Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Volume 23, Issue 5
Displaying 1-35 of 35 articles from this issue
  • Shozo YAMASHITA, Akiko SUZUKI, Tamiko YANAGITA, Shunsei HIROHATA, Masa ...
    2000 Volume 23 Issue 5 Pages 519-522
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
    JOURNAL FREE ACCESS
    Protein changes in the neutrophils of patients with Behcet's disease were analyzed by high resolution two-dimensional gel electrophoresis to investigate the pathological features of Behcet's disease. Two clear protein spots were found to be differently expressed between healthy volunteers and patients with Behcet's disease. One was a 53 kDa protein with pI 5.2 that was detected in healthy volunteers but was nearly absent in the patients. The other was a 40 kDa protein with pI 5.2 that was detected in the patients but nearly absent in the healthy volunteers. Analysis of the N-terminal amino acid sequence of the 40 kDa protein revealed that it was a truncated actin with an N-terminus of Met-44. The presence of the truncated actin in the neutrophils of patients was confirmed by Western blot analysis using an antibody to the C-terminus of actin. The 53 kDa protein could not be identified because its N-terminus was blocked. The presence of the truncated actin in the neutrophils of the patients may be important in understanding the pathology of Behcet's disease.
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  • Jin-Ju NAH, Jae-Young SONG, Seok CHOI, Seok-Chang KIM, Hye Won RHIM, T ...
    2000 Volume 23 Issue 5 Pages 523-526
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    A rapid and sensitive indirect competitive enzyme immunoassay method has been developed for quantitating ginsenoside Rf (Rf) in crude total Panax ginseng saponins and in rat plasma using high titer mouse monoclonal antibody (mAb) raised against a conjugate of Rf and bovine serum albumin (BSA). The isotype of mAb against Rf was IgG3 with a κ chain. The presence of Rf inhibited the binding of the mouse anti-Rf mAb to a Rf-BSA solid phase coating antigen. The working range was 0.01-10 ng/assay and detection limits were 20 pg in various ginseng extract fractions or 34 pg in rat plasma per assay. The anti-Rf mAb cross-reacted with ginsenoside Rg2 by 57.5%, but not with other ginsenosides. However, this anti-Rf mAb did not cross-react with BSA or cellubiose, which is a carbohydrate component of Rf. Using this standard curve, we could measure the amount of Rf in ginseng total extract, ginseng total saponins, protopanaxadiol saponins, and propanaxatriol saponins. We could also measure the amount of Rf in rat plasma after the oral administration of Rf and found that Rf reached a maximum level in rat plasma after 16 h. These results indicate that the anti-Rf mAb could be useful for the quantitation of Rf in crude ginseng fractions and in body fluids.
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  • Mio SENBA, Nobuhiro KASHIGE, Yukihiko NAKASHIMA, Fumio MIAKE, Kenji WA ...
    2000 Volume 23 Issue 5 Pages 527-531
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    The gene encoding β-N-acetylglucosaminidase (GlcNAcase) of Lactobacillus casei ATCC 27092 was cloned and expressed in Escherichia coli. The gene consisted of 1581 nucleotides, and had a promoter, Shine-Dalgarno, and rho-independent type transcription termination sequences typical in bacteria. The protein deduced from the sequence consisted of 526 amino acids, and had a putative signal peptide of 14 amino acids and 5 possible asparagine-linked glycosylation sites. A conserved sequence was homologous to the 12 other hexosaminidases from different origins.The recombinant GlcNAcase (r-GlcNAcase) purified from the transformed E. coli had a MW of 39 kDa and lacked oligosaccharide chains. The isoelectric point and the optimum pH for the activity of r-GlcNAcase were similar to those of original GlcNAcases (o-GlcNAcase). However, the thermal stability was lower, and sensitivity to Cd2+, Fe2+, Cu2+ and sodium dodecyl sulfate (SDS) was higher than those of o-GlcNAcases, suggesting that the oligosaccharide moieties of the enzyme contribute to their stability. The Km value for p-nitrophenyl-N-acetyl-β-1, 4-D-glucosamine (PNP-GlcNAc) of r-GlcNAcase (6.4 μM) implied that the affinity of r-GlcNAcase for the substrate was 200-fold higher than that of the original ones.
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  • Genichiro OSHIMA, Manabu KUNIMOTO, Yasuhito NAKAGAWA
    2000 Volume 23 Issue 5 Pages 532-536
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    Glutathione peroxidase (GPx) activity was detected in the ascite fluid of rats injected intraperitoneally with 2.5% heat-denatured casein solution. Activity in the ascite fluid increased with time after the injection of casein, and reached a maximum at 24 h. The active component was concentrated with successive 35% ammonium sulfate precipitation and Activated Thiol-Sepharose 4B column chromatography from the ascite fluid of rats at 24 h after the injection of casein. No N-terminal amino acid of the protein corresponding to GPx was detected by automatic amino acid sequence analysis following separation with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transfer to a polyvinyl difluoride (PVDF) membrane. Following BrCN treatment of the protein, the N-terminal amino acid sequences of two 14 and 2.6 kDa peptide fragments were found to be S-G-T-I-Y-E-Y-G-A-L and K-I-H-D-I-R-W-N-F-E, respectively. The former and the latter fragments corresponded to sequences beginning at the 37th and 176th amino acid residues of rat extracellular GPx (eGPx), respectively. The exclusive presence of eGPx in the ascite fluid of rats elicited by casein was confirmed immunologically by ELISA, immuno-precipitation and Western blotting assays. No other GPx isozymes such as cytosolic GPx (cGPx), phospholipid hydroperoxide GPx (PHGPx) or intestinal GPx (iGPx) were detected. eGPx activity and protein were also detected in the pleuritic fluid of rats following injection of 2% carrageenan. These findings indicate that eGPx apears at various sites of acute inflammation in rats. This pattern is due to leakage from circulation as a result of the increased capillary permeability at inflammation sites elicited by chemotactic factors.
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  • Naoko ABE, Naoki OKAMURA, Sadahiko ISHIBASHI
    2000 Volume 23 Issue 5 Pages 537-541
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    Both the plasma membrane-rich fraction and specific granule-rich fraction prepared from human neutrophil lysate by Percoll centrifugation have been reported to contain cytochrome b558, a membrane activation factor for NADPH oxidase. In this study, the plasma membrane-rich fraction and specific granule-rich fraction of guinea pig neutrophils were prepared, and the abilities of both fractions to activate NADPH oxidase in a cellfree system consisting of either fraction, cytosol and arachidonate were compared. There was no difference in the Km value for NADPH between NADPH oxidase activated by specific granules or by plasma membranes. Optimum concentrations of arachidonate for the activation of NADPH oxidase in both the fractions were also the same. However, after freeze-thawing, the specific granules markedly lost the ability, compared to plasma membranes. Such instability of specific granules was also observed on hypotonic- or deoxycholate-treatment. The inactivation by freeze-thawing was not suppressed by proteinase inhibitors, and gp91-phox, a large subunit of cytochrome b558, was not degraded by freeze-thawing. Freeze-thawed specific granules did not affect the ability in plasma membranes, indicating the absence of an inactivating factor in specific granules. The increase in the amount of cytosol in the cell-free assay mixture did not compensate for the markedly decreased ability of freezethawed specific granules. Translocation of p47-phox, one of the cytosolic activation factors, to specific granules was not affected by freeze-thawing. We found that the ability of specific granules to activate NADPH oxidase was fragile, though it is unclear what is responsible for the instability, at present.
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  • Xiao Ping PU, Jia Zheng WANG
    2000 Volume 23 Issue 5 Pages 542-544
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    Protein purification is a key technique for the identification of novel sensory nerve-specific proteins. A 35 kDa protein, the sensory neuronal specific protein, was isolated and purified from rabbit spinal ganglia and sensory fibers by homogenizing and deriving soluble extracts, followed by ion exchange chromatography using DEAE-Sephacel and gel filtration HPLC. Western blot analysis showed that the protein was present in spinal sensory ganglia but not in spinal motor neurons with anti-rat 35 kDa protein polyclonal antibody. The sensory-specific protein 35 kDa is termed SSP-35 in this paper. We found that the purified SSP-35 promoted axonal growth of the dorsal root ganglia of chick E8 embryos. Our data reveal that the protocol is an effective method for the purification of SSP-35. The protein may not only be a useful marker for sensory neurons, but also a possible tool to study the regeneration and function of sensory neurons.
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  • Takashi KOSUGE, Satoshi TOYOSHIMA
    2000 Volume 23 Issue 5 Pages 545-548
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    As described previously (Kosuge T., Toyoshima S., Biol. Pharm. Bull., 23, 1-5 (2000)), inhibitors of the glycoprotein processing enzymes, glucosidase I and II, induce decreased secretion of interferon-γ (IFN-γ) into culture supernatants of anti CD3-stimulated lymphocytes, and in the present study the mechanism has been investigated in further detail.The processing inhibitors did not affect intracellular levels of IFN-γ but enhanced the degradation of newly synthesized IFN-γ-in anti CD3-stimulated lymphocytes. Furthermore, since the stability of N-glycosylatd proteins is known to be regulated by lectin family chaperones, such as calnexin, a type I transmembrane protein located in the endoplasmic reticulum (ER), and calreticulin, a soluble protein in the ER lumen, the effect of the processing inhibitors on the interaction of IFN-γ with calnexin and calreticulin was investigated. It was found that IFN-γ formed complexes with calnexin and calreticulin in anti CD3-stimulated lymphocytes. Total binding of IFN-γ to calnexin was not affected but that to calreticulin was increased in anti CD3-stimulated lymphocytes treated with the processing inhibitors. However, binding of newly synthesized IFN-γ to calreticulin was decreased in the lymphocytes under the same conditions as above. These results suggest that these glycoprotein processing inhibitors block the release of IFN-γ from already formed calreticulin complexes, which prevents the binding of newly synthesized IFN-γ to calreticulin and results in the enhancement of IFN-γ degradation.
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  • Tetsuo MORITA, Akira FUJIWARA, Hiroshi UEKI, Asako KANAGAWA
    2000 Volume 23 Issue 5 Pages 549-554
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
    JOURNAL FREE ACCESS
    Ruthenium Red (RuR; ruthenium oxychloride ammoniated) stimulated the release of hepatic lipase (HTGL) activity from primary cultured rat hepatocytes into medium in a time- and dose-dependent manner. The RuR-stimulated release of HTGL activity was suppressed by tyrosine kinase (TK) inhibitors (ST-638 and biochanin A). The activity of partially purified TK preparation from hepatocytes was found to be increased by incubation with RuR. In addition, treatment of the hepatocytes with H-89, a potent inhibitor of cAMP-dependent protein kinase (PKA), decreased the stimulatory release of HTGL activity by RuR. Moreover, cAMP content in RuR-incubated hepatocytes was rapidly increased, and activation of PKA was observed. The RuR-stimulated release of HTGL activity is also inhibited by uncouplers and glycosylation inhibitors. In addition, incorporation of [3H]leucine into protein was increased in the present of RuR. Under marked inhibition of protein synthesis by cycloheximide, RuR still showed a full effect on the release of HTGL activity. These results suggest that RuR stimulates the release of HTGL activity through mechanisms of aciton involving TK- and PKA-activating pathways, which require a metabolic energy-sensitive process rather than elevation of enzyme molecule synthesis.
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  • Alejandra O.M. MARIA, Osvaldo DONADEL, Graciela H. WENDEL, Jorge A. GU ...
    2000 Volume 23 Issue 5 Pages 555-557
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
    JOURNAL FREE ACCESS
    The gastric cytoprotective activity of several molecules containing an α, β-unsaturated carbonyl system is reported. We attributed this gastroprotective activity to the presence of a non-hindered Michael acceptor in the molecules assayed and suggested that the mechanism of protection would involve, at least in part, a nucleophilic attack of the sulphydryl group of the gastric mucosa to the β carbon of the Michael acceptors of the compounds assayed.
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  • Francisco ORALLO, Helena TRISTAN, Tomas GARCIA-FERREIRO, Sonia DE FRAN ...
    2000 Volume 23 Issue 5 Pages 558-565
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    The in vivo and in vitro cardiovascular effects of the novel 5-HT2A1/H1 antagonist ketanserin analogues QF 0303B, QF 0307B, QF 0311B, QF 0313B were studied in anaesthetized normotensive rats (ANR) and in isolated rubbed rat aorta (IRRA). In ANR, 0.2mg·kg-1 i.v. of each compound produced a rapid, remarkable but short-lasting fall in mean arterial blood pressure (MAP) accompanied by bradycardia. All compounds significantly modified the pressor effects induced by 5-hydroxytryptamine (5-HT) and noradrenaline (NA). In IRRA, the compounds inhibited NA- and 5-HT-induced contractions in a competitive fashion. Furthermore, the analogues displayed lower H1-antagonist activity than ketanserin. Compounds tested showed low 5-HT2B affinity and no activity at muscarinic, nicotinic, or 5-HT3 receptors, nor any marked ability to produce smooth muscle relaxation via calcium entry blockade. There is a significant correlation between hypotension reached and inhibition of the 5-HT-induced pressor responses (but not for NA). A certain degree of correlation was observed between hypotensive effect endurance vs. α1-adrenoceptor blockade (but not for serotonin). These results indicate that in this series the brief hypotensive activity in ANR is attributed to a 5-HT2A receptor blockade and the duration of the effect is better attributed to an α1 adrenoceptor blockade.
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  • Miho TAKADA, Tamao YAMADA, Hiroto NAKAHARA, Yukio SUGIMOTO, Keiji IZUS ...
    2000 Volume 23 Issue 5 Pages 566-569
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    We report a new experimental allergic conjunctivities with Japanese cedar pollen as antigen in guinea pigs, and the immunological characteristics of this model were also elucidated. Allergic conjunctivitis was developed by immunization in guinea pigs with a mixture containing Japanese cedar pollen and killed Bordetella pertussis. When local application of Japanese ceder pollen suspension 14 d after systemic immunization was performed every 3 d, remarkable conjunctivitis was observed from 20 to 35 d. Increase in vascular permeability and decrease in histamine contents of the conjunctiva were also observed after local application of antigen. Passive cutaneous anaphylactic (PCA) reactions revealed that both IgG- and IgE-rich antibodies were produced in this model. Chlorpheniramine, ketotifen and levocabastine were effective in inhibiting cedar pollen-induced conjunctivities. Although a high concentration was needed, tranilast and amlexanox also showed significant inhibition of conjunctivitis induced by cedar pollen.
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  • Mikiko YASUHARA, Kuniharu SUZUMURA, Keiko TANAKA, Masakatsu TAKAHASHI, ...
    2000 Volume 23 Issue 5 Pages 570-574
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    The antioxidative effect of fluvastatin sodium (fluvastatin) on low-density lipoprotein (LDL) was evaluated in vivo and in vitro. Since ex vivo measurement of the LDL oxidizability is reported to reflect the response of the atherosclerotic process, LDL isolated from rabbits fed a high cholesterol diet for 4 weeks with or without fluvastatin, pravastatin or α-tocopherol administration was oxidized by copper ions to estimate conjugated diene formation. Fluvastatin but not pravastatin significantly prolonged the lag time of LDL oxidized by copper ions ex vivo without affecting plasma cholesterol levels at a dose of 3 mg/kg after four weeks of treatment. α-Tocopherol-treated rabbits showed dramatically elongated LDL oxidation lag time at a dose of 150 mg/kg. In order to assess the mechanism, the content of α-tocopherol, a major endogenous antioxidant in LDL was measured, and we found that only LDL isolated from α-tocopherol-treated rabbits contained a significantly larger amount of α-to-copherol than that from high cholesterol control rabbits. To elucidate the mechanism further, the effect of fluvastatin on conjugated diene formation during copper-induced LDL oxidation in vitro was studied. Fluvastatin not only prolonged lag time, but also suppressed the rate of LDL oxidation, both in a dose dependent manner above 1μM, while pravastatin showed no effect. These results suggest the direct antioxidative effect of fluvastatin on LDL oxidation in vivo. Since oxidation of LDL is an important step in the initiation and progression of atherosclerosis, fluvastatin may reduce the risk of this condition not only by lowering plasma cholesterol but also by protecting LDL from oxidation.
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  • Ji-E. KIM, Yhun Y. SHEEN
    2000 Volume 23 Issue 5 Pages 575-580
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    Since it is known that hypoxia increases inducible nitric oxide synthase (iNOS) gene expression through the hypoxia responsive element, it was hypothesized that nitric oxide could be a mediator of hypoxia to inhibit Cyp1a1 promoter activity. In order to test this hypothesis, we have undertaken a study to examine the effects of hypoxia and nitric oxide on Cyp1a1 promoter activity in Hepa I cells. Mouse Cyp1a1 5' flanking DNA, 1.6kb, was cloned into pGL3 expression vector in order to construct pmCyp1a1-Luc. Hepa I cells were tranfercted with pmCyp1a1-Luc and were treated with 10-9M 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) in the presence or absence of various hypoxic agents such as 10-6-10-4M cobalt chloride or 10-6-10-4M picolinic acid or 10-6-10-4M desferrioxamine. The luciferase activity of the reporter gene was measured from pmCyp1a1-Luc transfected Hepa I cell lysate which contains 2μg total protein using luciferin as a substrate. Hypoxic agents such as cobalt chloride, picolinic acid, and desferrioxamine showed inhibition of luciferase activity that was induced by 10-9M TCDD treatment in a dose dependent manner. Concomitant treatment of 1mM NG-nitro-l-arginine with 10-6-10-4M cobalt chloride or 10-6-10-4M desferrioxamine or 10-6-10-4M picolinic acid or 10-6-10-4M sodium nitroprusside recovered luciferase activity from the TCDD induced luciferase activity that was inhibited by hypoxic agents. These data demonstrated that nitric oxide might be a mediator of iron chelating agents and hypoxic agents to inhibit dioxin induced Cyp1a1 promoter activity in Hepa I cells.
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  • Hidetoshi MURAO, Naomasa SAKAGAMI, Tomomi IGUCHI, Tomomi MURAKAMI, Yas ...
    2000 Volume 23 Issue 5 Pages 581-584
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    In our previous experiment using rats, fluoride was reported to cause renal calcification, whose mechanism was deduced to be due to an increase in parathyroid hormone (PTH) secretion. However fluoride-induced renal calciffication that was independent of PTH has not been nuderstood well in the nephron of fluoride-treated animals. Thus, we examined the effect of sodium fluoride on intracellular calcium mobilization in a normal rat kidney epithelial cell line (NRK-52E cells). The calcium accumulation was found to be remarkably increased by the addition of sodium fluoride (NaF). The elevation of [Ca2+]i was demonstrated to be due to calcium entry through nifedipine-sensitive calcium channels. In addition, fluoride activates phospholipase C, but inositol 1, 4, 5-triphosphate (IP3) didn't induce Ca2+ release from the endoplasmic reticulum (ER). Moreover, fluoride alone was deduced to enhance the activity of ER-type Ca2+-ATPase.Finally, on the mechanism of fluoride-induced calcium accumulation in NRK-52E cells, fluoride may activate phospholipase C to generate IP3 and diacylglycerol, and these increases can be elucidated to induce calcium entry through dihydropiridine-sensitive calcium channels. Moreover, fluoride was found to stimulate calcium accumulation through ER-type Ca2+-ATPase into the endoplasmic reticulum. The elevation of ER-type Ca2+-AT-Pase activity by fluoride was elucidated to operate as a regulatory system to protect against abnormally higher increases in cytosolic calcium concentration via an increase of calcium influx into the endoplasmic reticulum.
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  • Yiquan WANG, Kaiya ZHOU, Luoshan XU, Tina T.X. DONG, Karl W.K. TSIM
    2000 Volume 23 Issue 5 Pages 585-588
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    A pair of diagnostic primers for distinguishing the Chinese crude drug Zaocys (Zaocys dhumandes) from its substitutes was designed based on the sequence data of the original animal of the drug and substitutes. Total DNAs were extracted from genuine crude drug and 5 of its substitutes, as well as from 12 species of original animal of the snake crude drug. Diagnostic PCRs were performed using the primers with these total DNAs as a template, annealing at 60-65°C. Positive amplifications were obtained from all DNA templates of Zaocys, whereas negative amplifications were obtained from that of others. The results indicate that Zaocys samples could be definitely distinguished from its substitutes by diagnostic PCR, and no incorrect discrimination was found under the same reaction condition. The advantages of the method in the authenticaiton of crude drugs are also discussed in the present paper.
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  • Hajime MIZUKAMI, Yuka OKABE, Hiroshi KOHDA, Noboru HIRAOKA
    2000 Volume 23 Issue 5 Pages 589-594
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    Novel methods for molecular authenticaiton of Atractylodes-derived crude drugs (Jutsu) were established based on PCR-restriction fragment length polymorphism (RFLP) and direct sequencing of chloroplast trnK. Two regions inside the chloroplast trnK were selected as molecular markers for identification and discrimination of Atractylodes Rhizome (Byaku-jutsu) and Atractylodes Lancea Rhizome (So-jutsu). The Region 1 fragment (260 bp) amplified from So-jutsu and Wa-byaku-jutsu (Atractylodes Rhizome derived from A. japonica) gave 2 bands of 180 bp and 80 bp on agarose gel electrophoresis after digestion with a restriction endonuclease HinfI, whereas the fragment amplified from Kara-byaku-jutsu (Atractylodes Rhizome derived from A. ovata) remained undigested, which allowed unambiguous identification of Kara-byaku-jutsu. By direct sequencing of Region 2 (436 bp) and comparison of the nucleotide sequence data sets we could not only discriminate Byaku-jutsu and So-jutsu but also identify the original plant species of each crude drug specimen. A simple and reliable protocol for rapid preparation of DNA suitable for PCR from as little as 1 mg of Atractylodes-derived crude drugs was also described.
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  • Takayuki MATSUNAGA, Chika HASEGAWA, Toru KAWASUJI, Hideyo SUZUKI, Haru ...
    2000 Volume 23 Issue 5 Pages 595-598
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    Essential oil from the leaves of Tateyamasugi (Cryptomeria japonica) exhibited strong inhibitory activity on ulceration induced by HCl/ethanol, HCl/aspirin, water-immersion stress and pylorus-ligation. We separated the antiulcer compounds from cedar essntial oil by use of distillation and chromatography. As a result, terpinen-4-ol, a monoterpene, and elemol, a sesquiterpene, were isolated as active compounds. The antiulcer activity of the former was more potent than that of the latter. Terpinen-4-ol was a mixture of optical isomers and each possessed potent antiulcer activity. Secretion of gastric juice and output of acid and pepsin activity were lowered by terpinen-4-ol.These results suggest that terpinen-4-ol isolated from cedar essential oil could be a valuable antiulcer agent.
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  • Chihiro TOHDA, Yumiko KAKIHARA, Katsuko KOMATSU, Yasushi KURAISHI
    2000 Volume 23 Issue 5 Pages 599-601
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    In a search for new anti-pruritic drugs we screened methanol extracts of 33 harbal medicines which have been used for cutaneous diseases for their antipruritic activity using substance P(SP) as a pruritogen in mice. When administered perorally 30 min before SP injection, methanol extracts of 6 of these herbal medicines, the root of Scrophularia ningpoensis HEMSL, the root of Patrinia villosa (THUNB.) JUSS, the fruit of Forsythia suspenna VAHL, the rhizome of Cimicifuga dahurica (TURCZ.) MAXIM., the aerial part of Schizonepeta tenuifolia BRIQ. and the fruit of Cnidium monnieri (L.) CUSS, inhibited SP-induced itch-scratch response at a dose of 200 mg/kg without affecting locomotor activity. Dose dependence of these 6 extracts (50-500mg/kg) was investigated and all of them inhibited SP-induced itch-scratch response, with extracts from Scrophularia ningpoensis, Schizonepeta tenuifolia and Cnidium monnieri showing particularly significant inhibition. The results suggest that these 6 methanol extracts have inhibitory activity against SP-induced itching.
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  • Hiroaki HAYASHI, Noriko HOSONO, Mieko KONDO, Noboru HIRAOKA, Yasumasa ...
    2000 Volume 23 Issue 5 Pages 602-606
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    The nucleotide sequences of ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit gene (rbcL) of Glycyrrhiza glabra, G. uralensis, G. inflata, G. echinata, G. macedonica and G. pallidiflora have been determined to construct their phylogenetic tree. Based on these sequences, the six Glycyrrhiza species were divided into two groups : three, G. glabra, G. uralensis, and G. inflata, which produce glycyrrhizin as a major saponin, and the others, G. echinata, G. macedonica and G. pallidiflora, which produce macedonoside C as a major saponin. Among the three glycyrrhizin-producing species, only two nucleotide substitutions were observed between the rbcL sequences of G. glabra and G. uralensis, and the sequence of G. uralensis was identical to that of G. inflata, indicating that G. uralensis and G. inflata are closely related. Among the three macedonoside C-producing species, only one nucleotide substitution was observed between those of G. echinata and G. macedonica, indicating that these two species are also closely related.
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  • Koji WADA, Satoshi ISHIZUKI, Takao MORI, Eiichi FUJIHIRA, Norio KAWAHA ...
    2000 Volume 23 Issue 5 Pages 607-615
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    Aconitum alkaloids of the C20-diterpenoid type, kobusine (1) and pseudokobusine (2), their anisoyl, veratroyl, p-nitrobenzoyl, nicotinoyl or pivaloyl derivatives, and dehydrokobusine and N, 6-seco-6-dehydro-pseudokobusine derivatives were examined for their peripheral vaso-activities by laser-flowmetrical measurement of the cutaneous blood flow in the hind foot of mice after intravenous administraiton. Kobusine 15-anisoate (4), 11-veratroate (5), 15-veratroate (6), 11-pivaloate (9) and 15-pivaloate (10)-were significantly effective at a low dose of 0.5 or 0.05 mg/kg. Pseudokobusine derivatives were all avtive at 1, 0.5 or 0.05 mg/kg, and the effects of pseudokobusine 15-anisoate (13), 15-veratroate (16) and 15-p-nitrobenzoate (19) at 0.1 mg/kg were remarkable. Yesoline (26) and alkaloid (28) were significantly effective at a low dose of 1 mg/kg, whereas yesonine (25) and N-acetyl-N, 6-seco-6-dehydropseudokobusine (27) were inactive. Dehydrokobusine derivatives (29, 30) were significantly effective at a low dose of 0.5 or 0.1 mg/kg. It is thought that the hydroxyl groups of alkaloids, espectially a free OH group of 2 at C-6, are important for action on the peripheral vasculature leading to dilatation, and the results indicated that esterificaiton of the hydroxyl group at C-15 with either anisoate, veratroate or p-nitrobenzoate may contribute to enhancement of the activity of the parent alkaloids.
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  • Tomohiro NABEKURA, Yoshimasa ITO, Hong CAI, Motome TERAO, Ryohei HORI
    2000 Volume 23 Issue 5 Pages 616-620
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    Disulfiram, a dimer of diethyldithiocarbamate (DDC) which is a strong radical scavenger, is known to prevent cataract development. However, disulfiram is hardly absorbed from the cornea and its bioavailability is extremetly low. In this study, we attempted to prepare disulfiram solid dispersion for the improvement of ocular bioavailability.Solid dispersions of disulfiram were prepared by either an evaporation method or a spray-drying method, using polyvinylpyrrolidone (PVP) as a carrier. Preparations were analyzed by scanning electron microscopy, powder X-ray diffractometry and differential scanning calorimetry, and confirmed to be a solid dispersion. The particle size of the solid dispersion prepared by the spray-drying method was smaller than the preparaiton by the evaporation method (spray-drying : 3.3±0.04μm, evaporation : 34.3±18.0μm). An in vivo ocular absorption experiment was conducted by instilling solid dispersions to rabbit eye and measuring the DDC in the aqueous humor. After instillation of disulfiram and PVP physical mixture, DDC was not detected in the aquenous humor. On the other hand, DDC appeared in the aqueous humor after the instillation of a solid dispersion. Maximal concentration and the area under the aqueous humor concentration-time curve were greater in the solid dispersion prepared by the spray-drying method than the preparation by the evaporation method.Disulfiram solid dispersion, especially prepared by the spray-drying method, improved ocular bioavailability.
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  • Hideo NOGUSA, Keiji YAMAMOTO, Toshiro YANO, Masahiro KAJIKI, Hiroshi H ...
    2000 Volume 23 Issue 5 Pages 621-626
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    Plasma and tissue distribution of conjugates of carboxymethylpullulan (CMPul) and doxorubicin (DXR), either bound directly or through three types of tetrapeptide spacers, was studied after intravenous injection to rats bearing Walker 256 carcinosarcoma and compared with that of DXR. In contrast to DXR, each conjugate retained high levels of DXR in the conjugated form in plasma and displayed high accumulation in the tumor at 6 h after the administration. Disposition characteristics of [3H]CMPul in rats bearing Walker 256 carcinosarcoma indicate that pullulan, which had molecular weight over 50 kDa, is a suitable macromolecular carrier for tumor targeting in cancer chemotherapy by carboxymethylation. We find that the in vivo antitumor effect of the conjugates depends on the tumor AUC of free DXR released from the conjugates. CMPul-DXR conjugates were also distributed in the reticuloendothelial organs, such as liver, spleen and bone marrow; however, the tissue concentrations of the conjugates in the heart, lung and muscle were lower than those of DXR. We next investigated the effect of the DXR contents of CMPul-DXR conjugates on their body distribution in rats bearing Walker 256. The half life of CMPul-DXR conjugates in plasma were shorter and the congutates had greater accumulation in the reticuloendothelial system, while they showed lower concentrations in the tumor with increasing DXR contents. Antitumor activity of CMPul-DXR conjugates were reduced and the lethal toxicities of CMPul-DXR conjugates were amplifid with increasing DXR contents.
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  • Hugues MALONNE, Jeanine FONTAINE, Andre MOES
    2000 Volume 23 Issue 5 Pages 627-631
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    Various monoolein-water systems containing tramadol HCl, a potent analgesic, were formulated to obtain sustained-release dosage forms which could be administered by subcutaneous, intramuscular or intrathecal injections. They were examined for their in vitro drug-release profiles and in vivo analgesic properties in rats in a 14 h period following intramuscular administration. In order to obtain a lower viscosity, we have substituted a part of monoolein by oleic acid and phospholipids. Both binary (monoolein-water) and quaternary (oleic acid-phospholipid-monoolein-water) formulations exhibited controlled drug-release profiles which were accelerated by surfactant adjunction. This surfactant action was probably due to structural changes in the lipid arrangement and was much more pronounced for the modified formulations. According to the results obtained in vitro, formulations with slower drug release (i.e. the native formulation and the modifird one without surfactant ) were selected for assessment of their in vivo properties. Both formulations demonstrated prolonged analgesic activities in the rat tail flick test manifested by stable pain relief during more than 10 h compared with the 3 to 4 h analgesia obtained with the commercially available tramadol HCl solution. The sustained-release capabilities were evaluated by using a modified half value duraiton (HVD) ratio and all sustained-released formulations exhibited a HVD ratio equal or superior to 3.9.
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  • Yoshikazu TASHIRO, Yasuki KATO, Eiji HAYAKAWA, Kunio ITO
    2000 Volume 23 Issue 5 Pages 632-636
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    The objective of our study is to establish a novel method for the in vivo evaluation of transdermal delivery. In this study, cathodal iontophoresis of ketoprofen, a nonsteroidal anti-inflammatory drug, was performed in the thoracic area of rats at a constant direct current, and blood samples were collected from cutaneous vein passing through the thoracic part of the body. After the iontophoresis, the plasma ketoprofen concentration in cutaneous vein ipsilateral to the application site was significantly higher than that in systemic vein. On the other hand, the plasma concentration in cutaneous vein contralateral to the application site was not significantly different from that in systemic vein. A comparison of the time-course curves demonstrated that, for the duration of iontophoresis, the plasma ketoprofen concentration in cutaneous vein ipsilateral to the application site increased with the amount of ketoprofen absorbed in the skin. These results suggest that the plasma concentration in the cutaneous vein ipsilateral to the application site is related with the transfer of drug from skin to cutaneous blood circulation. Therefore, the measurement of plasma concentration in cutaneous vein close to the application site would allow us to directly quantify the local behavior of iontophoretic transdermal absorption.
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  • Shuichi KISHIMOTO, Toshihiro NOGUCHI, Takashi YAMAOKA, SHoji FUKUSHIMA ...
    2000 Volume 23 Issue 5 Pages 637-640
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    SM-11355, cis[((1R, 2R)-1, 2-cyclohexanediamine-N, N')bis(myristato)] platinum(II) is a lipophilic platinum complex. SM-11355 suspended in Lipiodol (SM-11355/Lipiodol) was previously shown to have antitumor effects against rat hepatic tumors after intra-arterial administration. In the present study, the in vitro release of platinum compounds from SM-11355/Lipiodol was examined. A test tube containing 10 ml of saline and 1 ml of SM-11355/Lipiodol was rotated at 5 rmp in a vertical orientation. The platinum concentration in saline gradually increased for 28 d. From HPLC analysis, cyclohexane-1, 2-diamineplatinum(II) dichloride (DPC) and cyclohexane-1, 2-diamineplatinum(II) chloroiodide (DPCI) were detected in the saline, and the sum of these two compounds was equivalent to the total platinum amount in the saline determined by atomic absorption spectrophotometry at days 21 and 28. DPC showed significant growth inhibitory activities, with IC50 values of 0.1-0.7 nmol/ml in rat hepatoma AH-109A cells and 5 human tumor cell lines, as effective as cisplatin. These findings suggest that SM-11355/Lipipdol exerts antitumor effects by releasing active platinum compounds, and that DPC is one of the candidates of the active compounds.
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  • Ken-ichiro MATSUMOTO, Kazutoyo ENDO, Hideo UTSUMI
    2000 Volume 23 Issue 5 Pages 641-644
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    In vivo reducing capacity and cytosolic glutathione peroxidase (GSH-Px) activity in liver homogenate were evaluated in 6 weeks old Se-deficient and normal rats. GSH-Px was significantly lower in Se-deficient rats than in normal rats. In vivo reducing capacity in head and liver parts, estimated from in vivo signal decay of a nitroxyl spin probe using a low frequency (300 MHz) ESR spectrometer, was significantly decreased in Se-deficient rats, suggesting a decrease of antioxidant capacity in Se-deficient rats.
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  • Yoshikazu SAKAGAMI, Yoshihiko INAMORI, Nami ISOYAMA, Hiroshi TSUJIBO, ...
    2000 Volume 23 Issue 5 Pages 645-648
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    β-Dolabrin and γ-thujaplicin isolated from Thujopsis dolabrata Sieb. et Zucc. var hondai MAKINO, like hinokitiol, showed strong phytogrowth-inhibitory activities, and their growth-inhibitory activities were as high as that of sodium 2, 4-dichlorophenoxyacetate used as a positive control. In particular, the phytogrowth-inhibitory activity of γ-thujaplicin was strong and it completely inhibited the germination of this seed ob Brassica campestris L. subsp. rapa HOOK f. et ANDERS at the concentration of 30 ppm. Both compounds exhibited inhibitory activities on B. campestris L. subsp. rapa HOOK f. et ANDERS and Sesamum inducum LINNE, even at the low concentraiton of 10 ppm. At 7 d after treatment with β-dolabrin and γ-thujaplicin, the amount of chlorophyll in the cotyledons of B. campestris L. subsp. rapa HOOK f. et ANDERS treated with both compounds was greatly decreased as compared with the control. The findings in dicate that the phytogrowth-inhibitory action might be a common biological activity of hinokitiol-related compounds, suggesting that at least a part of their phytogrowth-inhibitory actions seems to be related to a decrease in chlorophyll content.
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  • Shigeo YAMAMOTO, Nobuo MUTOH, Daisuke TSUZUKI, Hisato IKAI, Hiroshi NA ...
    2000 Volume 23 Issue 5 Pages 649-653
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    L-2, 4-Diaminobutyrate decarboxylase (DABA DC) catalyzes the formation of 1, 3-diaminopropane (DAP) from DABA. In the present study, the ddc gene encoding DABA DC from Enterobacter aerogenes ATCC 13048 was cloned and characterized. Determination of the nucleotide sequence revealed an open reading frame of 1470 bp encoding a 53659-Da protein of 490 amino acids, whose deduced NH2-terminal sequence was identical to that of purified DABA DC from E. aerogenes. The deduced amino acid sequence was highly similar to those of Acinetobacter baumannii and Haemophilus influenzae DABA DCs encoded by the ddc genes. The lysine-307 of the E. aerogenes DABA DC was identified as the pyridoxal 5'-phosphate binding residue by site-directed mutagenesis. Furthermore, PCR analysis revealed the distribution of E. aerogenes ddc homologs in some other species of Enterobacteriaceae. Such a relatively wide occurrence of the ddc homologs implies biological singificance of DABA DC and its product DAP.
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  • Carmela SATURNINO, Bruno FUSCO, Paola SATURNINO, Giovanni DE MARTINO, ...
    2000 Volume 23 Issue 5 Pages 654-656
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    We have examined the in vivo anti-inflammatory and analgesic activity of a new series of monocyclic β-lactams (azetidinones), similar to others which have been demonstrated to be inhibitors of human leukocyte elastase (HLE), an enzyme involved in degradation processes of connective tissue. Our new compounds have been administered orally (15 mg/kg) to albino rats 30 min before injecting carrageenin in the plantar aponeurosis. Tested compounds have demonstrated a certain activity and stability to gastric hydrolysis, in particular two of them markedly reduced paw efema formation, even if slightly less effectively than indomethacin (reference compound, 5 mg/kg). To eveluate the analgesic activity we carried out the acetic acid writhing test, pretreating rats orally with our compounds 30 min before injecting the acid solution i.p. Ths same two molecules which showed the anti-inflammatory activity demonstrated a very light analgesic activity. These results suggest the possibility of carrying out further studies, particularly in vitro, on the mechanism of action of our compounds, mechanism which could be the HLE inhibition.
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  • Kazuhiko TSUSUMI, Sanae KISHIMOTO, Osamu KOSHITANI, Hideaki KOHRI
    2000 Volume 23 Issue 5 Pages 657-659
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    In an attempt to create an animal model of constipation in monkeys, amitriptyline was administered to cynomolgus monkeys at doses of 10-160 mg/kg body weight via a nasogastric tube. Normal control monkeys excreted feces frequently throughout the day. Monkeys treated with amitriptyline at doses of 10-40 mg/kg showed delays in feces excretion. The 60 mg/kg treated monkeys for the most part did not excrete feces during the 24 h after amitriptyline administration. The 80 and 120 mg/kg treated monkeys did not excrete feces until 24 h from administraiton of amitriptyline, and also showed prolonged crouching and lethargy. On the other hand, 160 mg/kg treated monkeys died within 24 h after administration. We therefore felt that the optimal dose for creating constipation in the monkeys was 60 mg/kg. We tested the appropriateness of this amitriptyline-induced constipated monkey model by observing the effects of a new laxative, the herbal medicine ND-10 and the commercially available laxative bisacodyl. Control monkeys (those not receiving ND-10 or bisacodyl) treated with 60 mg/kg amitriptyline did not excrete feces up to 32 h after amitriptyline administration in 2 of 3 monkeys. However, all monkeys treated with one tablet of ND-10 excreted feces. Also, in 4 monkeys administrated with bisacodyl, 3 excreted feces. In this study, we confirmed that constipation can be caused in cynomolgus monkeys by oral administration of amitriptyline. This model may also be useful for the evaluation of laxatives.
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  • Masaki BABA, Masayoshi OHMURA(Nee Matsuda), Naoki KISHI, Yoshihito OKA ...
    2000 Volume 23 Issue 5 Pages 660-662
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    Investigation of the Chinese crude drug "Xiebai, " the bulbs of Allium chinense G. DON (Liliaceae), led to the isolation of 2 saponins, xiebai-saponin I (laxogenin 3-O-β-xylopyranosyl (1→4)-[α-arabinopyranosyl (1→6)]-β-glucopyranoside) (1) and laxogenin 3-O-α-arabinopyranosyl (1→6)-β-glucopyranoside (2), and the aglycone, laxogenin (3), together with 2 chalcones, isoliquiritigenin (4) and isoliquiritigenin-4-O-glucoside (5), and β-sitosterol glucoside (6). Compounds 1-5 were tested in vitro for their inhibitory effect on the 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated 32Pi-incorporation into phospholipids of HeLa cells. In addition to this, laxogenin (3) was proven to have an antitumor-promoting activity in a two-stage lung carcinogenesis experiment.
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  • Fumio TSUJI, Hidehito MATSUOKA, Hiroyuki AONO, Miwa TAKAI, Masato HORI ...
    2000 Volume 23 Issue 5 Pages 663-665
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    We investigated the effects of various sulfhydryl compounds on interleukin-1 (IL-1)-induced vasucular endothelial growth factor (VEGF) production in human synovial stromal cells (HSSC). HSSC stimulated by IL-1β(100 ng/ml) produced VEGF and interleukin-6 (IL-6) in vitro. Monosulfhydryl compounds, N-acetylcysteine, D-penicillamine, tiopronin and the bucillamine-like disulfhydryl compound, compound A scarcely affected VEGF or IL-6 production at concentraitons of 10-5 and 10-4M. However, the disulfhydryl compound, budillamine in hibited VEGF production but not IL-6 production at concentrations of 10-5 and 10-4M. These results suggest that bucillamine may be a selective inhibitor of IL-1-induced VEGF production in HSSC, and that inhibition of VEGF production may require not only SH groups but also a specific chemical structure.
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  • Sang Hyun SUNG, Eun Ju LEE, Jung HEE CHO, Hyr Soo KIM, Young Choong KI ...
    2000 Volume 23 Issue 5 Pages 666-668
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    We used primary cultures of rat hepatocytes injured by the hepatotoxin, carbon tetrachloride (CCl4), as a test system to screen for hepatoprotective compounds from natural products. Sauchinone was isolated from the aerial parts of Saururus chinensis (Saururaceae) by this method. At a concentration of 50 μM, sauchinone significantly reduced the release into the culture medium of glutamic pyruvic transaminase from CCl4-damaged cultures of rat hepatocytes. It has been determined that glutathione, superoxide dismutase and glutathione peroxidase all play important roles in the cellular defense against oxidative stress. Sauchinone appeared to protect primary cultured rat hepatocytes exposed to CCl4 from significant drops in the levels of each of these three specific markers, respectively. Sauchinone also seemed to ameliorate lipid peroxidation as demonstrated by a reduction in the production of the oxidized lipid byproduct, malondialdehyde. These results suggest that sauchinone may exert hepatoprotective activity through antioxidant activity.
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  • Ryoko SAWAMURA, Hitoshi SATO, Junichi KAWAKAMI, Tatsuji IGA
    2000 Volume 23 Issue 5 Pages 669-671
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    Azole antifungal agents (azoles) have inhibitory effects on the cytochrome P450. However, the effect of azoles on conjugative metabolism has not been given much attention. Lorazepam (LZP), a benzodiazepine sedative agent, is known to be metabolized by uridine 5'-diphosphate (UDP)-glucuronyltransferase. Herein we report investigation of the effect of azoles on the enzyme-kinetics of glucuronidation of lorazepam using rabbit liver microsomes in vitro. The Km and Vmax for LZP glucuronidation were determined to be 0.26±0.08 mM and 1.25±0.21 nmol/min/mg protein, respectively, when evaluated in the presence of a detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-prooanesulfonate (CHAPS) (0.8 mg/mg protein). Azoles fluconazole, miconazole, and ketoconazole competitively inhibited the glucuronidation of LZP, with Ki values of 7.17±4.78 mM, 0.17±0.08 mM, and 0.092±0.026 mM, respectively. These results are comparable to the previously reported Ki values of azoles with zidovudine (AZT) glucuronidation (1.4, 0.18, and 0.08 mM for fluconazole, miconazole, and ketoconazole, respectively) [Sampol et al., Br. J. Clin. Pharmacol., 40, 83-86, 1995]. Therefore, in order to avoid possible side effects of LZP, the concomitant administration of LZP and azles should be carefully evaluated.
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  • Michiharu UCHIYAMA, Naohito OHNO, Noriko N. MIURA, Yoshiyuki AdACHI, H ...
    2000 Volume 23 Issue 5 Pages 672-676
    Published: May 01, 2000
    Released on J-STAGE: April 10, 2008
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    Antibody to β-glucan is generally difficult to produce in mice. We have recently developed a protocol to obtain a soluble Candida spp. β-(1→3)-D-glucan (CSBG) by sodium hypochlorite (NaClO) oxidation and subsequent dimethyl sulfoxide (Me2SO) extraction. CSBG is composed mainly of β-(1→3) and β-(1→6)-glucosidic linkages with a small amount of branch. In this paper, mice were immunized with Candida albicans and the specificity of the resulting sera to CSBG was examined by ELISA. Using CSBG coated plate, sara of the Candida immune mice showed higher reactivity than non-immune, normal mice and the reactivity was neutralized by adding soluble CSBG as a competitor. However, the reactivity could not be neutralized by a β-(1→6) branched β-(1→3)-glucan, grifolan. Similar specificity of the sera was obtained by commercially available β-glucan particle, zymosan or zymocel, immune mice. These facts strongly suggested that CSBG included epitopes of the specific antibody in Candida immune mice.
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