied with the bases used (30.5-97.5%), but every value was higher than that in the case of oral administration (13.5%). Witepsol bases were preferable to macrogol base for the rectal absorption of morphine. In particular, Witepsol S-55 or W-35 gave a higher plasma peak level than H-15 or E-75, whereas the difference in the mean residence times obtained from these bases could not be regarded as significant. Sustained-release suppositories of morphine could be prepared simply by mixing alginic acid (Alg) with morphine in a suppository base. Further, prolonged rectal absorption could be obtained by using these sustained-release suppositories, and the absorption rate was controlled by the amount of Alg added. It seems likely that the sustained release was due to the binding of morphine to Alg from the results of partition coefficient and binding ratio measurements in aqueous solution. The rapid initial absorption and the subsequent prolonged absorption of morphine simultaneously obtained from the morphine-Alg suppository may be useful in the clinical context.

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Gel was prepared from 6-O-carboxymethyl-chitin (CM-chitin) by the addition of iron(III) chloride under mild conditions without any organic solvent. The optimal conditions for the gel formation were 15 to 30 mM iron(III) chloride and 0.5 to 0.8 degree of substitution in CM-chitin. The amounts of bovine serum albumin (BSA) and the anticancer drug doxorubicin (DOX) incorporated into CM-chitin gels were more than 80% and 30%, respectively under the conditions described above. The release of BSA or DOX from the gesl was observed to be increased by lysozyme digestion in a time-dependent manner. This result indicates that CM-chitin might prove useful as a carrier gel for the sustained release of drugs and cytokines, including vaccines.

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Phenytoin (PHT) is a commonly used drug in children and abults, but it is often difficult to adjust the dosage to attain therapeutic drug concentrations due to the nonlinear nature of PHT metabolism. Therefore, many techniques have been proposed to aid dosage adjustment based on single-point PHT concentration determined at the steady state. We have derived a simple equation to predict PHT dosage in a patient in whom one steady-state serum concentration is known. This equation is based on a power relationship between PHT clearance (CL; l/d) and PHT concentration determined at the steady state (C_ss; μg/ml).CL=D/C_ss=a·C_ss^b (1)D_n=D_o·C_o^-(b+1)·C_d^(b+1) (2) where D is the PHT dose (mg/d), D_o is the original dose, D_n is the new dose, C_o is the original C_ss, and C_d is the desired C_ss.We retrospectively investigated the values of a and b in a population of 40 outpatients who had three or more reliable measurements of C_ss in serum, measured while they were taking different daily doses. The a and b values were estimated to be 114.34 and -0.6786, respectively (n=126; r=0.871). Graves et al. have previously derived a similar equation based on an exponential relation between CL of PHT and C_ss in 59 patients (a=133.03, b=-0.804). The predictive ability of this equation was compared with the results of Graves's method and the Bayesian feedback method using retrospective data from 70 patients. This equation allowed the prediction of a dose needed to produce a desired steady-state concentration with satisfactory error, being as effective as the Bayesian feedback method for therapeutic drug monitoring.

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In a previous paper, we reported that methanol extracts obtained from 13 Chinese traditional medicines showed remarkable choleretic effects in normal rats. This paper examines the protective effects against experimental cholestasis induced by carbon tetrachloride (CCl₄) or α-naphthylisothiocyanate (ANIT) in rats. No medicines, including sodium dehydrocholate and 1-phenylpropanol which are used clinically as choleretic drugs, inhibited the decrease of bile flow induced by CCl₄. On the other hand, Intinko-to, Saiko-seikan-to and Bohu-tusyo-san revealed marked improvement of the dysfunction in bile secretion induced by ANIT. These three medicines inhibited the decrease of excretion of bile acid or bilirubin in the bile. They also exerted a protective effect against the alterations of serum components induced by ANIT, i.e., of glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, alkaline phosphatase and the concentration of serum bilirubin.These results indicate that methanol extracts of Intinko-to, Saiko-seiken-to and Bohu-tusyo-san demonstrate not only choleretic effects but also improvement of cholestasis and liver parenchymal injury in rats.

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