Fullerence (C₆₀) was determined by high-performance liquid chromatography using both ultraviolet and mass spectrometric detection. The detection limit for each method was 0.05 and 2.0ng (signal-to-noise ratio (S/N=2)) per injection, respectively. Rat plasma spiked with C₆₀ (10μg/ml) was extracted using solid phase extraction with a recovery of 62.1% and the coefficient of variation (c.v., n=5) between intra-day assays was 4.0%. The calibration curve for peak area and plasma C₆₀ concentration with ultraviolet detection showed good linearity (r=0.996) over the range 0.5-60 μg/ml. This newly developed method was applied to rat plasma samples after intravenous administration of C₆₀ solubilized with polyvinylpyrrolidone.

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A geniposide-hydrolyzing β-glucosidase was discovered in Eubacterium sp. A-44, a human intestinal anaerobe. The enzyme was intracellularly distributed in the bacterium, and purified to homogeneity from the extract using Butyl-Toyopearl 650M, Sephacryl S-300, hydroxyapatite and chromatofocusing column chromatography. The enzyme was a single polypeptide chain with the molecular weight of 90 kDa and the N-terminal amino acid sequence initiated from methionine up to the 29th residue did not show more than 50% homology against known protein sequences. A broad substrate specificity was shown for the β-glucosidase to hydrolyze aryl β-D-glucosides (p-nitrophenyl β-D-glucopyranoside-pNPG, esculin and salicin), alkyl β-D-glucosides (geniposide and amygdalin) and cellobiose. The K_m values (mM) for various β-D-glucosides were 0.068 for geniposide, 0.10 for pNPG, 0.21 for esculin, 0.22 for salicin, 2.9 for amygdalin, and 0.91 for cellobiose. The pH optimum with pNPG and geniposide as the substrates was 6.0. The enzyme was inhibited by sulfhydryl reagents, Cu²⁺, and nojirimycin bisulfite.

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The mechanism of inactivation of cholinesterase (EC 3.1.1.8) by the Cu²⁺-ascorbic acid (AsA) system was investigated. Incubation of the enzyme with the Cu²⁺-AsA system under aerobic conditions resulted in an irreversible loss of enzyme activity. At low concentrations of Cu²⁺, the extent of inactivation showed the same dependence in accordance with the extent of oxidation of AsA. Saturation kinetics were observed with respect to the concentration of AsA. No change in the dissociation constant of the enzyme-AsA complex was observed at various concentrations of Cu²⁺. Catalase at a low concentration partially protected the enzyme from the inactivation, but did not affect the oxidation of AsA. In addition, catalase at a high concentration completely protected both the enzyme from inactivation and the AsA from oxidation. Both thiourea and thiocyanate completely protected the enzyme from the inactivation, while AsA was partially oxidized only in the initial phase. Our proposed mechanism for the inactivation of an enzyme by the Cu²⁺-AsA system is as follows. A ternary complex involving the enzyme, Cu²⁺ and AsA is formed. This is followed by a redox reaction within the complex which generates a superoxide (·O^-₂) and hydrogen peroxide (H₂O₂). The H₂O₂ then reacts with·O^-₂ in a Haber-Weiss reaction producing the hydroxyl radical (·OH). Another role of H₂O₂ is the conversion of the reduced Cu⁺ within the complex to Cu²⁺. Thus, repeated cycles of the redox reaction between the Cu²⁺ and AsA take place at the same locus, producing multiple·OH, which causes its complete inactivation.

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β-Glucuronidase was purified 360-fold from Escherichia coli HGU-3, an human intestinal bacterium. The specific activity of the purified enzyme was 17.78 units/mg protein. The enzyme (M.W. 290000) is composed of four subunits (M.W. 72000) with a pI and optimal pH of 4.8 and 6-7, respectively. The apparent K_m for p-nitrophenyl-β-D-glucuronide was found to be 0.22 mM. The enzyme was inhibited by saccharic acid 1, 4-lactone, glycyrrhizin, N-ethylmaleimide (NEM) and p-chloromercuriphenylsulfonic acid (PCMS). Using the bile containing bilirubin diglucuronide as a substrate, the purified β-glucuronidase was able to hydrolyze it to bilirubin. This hydrolyzed bilirubin formed calcium bilirubinate with a reaction mixture containing CaCl₂.

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In an attempt to clone a gene encoding the K⁺ uptake system from Vibrio alginolyticus, two plasmids, pKT2 and pKT4, were derived from pACYC184. These plasmids allowed the growth of K⁺ uptake-deficient mutant strains of Escherichia coli TK420 and V. alginolyticus FS181 in a low K⁺ medium. The pKT2 and pKT4 had an insertion about 7 and 6kb, respectively, from the genome of V. alginolyticus. We prepared deletion plasmids from both plasmids and found that the site of genes inserted in the two was not identical and that the active locus corresponded to the structural gene encoding the N-terminal quarter part of tetA (C) gene. The N-terminal region of tetA (C) gene was ligated in another vector plasmid pHG165 to produce pHGK23. pHGK23 complemented the growth of TK420 in the low K⁺ medium. It contained only 62bp from the genome of V. alginolyticus, and the open reading frame was composed of 98 amino acid residues from the N-terminal quarter part of tetA (C) and 5 amino acid residues attached by gene fusion. Using the Na⁺-loaded cells of TK420, pHGK23 was found to increase the activity of K⁺ uptake. These results show that the N-terminal side of tetA (C) gene product functions as a K⁺ uptake system.

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Intraduodenal administration of nattokinase (NK) at a dose of 80mg/kg, resulted in the degradation of fibrinogen in plasma suggesting transport of NK across the intestinal tract in normal rats. The action of NK on the cleavage of fibrinogen in the plasma from blood samples drawn at intervals after intraduodenal administration of the enzyme was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis with an anti-fibrinogen γ chain antibody. The 270 kDa fragment carrying antigenic sites for the binding of the anti-fibrinogen γ chain antibody appeared within 0.5h and was then degraded gradually to a 105kDa fragment via a 200 kDa fragment. This suggests that fibrinogen was degraded to a 105 kDa fragment via several intermediates (270 and 200kDa). In parallel with the degradation process, plasma recalcification times were remarkably prolonged. NK was also detected in the plasma from blood samples drawn 3 and 5 h after administration of the enzyme by SDS-PAGE and Western blotting analysis with an anti-NK antibody. The results indicate that NK is absorbed from the rat intestinal tract and that NK cleaves fibrinogen in plasma after intraduodenal administration of the enzyme.

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We examined the inhibitory effect of two saponin preparations from Red ginseng, 20 (R)-and 20 (S)-ginsenoside-Rg3, in comparison with that of ginsenoside-Rb2, on lung metastasis produced by two highly metastatic tumor cells, B16-BL6 melanoma and colon 26-M3.1 carcinoma, in syngeneic mice. In an in vitro analysis, both saponin preparations showed a significant inhibition of adhesion to fibronectin (FN) and laminin (LM) by B16-BL6 melanoma. Similarly, they significantly inhibited the invasion of B16-BL6 cells into the reconstituted basement membrane (Matrigel)/FN in a dose-dependent manner. In an experimental metastasis model using B16-BL6 melanoma, consecutive intravenous (i.v.) administrations of 100μg/mouse of 20 (R)- or 20 (S)-ginsenoside-Rg3 1, 2, 3 and 4d after tumor inoculation led to a significant decrease in lung metastasis. The inhibitory effect of i.v. administration of both ginseng saponins on the tumor metastasis of B16-BL6 melanoma was also recognized in a low dose of 10μg/mouse. The oral administration (p.o.) of both saponins (100-1000μg/mouse) induced a significant decrease in lung metastasis of B16-BL6 melanoma. Moreover, both ginseng saponins were effective in inhibiting of lung metastasis produced by colon 26-M3.1 carcinoma. When 20 (R)-or 20 (S)-ginsenoside-Rg3 was orally administered consecutively after tumor inoculation in a spontaneous metastasis model using B16-BL6 melanoma, both of them significantly inhibited lung metastasis. In the experiment involving neovasculization by tumor cells in vivo, both mice groups given each saponin preparation after tumor inoculation exhibited a significant decrease in the number of blood vessels oriented toward the tumor mass, with no repression of tumor size. These findings suggest that both ginseng saponins, 20 (R)-and 20 (S)-ginsenoside-Rg3, possess an ability to inhibit the lung metastasis of tumor cells, and the mechanism of their antimetastatic effect is related to inhibition of the adhesion and invasion of tumor cells, and also to anti-angiogenesis activity.

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The inhibitory effects of synthetic butylidenephthalide (BP) derivatives on 10% fetal bovine serum-stimulated proliferation were assayed by measuring the proliferative cell number at an interval of 12h in primary cultures of mouse aorta smooth muscle cells (SMC). Their potencies for the anti-proliferation effect were in the order BP-42 (4, 5-dihydroxy BP)>BP-92 (4, 5-dihydroxy butylphthalide)>BP-97 (6, 7-dihydroxy-3-(3-bromo-1-octenyl)-phthalide)>BP-82 (6, 7-dihydroxy BP)>BP-86 (5, 6-dihydroxy BP)>BP-87 (4, 5, 6-trihydroxy BP)>BP-85 (4, 7-dihydroxy BP)>BP-84 (5, 7-dihydroxy BP)>BP-4C₃ (4-methoxy propylphthalide)>BP-7 (4-hydroxy BP)>BP-40 (4, 5-dimethoxy butylphthalide)>BP-5C₃ (4-hydroxy propylphthalide). We divided these anti-proliferative effects into anti-competence and anti-progression effects by using a convenient assay. BP-42 had the greatest potency in used phthalides for competence inhibition of the SMC proliferation. BP-92 had small potency for competence inhibition. BP-97 had greater potency for competence inhibition than BP-82. These results demonstrated that the anti-proliferative effect of BP-42 was greatest in used phthalides in primary cultures of vascular SMC. The 4, 5-dihydroxy group and 3-butylidene or 3-(3-bromo-1-octenyl) group in these synthetic BP derivatives contributed to the anti-competence effect on SMC. BP-42 may become a prototype of an anti-atherosclerotic drug.

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The effects of yohimbine and desipramine on adrenal catecholamine (CA) release in response to splanchnic nerve stimulation (SNS) were examined in anesthetized dogs. SNS at 1 and 3Hz produced frequency-dependent increases in epinephrine (EPI) and norepinephrine (NE) output determined from adrenal venous blood. Yohimbine (30 and 100μg/kg, i.v.), a selective α₂-adrenoceptor antagonist, enhanced the SNS-induced increases in both EPI and NE output. Desipramine (100 and 300μg/kg, i.v.), an amine pump inhibitor, enhanced the SNS-induced increases in NE output, whereas no enhancement of EPI output was produced. After desipramine treatment, yohimbine further enhanced the SNS-induced increases in EPI and NE output. After yohimbine treatment, desipramine further enhanced the SNS-induced increase in NE output. These results suggest that the release of adrenal CA in response to SNS is inhibited by α₂-adrenoceptors, and that released NE, rather than EPI, is predominantly taken up into the dog adrenal medullary cells.

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Inhibitory effects of atrial natriuretic peptide (ANP) on angiotensin II (ANG II)-induced renal responses were examined in anesthetized dogs. ANG II (5ng/kg per min) was infused intravenously and changes in renal hemodynamics and urine formation were compared between the ANP (10ng/kg per min)-infused kidney and the contralateral vehicle-infused (control) kidney. ANG II reduced absolute and fractional urinary sodium excretion in both the ANP-infused kidney and the control kidney. ANG II also reduced glomerular filtration rate in the control kidney but not in the ANP-infused kidney. The ANG II-induced reduction in urinary sodium excretion in the ANP-infused kidney was smaller than the response in the control kidney, whereas ANP did not affect the reduction in fractional sodium excretion. These results suggest that ANP prevents hypofiltration and thereby attenuates the antinatriuresis induced by ANG II.

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Male F344 rats were pretreated with lead nitrate, nickel chloride, cobalt chloride or cadmium chloride, and their effects on the induction of cytochrome P450 (CYP) enzymes, mainly CYP1A2 enzyme, with 2-methoxy-4-aminoazobenzene (2-MeO-AAB) in the livers were comparatively examined by enzymatical, immunochemical, and molecular biological methods. When rats were pretreated with each ionic metal, the total CYP amount in the liver microsomes decreased, as compared with that of rats treated with 2-MeO-AAB alone. However, among the ionic metals used only lead reduced the levels of the mRNA and protein of CYP1A2 induced with 2-MeO-AAB in the rat liver, and decreased the microsomal activity (per CYP) for CYP1A2-mediated mutagenesis. Furthermore, ionic lead, but not other ionic metals, showed an ability to induce a placental form of glutathione S-transferase (GST-P). The level of CYP1A2 induced with 2-MeO-AAB was decreased along with increase in that of the induced GST-P.

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The effect of glutathione (GSH) isopropyl ester on cellular sensitivity to UVB irradiation was investigated in HeLaS3 cells. Pretreatment with 0.1-0.5 mM GSH isopropyl ester for 4h significantly inhibited the decrease of thymidine (TdR) incorporation caused by UVB irradiation at a dose of 500 J/m², whereas pretreatment with a high dose (1 mM) had no effect. The colony formation ability of the pretreated cells (0.3 mM) was significantly better than that of cells that received irradiation only. When the cells were treated with GSH isopropyl ester, their intracellular GSH level increased dose-dependently over a 4 h period, suggesting that GSH isopropyl ester was transported into the cells and there converted to GSH. Within 2 min of exposure, the intracellular GSH level depleted rapidly to about 75% of that in non-irradiated normal cells. In contrast, the GSH level in cells pretreated with 0.3 mM GSH isopropyl ester was maintained at the same level as that in normal cells, indicating that the maintenance of intracellular GSH level is due to converted GSH from GSH isopropyl ester. These results clearly show that intracellular GSH is involved in cell protection against photodamage, and that GSH isopropyl ester is a useful antioxidant for protection against photooxidative injury.

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To determine the mechanism involved, the incorporation rate (ICR) of drugs into hair was compared to melanin affinity, lipophilicity and membrane permeability. The following 20 drugs were tested ; amphetamine, methamphetamine, p-hydroxyamphetamine, p-hydroxymethamphetamine, cocaine, benzoylecgonine, ecgonine methyl ester, morphine, 6-acetylmorphine, phencyclidine, 1-(1-phenyl)-piperidinyl-cyclohexanol, methylenedioxyamphetamine, methylenedioxymethamphetamine, methoxyphenamine, O-desmethyl methoxyphenamine, benzphetamine, norbenzphetamine, deprenyl, lysergic acid diethylamide (LSD) and 11-nortetrahydrocannabinol-9-carboxylic acid (THCA). Their ICRs were represented as the ratios of the drug concentrations in rat hair to AUCs (the areas under the concentration vs. time curves) in rat plasma. Cocaine had the highest incorporation rate understood and there was a 3600 fold difference between the ICR of cocaine and that of THCA, the lowest drug. The melanin affinity of these drugs was determined by incubating a test solution with melanin at 36°C in the dark for 2h. After incubation and centrifugation, the drug concentration in the filtrate was determined by GC/MS or LC. The drug most affinitive to melanin was cocaine, followed by benzphetamine, phencyclidine, methylenedioxymethamphetamine and LSD. The correlation coefficient between ICR and melanin affinity of the 20 drugs was 0.947 (0.949 excluding THCA). Lipophilicity was calculated from the retention times of HPLC according to Kaliszan's method. Although the correlation coefficient between ICR and lipophilicity was very low (0.201), it rose to 0.770 by removing only THCA. The combination of melanin affinity and lipophilicity brought about a higher correlation (0.979) with the ICRs. Our data also suggested that the higher ICRs of basic drugs than neutral or acidic ones are strongly related to the membrane permeability of the drug based on the pH gradient between blood (pH 7.4) and hair matrix (acidic).

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Benzylphthalimide analogs (P1P's) and phenethylphthalimide analogs (P2P's) have been found to exhibit thalidomide-like activity on the production of tumor necrosis factor (TNF)-α by the human leukemia cell line, HL-60, stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA). Structure-activity relationships are discussed on the basis of the TNF-α production-enhancing activity. Benzylphthalimide (P1P-00) exhibited activity which is weaker than that of thalidomide, but introduction of a methyl group at the ortho-position of the benzyl moiety (P1P-10) resulted an increase to a level comparable with that of thalidomide. Phenethylphthalimide (P2P-00) is more potent than thalidomide, and its fluorinated derivative, 2-phenethyl-4, 5, 6, 7-tetrafluoro-1H-isoindole-1, 3-dione (FP2P-00), exhibited potent activity at very low concentrations.

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The antitumor activity of doxorubicin (DXR) which had been encapsulated in poly (ethylene glycol) (PEG)-coated long-circulating liposomes was examined in mice inoculated with colon 26 carcinoma cells. Six mol% of the distearoylphosphatidylethanolamine derivative of PEGs with different molecular weights was incorporated in liposomes (90-110nm, mean diameter) composed of distearoylphosphatidylcholine/cholesterol (1/1, molar ratio), and the encapsulating efficiency of DXR in liposomes was more than 98% by the pH gradient method. Each concentration of DXR in blood and tumor tissue was significantly greater after administration of the drug encapsulated in PEG-coated liposomes (DXR-PEG-liposome) compared to the non-coated control liposomes or non-encapsulated free drug. DXR-PEG-liposome prepared with PEG1000 (DXR-PEG1000-liposome) more effectively increased the level of DXR in blood and tumor than did the preparations with PEG5000 or PEG12000. A single treatment with DXR-PEG1000-liposome (10mg DXR/kg) resulted in increased survival time. Further therapeutic improvement in terms of tumor growth retardation and prolongation of survival time were observed following multiple treatments with DXR-PEG1000-liposome (3×5mg DXR/kg). Long-circulating liposome coating optimized PEGs should be useful for the delivery of chemotherapeutic agents for the treatment of solid tumors.

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The differences in gastrointestinal absorption behaviors of glycyrrhizin (GZ) between pure GZ and GZ in glycyrrhiza extract (GE) (equivalent dose as GZ) were examined in rats. Similarly to the case of pure GZ, both GZ and glycyrrhetic acid (GA) were detected in the plasma after oral administration of GE. However, the plasma concentration-time curves of GZ and GA after GE oral administration were much lower than those of pure GZ, indicating the marked reduction in bioavailability of GZ and as GA after this administration. To identify the GE components affecting the absorption of GZ, GZ was removed from GE and the effect of the remaining components on the gastrointestinal absorption process of GZ was examined. The lipophilic components of GE reduced the gastric emptying rate and the absorption of GZ from the small intestine, while these effects were not observed in the hydrophilic components. In contrast, the bioavailability of GZ as GA was increased by the hydrophilic components, but not by the lipophilic ones. At least some of the factors in GE altering the bioavailability of GZ were identified.

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(1→3)-β-D-Glucans exhibit a variety of biological and immunopharmacological activities, and the significance of these activities is dependent on the structure of the glucans such as molecular weight, degree of branching, and conformation. Based on the generally accepted evidence that the conformation of clinically used Sonifilan (SPG) is a triple helix, we prepared alkaline treated SPG (SPG-OH) as a single helix conformer. In this report, we examined (A) the antitumor effect on a solid form tumor in vivo, (B) hematopoietic response on cyclophosphamide induced leukopenia, (C) antagonistic effect for zymosan mediated-hydrogen peroxide synthesis on peritoneal macrophage (PM), (D) priming effect of lipopolysaccharide (LPS) triggered tumor necrosis factor (TNF) synthesis, (E) nitric oxide synthesis of PM in vivo, and (F) hydrogen peroxide synthesis of PM in vivo. Both SPG and SPG-OH showed a significant effect on (A) and (B). The activity on (C) was stronger in SPG than SPG-OH. The activities of (D), (E), and (F) were stronger in SPG-OH. These facts strongly suggested that the glucan-mediated immunopharmacological activities were dependent on the helical conformation, and the conformation dependency varied dependent on the assays used.

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Tetrameric carbonyl reductase of pig lung has been shown by cDNA cloning to be a member of the short-chain dehydrogenase family of enzymes. Construction of the carbonyl reductase cDNA in an expression vector yielded an abundant, enzymatically active enzyme in Escherichia coli. The recombinant enzyme was purified to homogeneity and shown to be structurally, immunologically and functionally similar to lung carbonyl reductase.

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Sialidases possessing enzyme activity were solubilized from mouse-adapted influenza viruses A/PR/8/34 (A/PR8, H1N1), A/Guizhou/54/89 (A/Guizhou, H3N2) and B/Ibaraki/2/85 (B/Ibaraki) by proteolytic digestion and purified by affinity chromatography and/or sucrose density gradient centrifugation. The purified sialidasese were observed as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH of purified sialidases from A/PR8, A/Guizhou and B/Ibaraki against sodium p-nitrophenyl-N-acetyl-α-D-neuraminate were 6.5, 7.5 and 5.5, respectively. The purified sialidase (N1) from A/PR8 and its original virus showed enzyme activity with similar substrate specificity, and preferentially hydrolyzed α (2→3) sialyllactose and bovine submaxillarymucin (BSM). Purified sialidase from B/Ibaraki hydrolyzed α(2→3) sialyllactose, α(2→6) sialyllactose and most glycoproteins, especially BSM, but the intact virus showed higher sialidase activity against sialyllactoses than against glycoproteins and gangliosides. These results indicate that the purified enzyme and the original virus of B/Ibaraki have different substrate specificities of sialidase activity. Purified A/Guizhou sialidase (N2) hydrolyzed α(2→3) sialyllactose and porcine stomach mucin but not α(2→6) sialyllactose and BSM. The original virus of A/Guizhou showed substrate specificity similar to its purified enzyme, except that the virus was active against BSM.

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Denuded oocytes obtained from the ovaries of ddY mice underwent spontaneous germinal vesicle breakdown (GVBD) and polar body formation (PBF) by cultivation for 3 and 16h, successively. To study the significance of protein synthesis in oocytes regarding their maturation, denuded oocytes were cultured in the presence of reversible (cycloheximide and puromycin) and irreversible (ricin) protein synthesis inhibitors. When denuded oocytes were cultivated in the continual presence of any inhibitor, only PBF but not GVBD was found to be inhibited. These inhibitions were dose dependent, ranging from 10^-9 to 10^-6M, and were relative to the protein synthesis inhibitions by them. The PBF inhibition was relieved by withdrawing cycloheximide for first 2, 3 and 4h in the course of the culture. Further, the PBF inhibition occurred in the presence of ricin for only the first 2 or 3h in the course of the culture. These results suggested that proteins synthesized during 2 to 4h in the course of culture may play important roles in PBF.

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Treatment of Klebsiella pneumoniae 109 with cefodizime, ceftazidime, cefbuperazone, cefotaxime or flomoxef at a sub-minimum inhibitory concentration (sub-MIC) (1/4 MIC) for 1 h altered its morphology. The bacteria treated with cefodizime at sub-MICs (1/8, 1/4 MIC) enhanced the chemiluminescencence (CL) of human polymorphonuclear leucocytes (PMNs), implying an increase in the production of active oxygen species in association with phagocytosis, whereas the cells treated with other cephalosporins at the same sub-MICs did not. Furthermore, a significant decrease in electrophoretic mobility was induced by the bacteria treated with cefodizime at sub-MICs (1/8, 1/4 MIC), but other cephalosporins neither increased nor decreased the electrophoretic mobility significantly. These findings suggested that cefodizime caused a morphological change, with a decrease in negative surface charge density of K. pneumoniae, more easily than ceftazidime, cefbuperazone, cefotaxime or flomoxef, followed by an increase in the phagocytic activity of PMNs.

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The interaction of zinc sulfate with thrombospondin (TSP) derived from bovine platelets, on the proliferation of bovine aortic smooth muscle cells was investigated using cell culture. Zinc alone has no effect but potentiates the stimulatory effect of TSP on the incorporation of [³H] thymidine into the acid-insoluble cell fraction. Other heavy metals including copper, lead, manganese and nickel did not exhibit a similar effect. In bovine aortic endothelial cells, [³H] thymidine incorporation was stimulated by zinc alone and suppressed by TSP alone ; the stimulation by zinc was significantly reduced by TSP. In human fibroblastic IMR-90 cells, [³H] thymidine incorporation was stimulated by either zinc or TSP ; a simultaneous treatment with zinc and TSP showed an additive effect. The present data suggest that zinc may be a unique heavy metal that potentiates the proliferation of vascular smooth muscle cells stimulated by TSP ; in addition, such an interaction was observed selectively in this cell type. Since a large amount of TSP is released from α-granules of aggregated platelets, zinc may augment TSP activity which is involved in the physiology of vascular tissues.

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Only a few biosynthetic pathways in fungal cells have been used as antifungal targets. Therefore, the number of antifungals has been limited, and a cross-drug resistance among them has emerged in the therapy of mycoses. Under such circumstances, the identification of an antifungal with a new mode of action is highly desirable. By infecting mice with a mutant of C. albicans deficient in the sulfate assimilation pathway, we have discovered a new target for the discovery of antifungal agents. We have proven that azoxybacilin inhibits the sulfate assimilation pathway by showing its inhibitory activity for [³⁵S] SO₄ incorporation into proteins. We have also demonstrated that azoxybacilin was taken up into fungal cells via an active transport system specific for methionine. This sharing of the uptake system with methionine may explain the mechanism by which the antifungal activity of azoxybacilin is antagonized by methionine, and led us to design azoxybacilin derivatives that lack the structural feature of amino acids and, at the same time, have increased hydrophobicity to give higher non-specific permeability through the cell membrane. As a result, we have found that ester derivatives of azoxybacilin were not antagonized by methionine in their uptake, and that they showed antifungal activity independent of methionine. The benzyl ester of azoxybacilin was the same as azoxybacilin in its mode of action, but was not markedly antagonized by methionine at concentrations up to 1 mg/ml. These results suggest that azoxybacilin may not merely interfere with the sulfate assimilation pathway.

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Metallothionein induction by thrombin was investigated using a culture system of vascular endothelial and smooth muscle cells. It was found that the protease induced metallothionein in endothelial cells derived from human aorta and umbilical vein as well as bovine aorta in a concentration-dependent manner. Thrombin also induced metallothionein in vascular smooth muscle cells derived from bovine aorta but not in cells derived from human, rabbit and rat aortas. In bovine aortic endothelial and smooth muscle cells, induction of metallothionein by thrombin was unaffected by the protein kinase C inhibitor staurosporine ; in addition, a protein kinase C activator, phorbol 12-myristate 13-acetate, failed to induce metallothionein, suggesting that the induction by thrombin occurred through pathway (s) other than protein kinase C activation. It is suggested that metallothionein may be induced during hemostasis in the vascular tissue through protein kinase C-independent pathway (s).

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The level of urinary thiobarbituric acid reactive substances (TBARS) consisting of bound forms of malonaldehyde and, to a lesser extent, other aldehydes is one of the indices of lipid peroxidation status. Levels of urinary TBARS in healthy subjects before and after supplementation with a high dose of d-α-tocopherol were measured using high performance liquid chromatography. Four healthy Japanese were given a supplement of 300mg d-α-tocopherol/d, about 40-fold higher than the normal intake recommended, for a period of 50d. Levels of urinary TBARS (nmol/kg body weight·h) within-day, before and after supplementation with d-α-tocopherol, exhibited similar behavior and levels of daily urinary TBARS (nmol/kg body weight·d) were unchanged by d-α-tocopherol supplementation. These results indicate that supplementation with a high dose of d-α-tocopherol does not affect the level of urinary TBARS.

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Monoclonal antibodies targeting c-erbB-2 protooncogene product p185 were conjugated with adriamycin via a pH-sensitive spacer. The resultant antibody-adriamycin conjugates showed immunoselective binding, internalization and cytotoxicity to p185-positive human breast cancer cell SKBr-3 and gastric cancer cell MKN-7, but not to normal human lymphocytes.

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We investigated the antihypertensive effect of sesamin, a lignan from sesame oil, using two-kidney, one-clip (2K, 1C) renal hypertensive rats. After clipping the left renal artery, animals were assigned to either a normal diet group (control group) or a sesamin-containing (1% (w/w)) diet group (sesamin group). The sham-operated rats (sham group) were fed a normal diet and tap water. The systolic blood pressure of the control group increased progressively in comparison with the sham group. This 2K, 1C-induced hypertension was markedly reduced by feeding the sesamin-containing diet. The systolic blood pressure after 4 weeks was 123.60±4.01 mmHg in the sham group, 187.43±5.69 mmHg in the control group and 145.57±6.78 mmHg in the sesamin group, respectively. There were significant increases in left ventricle plus septum weight-to-body weight ratio in the control group compared with the sham group. This rise was also significantly reduced in the sesamin group. When the thoracic aorta was histochemically evaluated, the wall thickness and wall-to-lumen ratio in the control group were significantly increased, compared with the sham group, indicating that vascular hypertrophy had occurred in the control group. The sesamin diet tended to ameliorate this vascular hypertrophy, although its effect was not statistically significant. These findings suggest that sesamin is useful as prophylactic treatment to combat the development of renal hypertension and cardiac hypertrophy.

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The effect of Panax ginseng extract on the learning performance of aged Fischer 344 rats using the 8-arm radial maze task and the operant discrimination task was examined. Aged rats showed significantly impaired learning performance in both tasks. Daily administration of ginseng extract (8g/kg/d, p. o. for 12-33 d) ameliorated the impairment of learning performance in the radial maze task but not in the operant discrimination task. These results suggest that subchronic treatment with ginseng extract improves spatial cognitive impairment in aged rats.

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The absorption of carbamazepine (CBZ) after rectal administration in the form of a suppository was studied in rats. CBZ suppositories were prepared with Witepsol H-15 (H-15), Witepsol S-55 (S-55) or polyethylene glycol 6000 (PEG) bases by moulding procedure. An in vitro investigation was undertaken using a dissolution apparatus. The in vitro dissolution rate of CBZ showed marked differences among the bases in the order PEG>H-15>S-55. An in vivo study demonstrated marked differences in the time required to reach peak plasma concentration (T_max) of CBZ after rectal and oral administration : the absorption of CBZ from PEG base was the most prolonged among these bases (in the order PEG>H-15>S-55>oral). Comparison of the area under the plasma concentration-time curve (AUC) of CBZ after rectal administration of the three different suppositories with those after intravenous and oral administration showed no significant differences in the AUC among the five preparations. These results suggest the possibility that CBZ suppositories can replace oral treatment for epilepsy.

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Pharmacokinetics of a very short-acting, a short-acting and two long-acting cholinesterase (ChE) inhibitors, edrophonium, neostigmine, pyridostigmine and ambenonium, respectively, were compared to elucidate the major determinant of their pharmacokinetics. No dose-dependency in pharmacokinetic behavior was observed within the range of 2-10 μmol/kg for edrophonium, 0.5-2 μmol/kg for pyridostigmine, 0.1-0.5 μmol/kg for neostigmine and 0.3-3 μmol/kg for ambenonium, respectively. Neostigmine has the shortest elimination half-life, and edrophonium, pyridostigmine and ambenonium follow in that. Four ChE inhibitors have similar Vd_ss values within the range of 0.3-0.7l/kg, which is similar to the muscle/plasma concentration ratio of these drugs. The liver or kidney to plasma concentration ratio of all ChE inhibitors at 20 min after i.v. administration ranged from 5 to 15. Small distribution volumes estimated from the plasma concentration profiles may reflect the distribution to muscle and to the extracellular space of other organs/tissues, while the rapid disappearance of ChE inhibitors from plasma may reflect the concentrative uptake to the liver and kidney.

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The effects of the serotonin_1A (5-HT_1A) receptor agonist buspirone on hypoglycemia elicited by tolbutamide were investigated in rats. Buspirone, at doses not affecting plasma glucose levels, inhibited the hypoglycemic effects of tolbutamide. The inhibitory effects of buspirone on tolbutamide-induced hypoglycemia were antagonized by the 5-HT_1A receptor antagonist pindolol. As tolbutamide is known to induce hypoglycemia by facilitating insulin release, the effects of buspirone on a tolbutamide-induced increase in serum insulin levels were also studied. However, buspirone did not affect tolbutamide-induced insulin release. Adrenodemedullation inhibited the effects of buspirone. These results suggest that buspirone inhibits tolbutamide-induced hypoglycemia mediated by the 5-HT_1A receptor, and adrenaline release may be involved in the effects of buspirone.

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A 5S-ribosomal gene spacer region was amplified by the polymerase chain reaction using a DNA preparation from the crude drug"Angelica Root"as a template. The nucleotide sequence of the amplified product was identical to that of Angelica acutiloba, the source plant of "Angelica Root, "but different from that of Bupleurum falcatum of Glehnia littoralis. The possibility of discriminating"Angelica Root"from other umbelliferous crude drugs based on a Hin dIII restriction site inside the spacer region was suggested.

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The role of leukotriene B₄ (LTB₄) in leukocyte infiltration in zymosan induced rat pleurisy was investigated by studying the effects of 5-lipoxygenase inhibitors, T-0757 and AA-861, and a cyclooxygenase inhibitor, indomethacin, on leukocyte infiltration and LTB₄ levels in the inflammatory exudate of rat pleurisy induced by intrapleural injection of zymosan (20 mg/rat). T-0757 and AA-861 inhibited the infiltration of leukocytes, mainly neutrophils, 3 h after injection of zymosan at a dose of 100mg/kg, p.o., but indomethacin did not do so at a dose of 5 mg/kg, p.o. LTB₄ was detected in the exudate 1h after zymosan injection, and its level peaked at 3h (45.4±8.6ng/rat) and decreased thereafter. These LTB₄ levels were depressed by T-0757 and AA-861 in a dose-dependent manner. T-0757 completely prevented LTB₄ production at a dose of 100 mg/kg. These observations suggest that the inhibition of leukocyte infiltration is mediated by the inhibition of LTB₄ production, and that LTB₄ is one of the main chemical mediators of leukocyte infiltration in zymosan-induced rat pleurisy.

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Depudecin, a microbial metabolite containing two epoxide groups, was tested for its anti-angiogenic activity in an in vivo assay system involving the chorioallantoic membrane of growing chick embryo. The microbial metabolite inhibited embryonic angiogenesis in a dose-dependent manner with an ID₅₀ of 320ng (1.5 nmol) per egg. It also affected the growth of vascular endothelial cells, a key event in the process of angiogenesis in vivo. These results suggest that depudecin could be promising as an anti-angiogenic agent and that its anti-angiogenic action involves an inhibitory effect on vascular endothelial cell growth.

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We formulated a new compressed tablet which is rapidly disintegrated and dissolved in the mouth without the need to drink water. The crushing strength increased with increasing compression force and maximum values (8-18 kgf) for the formulations comprising crystalline cellulose and low-substituted hydroxypropylcellulose (L-HPC) were obtained at the compression force of 300 kgf. Rapid disintegration (within 30 s) was obtained in vitro using various compounding ratios of crystalline cellulose to L-HPC. Tablets prepared with crystalline cellulose and L-HPC rapidly disintegrated in saliva (small amount of water) in the mouth of humans.

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