Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Volume 27 , Issue 11
Showing 1-35 articles out of 35 articles from the selected issue
Review
  • Toshio Narahashi, Shigeki Moriguchi, Xilong Zhao, William Marszalec, J ...
    2004 Volume 27 Issue 11 Pages 1701-1706
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    No strategies for curing Alzheimer's disease have been developed yet as we do not know the exact cause of the disease. The only therapy that is available for patients is symptomatic treatment. Since Alzheimer's disease is associated with downregulation of the cholinergic system in the brain, its stimulation is expected to improve the patients' cognition, learning, and memory. Four anticholinesterases have been approved in the U.S.A. for the treatment of Alzheimer's disease patients. However, because of the inhibition of cholinesterases, these drugs have side effects and their effectiveness does not last long. Thus new approaches are needed. One approach is to stimulate directly nicotinic acetylcholine (nACh) receptors in the brain, and another is to stimulate NMDA receptors which are also known to be downregulated in Alzheimer's patients. Nefiracetam has been shown to potentiate ACh currents in the α4β2 receptor of rat cortical neurons with a bell-shaped dose–response relationship and the maximum effect at 1 nM. This effect was exerted via Gs proteins. The α7 receptor was almost unaffected by nefiracetam. Nefiracetam also potentiated NMDA currents with the maximum effect at 10 nM via interaction with the glycine-binding site of the receptor. Galantamine had a moderate potentiating effect on the α4β2 receptor and potentiated NMDA currents with the maximum effect at 1 μM. However, galantamine did not interact with the glycine-binding site. Donepezil, a potent anticholinesterase, also potentiated NMDA currents at 1—10000 nM. In conclusion, these three drugs potentiate the activity not only of the cholinergic system but also of the NMDA system, thereby stimulating the downregulated nACh receptors and NMDA receptors to improve patients' learning, cognition, and memory.
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  • Naoshi Yamazaki
    2004 Volume 27 Issue 11 Pages 1707-1716
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    To characterize energy metabolism in rat brown adipose tissue (BAT), we carried out differential screening of a cDNA library of BAT with a cDNA probe of white adipose tissue and isolated one novel cDNA clone. It contained a single open-reading frame of 2316 bases, which encodes a protein of 88.2 kDa. The predicted amino acid sequence showed the highest homology (62.6%) with that of carnitine palmitoyltransferase I (CPTI) from rat liver. The transcript corresponding to this cDNA was found to be abundantly expressed not only in BAT but also in heart and skeletal muscle. CPTI is known to be a protein necessary for the β-oxidation of long-chain fatty acids in mammalian mitochondria, and it has been suggested that at least two isoforms, the liver type and muscle type, exist. From these observations, a cDNA clone isolated from rat BAT was concluded to be encoding muscle-type CPTI (M-CPTI). Characterization of a genomic DNA clone revealed that the gene for human M-CPTI consists of two 5′-noncoding exons, 18 coding exons, and one 3′-noncoding exon spanning approximately 10 kbp, and a gene encoding choline/ethanolamine kinase-β (CK/EK-β) was located only about 300 bp upstream from the M-CPTI gene with the same strand direction. Furthermore, we found that unordinary transcripts containing exons of both CK/EK-β and M-CPTI genes exist in human and rodent tissues. Although the physiologic role(s) of these transcripts is still unknown, it is interesting that such transcripts are produced from two tightly arranged and functionally unrelated genes.
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  • Kora Pattabhi Rama Chowdary, Yarraguntla Srinivasa Rao
    2004 Volume 27 Issue 11 Pages 1717-1724
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    Mucoadhesion is a topic of current interest in the design of drug delivery systems. Mucoadhesive micro-spheres exhibit a prolonged residence time at the site of application or absorption and facilitate an intimate contact with the underlying absorption surface and thus contribute to improved and/or better therapeutic performance of drugs. In recent years such mucoadhesive microspheres have been developed for oral, buccal, nasal, ocular, rectal and vaginal routes for either systemic or local effects. The objective of this article is review the principles underlying the development and evaluation of mucoadhesive microspheres and the research work carried out on these systems.
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Analytical Biochemistry
Regular Articles
  • Shinobu Fujii, Ayumi Yoshida, Saori Sakurai, Michio Morita, Kikuo Tsuk ...
    2004 Volume 27 Issue 11 Pages 1725-1729
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    We developed a convenient chromogenic assay method for the activity of sphingomyelinase (SMase) from Bacillus cereus. SMase reaction was quenched by Zn2+, and the released phosphocholine was converted into a choline by the action of alkaline phosphatase. After that, the choline was converted into a chromogenic dye by the actions of choline oxidase and peroxidase in the presence of EDTA to trap the added Zn2+ which could interfere with the choline oxidase/peroxidase reactions. Triton X-100 also was added to the reaction mixture, in order to remove turbidity generated from ceramide which had been produced by the SMase reaction. To test a large number of samples in a short period of time, this assay was performed using 96-well microtiter plates. This method proved to be applicable not only to the measurement of the hydrolysis of sphingomyelin but also to those of lysophosphatidylcholine (lysoPC) and lyso platelet-activating factor by B. cereus SMase. Using this method, the kinetic parameters (Km and kcat) for B. cereus SMase toward various types of substrates were then determined, and the effect of Triton X-100 on the hydrolysis of lysoPC was examined.
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Notes
  • Caiquan Zhou, Kaiya Zhou, Shaolai Zhang
    2004 Volume 27 Issue 11 Pages 1850-1858
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    Two pairs of allele-specific diagnostic primers (SL1L/SL1H and SL2L/SL2H) for distinguishing the Chinese crude drug Sailonggu (bone of plateau zokor, Myospalax baileyi) from its substitutes were designed based on complete sequences of mitochondrial 12S rRNA and cytochrome b genes of the original animals of Myospalacinae, bamboo rat Rhizomys sinensis and black lipped pika Ochotona curzoniae. Total DNA was extracted from crude drug samples and original animals. Allele-specific diagnostic PCRs were performed using these primers with the total DNA as a template annealing at 65 °C. Positive amplifications were obtained from all DNA templates of Sailonggu and M. baileyi, whereas negative amplifications resulted from those of other zokors, the bamboo rat and black lipped pika. These results indicate that Sailonggu samples can be definitely distinguished from their substitutes by diagnostic PCR, and no incorrect discrimination was found under the same reaction conditions. Each of the two diagnostic primer pairs can be used to distinguish crude drug Sailonggu from its substitutes or adulterants. The three Sailonggu samples studied were diagnosed as genuine Sailonggu. In addition, the results of sequence alignment and phylogenetic analysis are congruent with that of the allele-specific diagnostic PCR.
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Biochemistry/Molecular Biology
Regular Articles
  • Yaşar Demir, Hayrunnisa Nadaroğlu, Nazan Demir
    2004 Volume 27 Issue 11 Pages 1730-1734
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    In this study, the effects of omeprazole, famotidine, and ranitidine on bovine stomach carbonic anhydrase (EC 4.2.1.1.) isoenzymes have been investigated in vitro. Bovine stomach carbonic anhydrase (CA) was purified from four different cell localisations of bovine stomach using affinity chromatography by Sepharose 4B–L-tyrosine sulphanilamide. The inhibition or activation effects of three different medical drugs on CA isoenzymes were determined using esterase activity and the CO2–hydratase method by plotting activity % vs. [medical drug]. The Ki values for omeprazole, famotidine, and ranitidine were determined in all localization CA, respectively. The I50 values of the drugs exhibiting an inhibition effect were found by means of these graphs. It was observed that omeprazole, famotidine, and ranitidine showed inhibition of bovine stomach CA activity. In addition, in vivo studies were performed for these medical drugs in Sprague-Dawley rats. It was demonstrated that CA in erythrocytes was significantly inhibited by these drugs to 3 h.
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  • Ryoko Utsubo, Yoshiko Sonoda, Reiko Takahashi, Shiro Iijima, Eriko Aiz ...
    2004 Volume 27 Issue 11 Pages 1735-1741
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    We established several focal adhesion kinase (FAK) cDNA-transfected cells and found that FAK-transfected HL-60 (HL-60/FAK) cells are resistant to apoptosis induced with hydrogen peroxide, etoposide and radiation compared with the parental HL-60 or the vector-transfected (HL-60/Vect) cells. We carried out proteome analysis to study the mechanism of resistance to apoptosis in HL-60/FAK cells. Among 300 spots resolved in two-dimensional gels, ca. 10% of them were significantly increased in HL-60/FAK cells compared with HL-60/Vect cells, whereas ca. 2% of them were decreased or disappeared. These proteins were performed for further analysis by Western blots or N-terminal sequencing or mass spectrometry. Increased proteins included stress proteins such as hsp90, ribosomal proteins, and antioxidant enzymes such as peroxyredoxin 2. Some of these proteins are assumed to contribute to the antiapoptotic action of FAK.
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  • Tomoki Tatefuji, Chikako Arai, Takanori Okura, Tohru Kayano, Tetsuya M ...
    2004 Volume 27 Issue 11 Pages 1742-1749
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    We have established a monoclonal antibody K114 (mAbK114) against hamster keratinocytes. The mAbK114 recognizes a 50—95 kDa cell-surface protein that is expressed restrictedly in the dermal sheath cells near the bulge area of the hair follicle and in the differentiated sebocytes of the normal adult hamster skin. Upon being cultured in vitro, however, the keratinocytes strongly and transiently expressed this novel K114 antigen (AgK114) in spite of low expression level of AgK114 by the freshly prepared keratinocytes. The cDNA of AgK114 was isolated by expression cloning using mAbK114. Sequence analysis revealed that it had 242 amino acid residues with a signal peptide at the N terminus, potential six N-glycosylation sites, a characteristic repetitive threonine rich domain, and a possible glycosylphosphatidylinositol (GPI) anchoring site near the C terminus. We examined various conditions in which expression of AgK114 was enhanced in vivo. Interestingly, AgK114 molecule was expressed accompanying tissue damages of the skin. It was transiently induced in basal epidermal keratinocytes after UV exposure. In addition, AgK114 was also induced in elongating edge epidermal keratinocytes during tissue regeneration after an excised wounding. These results suggest that AgK114 is involved in the recovering process from injury.
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  • Kunio Ohyama, Tomomi Sano, Hiroo Toyoda
    2004 Volume 27 Issue 11 Pages 1750-1757
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    We have been investigating an apoptosis induction in human fetal membrane cells by influenza virus (IV) infection and the contribution of apoptosis induction to the viral infection-defense response between a fetus and the maternal body. For studying any role of uterine cells in the anti-viral response, we investigated the molecular mechanism of the apoptotic induction in human uterine cervical fibroblast cell line (HCF) by IV infection. IV type A and B infection induced DNA fragmentation in HCF. In IV-infected HCF, gene mRNA expression levels of interleukine (IL)-1β, IL-6, tumor necrosis factor (TNF) α, Fas ligand, interferon regulatory factor (IRF)-1, interferon (IFN) α and IFN β increased as compared with those in mock treatment cells, and the induction of mRNAs for double stranded RNA dependent protein kinase (PKR), indolamine 2,3-deoxygenase (IDO) and 2′-5′ oligoadenylate synthetase (2-5 OAS) were indicated, which had a role for a host defense response induced by IFN-β. The amount of IFN-β protein increased by IV-infection, and DNA fragmentation was inhibited with anti-IFN-β antibody and PKR inhibitor (2-aminopurine). Furthermore, a synthetic double stranded RNA, poly I : C, could induce almost the same phenomena as that induced by virus infection. We conclude that IV-infection induces the apoptosis in HCF cells through the IFN-β expression regulated by double stranded RNA and IRF-1 induction, and suggest that the IFN-β induction may be the predominant contribution to the IV infection induced HCF apoptosis.
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  • Yosuke Ohtake, Akiko Maruko, Soichi Kojima, Tomomi Ono, Tomomi Nagashi ...
    2004 Volume 27 Issue 11 Pages 1758-1762
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    We investigated a relationship within zonal differences in DNA synthesis and in transglutaminase (TGase) activity between perivenous versus periportal regions of regenerating rat liver. Using the digitonin/collagenase perfusion technique, hepatocyte subpopulations were isolated from each region at various time points after partial hepatectomy. The amounts of DNA synthesis as well as the levels of TGase mRNA and activity in each subpopulation were measured. Although increased DNA synthesis was observed in both subpopulations with a peak at 24 h after partial hepatectomy, the amount of DNA synthesis in periportal hepatocytes (PPH) was twice as much as that in perivenous hepatocytes (PVH). In PVH, TGase activity peaked at 24 h after partial hepatectomy with a preceding increase in its mRNA expression at 12 h, whereas TGase activity in PPH at 24 h was one-half of that in PVH. As TGase is known to have a growth-arresting activity, our data indicate that relatively higher TGase activity in PVH at 24 h after partial hepatectomy might correlate with relatively lower DNA synthesis in this region compared to periportal region.
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  • Toshiyuki Kaji, Shigeru Sakurai, Chika Yamamoto, Yasuyuki Fujiwara, Sh ...
    2004 Volume 27 Issue 11 Pages 1763-1768
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    Pericytes associate with the outside of endothelial cells in microvessels. Previous studies have shown that these cells synthesize glycosaminoglycans (GAGs) but the nature of the core proteins to which these GAGs are attached is unknown. In the present study, cultured bovine retinal pericytes were metabolically labeled with [3H]glucosamine, [35S]sodium sulfate or 35S-labeled amino acids and the proteoglycans synthesized by these cells were purified by DEAE-Sephacel ion exchange and molecular sieve Sepharose CL-4B chromatography. Separated proteoglycans were digested with papain, heparitinase or chondroitin ABC lyase and the GAGs characterized by Sepharose CL-6B chromatography. Proteoglycans were also assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after digestion with chondroitin ABC lyase. Pericytes predominantly synthesize and secrete chondroitin or dermatan sulfate proteoglycans (CS/DS PGs) rather than heparan sulfate proteoglycans (HSPGs). Two subclasses of CS/DS PGs are synthesized by pericytes; one is a high Mr subclass with high charge density. This subclass eluted at the void volume of a Sepharose CL-4B molecular sieve column, was susceptible to chondroitin ABC lyase, and contained core proteins of ca. 550 and 450 kD which were recognized by antibody to versican. The other major subclass eluted at a Kav ca. 0.45 on a Sepharose CL-4B molecular sieve column, was susceptible to chondroitin ABC lyase, and contained core proteins recognized by antibodies to either biglycan or decorin that separated as a broad band of ca. 50 kDa in SDS-PAGE. A small amount of HSPG was also synthesized by these cells and could be separated from the CS/DS PGs by DEAE-Sephacel chromatography using a linear gradient of 0.1—0.7 M NaCl. Release of GAG chains by protease digestion indicated that the length of GAG chains was approximately Mr 45000 in biglycan and decorin, approximately Mr 48000 in the small amount of HSPGs and approximately Mr 66000 in versican. These proteoglycans resemble those synthesized by vascular smooth muscle cells but differ markedly from those synthesized by vascular endothelial cells.
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Notes
  • Aya Suenaga, Hiroshi Ueki
    2004 Volume 27 Issue 11 Pages 1859-1863
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    Orthovanadate (vanadate) inhibited the platelet aggregation induced by platelet-activating factor (PAF) in a dose-dependent manner. Propranolol, a nonspecific β-adrenergic receptor antagonist, and H-8, a selective inhibitor of cAMP-dependent protein kinase (PKA), suppressed the inhibition of the PAF-induced platelet aggregation by vanadate. Vanadate increased the cAMP content in platelets accompanied by the activation of PKA. The β-adrenergic receptors of platelets have been reported to be abundant in the β2 isoform, coupled to adenylyl cyclases (R. Kerry and M. C. Scrutton, Br. J. Pharmacol., 79, 681—691 (1983)). When the washed platelets were preincubated with vanadate, salbutamol, a selective β2-adrenergic receptor agonist, or 8-Br-cAMP, the latter two mimicked the vanadate-induced anti-platelet aggregation and prolongation of clotting time of plasma, suggesting involvement of the increased intracellular cAMP content in both actions of vanadate. Butoxamine, a selective β2-adrenergic receptor antagonist, suppressed both actions of vanadate. The vanadate-induced increase in cAMP content was inhibited in part by butoxamine or genistein. These results suggest that vanadate inhibits the PAF-induced platelet aggregation by the stimulation of a cAMP/PKA-dependent process via the β2-adrenergic receptor and receptor tyrosine kinases, and that the anti-platelet aggregation is involved in part in mechanisms of the anticoagulant action of vanadate.
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  • Shingo Niimi, Masashi Hyuga, Mizuho Harashima, Taiichiro Seki, Toyohik ...
    2004 Volume 27 Issue 11 Pages 1864-1866
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    We recently showed that annexin III is expressed in isolated small rat hepatocytes but, not in parenchymal hepatocytes. In the present study, we used reverse transcription polymerase chain analysis to examine the annexin III mRNA level in isolated small rat hepatocytes and parenchymal hepatocytes. Annexin III mRNA was detected in isolated small hepatocytes, but not in isolated parenchymal hepatocytes, confirming the presence of annexin III expression in isolated small rat hepatocytes at the mRNA level and indicating that the absence of annexin III expression in isolated parenchymal hepatocytes is due to the absence of annexin III mRNA. Furthermore, we examined the mRNA level of tyrosine aminotransferase and tryptophan oxygenase, two terminally differentiated hepatocyte markers. mRNA for these markers was detected in both parenchymal hepatocytes and small hepatocytes.
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  • Shang-Feng Liu, Shan He, Bo-Wen Liu, Ying Zhao, Zhao Wang
    2004 Volume 27 Issue 11 Pages 1867-1870
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    Rat SPATA4 gene, homologue to the human and mouse SPATA4 gene, expressed specifically in the rat testis was cloned by informatics analysis. The cDNA mapped to chromosome 16 in the rat genome is made up of 6 exons and the exon-intron boundaries obey to the AG/GT rule. The gene contains a 972 bp open reading frame encoding 323 amino acid sequences with theoretical molecular weight of 36.64 KD and isoelectric point of 9.65. One CpG island is located in the gene from site −200 to +198. A typical promoter is also predicted from site −630 to +101. According to the computer-aided analysis of the putative protein encoded by the rat SPATA4, no transmembrane region and no signal peptides are found in the protein. Multi-tissue RT-PCR results show that the SPATA4 gene is expressed specifically in the testis only. Moreover, the expression of SPATA4 occurs in a development stage-dependent pattern. According to the RT-PCR results, no expression of SPATA4 is detected until the rat is 30 d old after birth. The amount of SPATA4 mRNA increases from 30-d to 65-d-old rat and then keeps stable after that. In conclusion, this study proves the conservation of SPATA4 in mammalian animals and predicts its important role in spermatogenesis.
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Pharmacology
Regular Articles
  • Zhao-Qiu Wu, Qing-Long Guo, Qi-Dong You, Li Zhao, Hong-Yan Gu
    2004 Volume 27 Issue 11 Pages 1769-1774
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    We determined the in vivo and in vitro antitumor activities of gambogic acid (GA) and one of the possible mechanisms for its inhibitory activities. In vivo antitumor activity of GA was evaluated by the relative tumor growth ratio (T/C) in nude mice, and in vitro inhibition of SPC-A1 cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and trypan blue exclusion assay. Telomere repeats amplification protocol (TRAP)-polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (ELISA) and RT-PCR were used to quantitatively detect telomerase activity and the expression of human telomerase reverse transcriptase (hTERT) mRNA, respectively. Results from our in vivo study showed that transplantable tumor growth remained suppressed for up to 21 d with minimal animal weight loss in nude mice treated with gambogic acid (i.v.). Proliferation of SPC-A1 cells cultured in vitro was significantly inhibited (p<0.01), showing time-dependent and dose-dependent inhibition. Telomerase activity and hTERT mRNA expression were both decreased significantly, when cells were exposed to gambogic acid for 24, 48 and 72 h (for 24 h p<0.05, and for 48, 72 h, p<0.01). These results suggeste that gambogic acid could inhibit the growth of SPC-A1 cells and its tumor xenografts, and when treated with gambogic acid for a period of time, telomerase activity and expression of hTERT mRNA in the tumor cells were both inhibited significantly. It is safe, at least in part, to conclude that the down-regulating telomerase activity of GA by modifying partly the expression of hTERT mRNA in SPC-A1 cells may be one possible mechanism for the inhibitory activity of GA in the cells.
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  • Kaku Nakagawa, Hideyuki Kishida, Naoki Arai, Tozo Nishiyama, Tatsumasa ...
    2004 Volume 27 Issue 11 Pages 1775-1778
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    Licorice, the root of the Glycyrrhiza species, is one of the most frequently employed botanicals in traditional medicines. In this study, we investigated the effects of hydrophobic flavonoids from Glycyrrhiza glabra LINNE on abdominal fat accumulation and blood glucose level in obese diabetic KK-Ay mice. In order to enrich a fraction of hydrophobic flavonoids, licorice flavonoid oil (LFO) was prepared by further extracting licorice ethanolic extract with medium-chain triglycerides (MCT), and adjusting the concentration of glabridin, the major flavonoid of licorice, to 1.2% in oil. KK-Ay mice aged 6 weeks were assigned to 5 groups (n=6 each), and fed a high-fat diet containing 0 (control), 0.5%, 1%, or 2% LFO, or 0.5% conjugated linoleic acid (CLA) for 4 weeks. Compared with the control, body weight gain and weights of abdominal adipose tissues were suppressed (p<0.05) by feeding the diet containing 2% LFO, and blood glucose levels after 2 and 4 weeks were suppressed by all of the diets containing LFO. Although CLA feeding suppressed (p<0.05) body weight gain, it increased (p<0.05) blood glucose level after 2 weeks compared with the control level. Furthermore, LFO and licorice ethanolic extract stimulated human adipocyte differentiation in vitro. These results indicate that licorice hydrophobic flavonoids have abdominal fat-lowering and hypoglycemic effects, possibly mediated via activation of peroxisome proliferator-activated receptor-γ (PPAR-γ).
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  • Dae Gill Kang, Mi Kyoung Moon, Eun Jin Sohn, Dae Ho Lee, Ho Sub Lee
    2004 Volume 27 Issue 11 Pages 1779-1783
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    High fructose (HF) feeding induces a moderate increase in blood pressure in rats, which is associated with insulin resistance, hyperinsulinemia, and hypertriglyceridemia. In the present study, we examined the chronic effect of morin, a flavonoid isolated from medicinal plants, on blood pressure, lipid profiles, and serum insulin and glucose in HF-induced hypertensive rats. Rats were divided into control group and HF-fed group during the first three weeks of experiments. Then, rats were further divided into four groups and treated for 4 more weeks as follows: 1) control group; 2) morin-treated (intraperitoneal 5 mg/kg/d) control group; 3) HF-fed group; 4) morin-treated, HF-fed group (n=8, each group). Morin-treated HF-fed group showed lower systolic blood pressure (SBP) (132.0±2.5 mmHg vs. 142.8±2.2 mmHg, p<0.05), lower serum insulin level (1.21±0.27 vs. 2.73±0.30 μIU/dl, p<0.05), and lower plasma triglycerides (47.8±5.0 vs. 65.5±5.0 mg/dl, p<0.05) than those of HF-fed group. Morin treatment also suppressed mRNA expression of endothelin-1 (ET-1) in the thoracic aorta from HF-induced hypertensive rats. Moreover, decreased renal sodium excretion in HF-induced hypertensive rats was ameliorated by morin treatment. In conclusion, the results of this study demonstrate that morin has an anti-hypertensive effect in HF-induced hypertensive rats. This effect of morin may be associated with the suppression of serum insulin and plasma triglyceride level, with the down-regulation of ET-1 in the thoracic aorta, and with the partial amelioration of renal dysfunctions in HF-induced hypertensive rats.
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  • Mitsuhiko Miyamura, Saburo Yoshioka, Atsuhide Hamada, Daisuke Takuma, ...
    2004 Volume 27 Issue 11 Pages 1784-1787
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    Using surface and deep seawater collected in the sea area of Muroto Cape (Kochi, Japan), desalinated drinking samples of about 1200 hardness were prepared and examined for the effects on the prevention of atherosclerosis in dietary induced hyperlipidemia rabbits. The plasma LDL cholesterol level was lower in the deep seawater group than in the surface seawater group. GPx activity was significantly higher in the deep seawater group than in the control group, while there was no difference between the surface seawater and control groups. The level of LPO was also significantly lower in the deep seawater group than in the control group. The Sudan IV lipid stained area ratio on the inner surface of the aorta was significantly lower in the deep seawater groups than in the control group, while there was no difference between the surface seawater and control groups. The oil red O stained cross section of the aorta in the control and surface seawater administration group foam cells had accumulated to form thick layers, while in the deep seawater administration group, the degree of their accumulation was very low. These results suggested that the deep seawater was useful for the prevention of hyperlipidemia and arteriosclerosis compared to the surface seawater, and it was found that reduction of the LDL cholesterol level and enhancement of GPx activity were involved in its effects.
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  • Hiromichi Takano, Masunobu Sugimura, Yoshito Kanazawa, Toshihiro Uchid ...
    2004 Volume 27 Issue 11 Pages 1788-1791
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    An excessive elevation of intracellular Ca2+ levels is known to play a key role in the pathological events following cerebral ischemia. DY-9760e, 3-[2-[4-(3-chloro-2-methylphenylmethyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate, is a potent calmodulin antagonist that attenuates brain damage in focal ischemia models. In the present study, we investigated the effect of DY-9760e on neuronal cell death induced by a variety of cell-toxic stimuli that increase intracellular Ca2+. Cell death was induced by the exposure of primary cultured neurons to excitotoxic agents such as glutamate and N-methyl-D-aspartate, membrane-depolarizing agents such as veratridine and high KCl, or thapsigargin an endoplasmic reticulum Ca2+-ATPase inhibitor. Treatment with DY-9760e resulted in a dose-dependent prevention of neuronal cell death elicited by excitotoxicity, voltage-gated channel opening, and inhibition of endoplasmic reticulum Ca2+-ATPase. These results indicate that DY-9760e can rescue neurons from various types of cell-toxic stimuli, which may contribute to attenuation of brain injury after cerebral ischemia.
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Notes
  • In-Young Choi, Hyuk-Sang Jung, Hyung-Ryong Kim, Eon-Jeong Lee, Eun-Hee ...
    2004 Volume 27 Issue 11 Pages 1871-1874
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    OK205 is a traditional Korean prescription containing water-soluble chitosan, glucosamine HCl, chondroitin sulfate, and extract of herbal medicine, and has been used commercially to treat rheumatoid arthritis (RA). Because infiltrated mast cells and their mediators may contribute to the initiation and progression of the inflammatory process and matrix degradation of RA, we tested the inhibitory effects of OK205 on cytokine production in a human mast cell line (HMC-1 cells). Production of tumor necrosis factor-α was significantly decreased to 0.091±0.010 ng/ml after treatment of HMC-1 cells with OK205 100 μg/ml. The inhibition rate was about 43.57%. In addition, production of interleukin-6 in OK205 1 pg/ml-treated cells was 2.779±0.071 ng/ml, and the inhibition rate was about 50.22%. However, OK205 did not significantly inhibit the production of interleukin-8. These findings may help in understanding the mechanism of action of OK205, leading to control of mast cells in inflammatory conditions like RA.
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  • Yuhki Sato, Fumihiko Katagiri, Shin Inoue, Hiroki Itoh, Masaharu Takey ...
    2004 Volume 27 Issue 11 Pages 1875-1877
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    Sensory afferent neurons in the gastrointestinal mucosa regulate neuropeptides [calcitonin gene-related peptide (CGRP), substance P, etc.], which play various physiologic roles and are gastroprotective. To determine whether the pharmacologic effects of Dai-kenchu-to (DKCT) on the gastrointestine are due to changes in gastrointestinal mucosa regulatory peptide levels, we examined the effects of the DKCT on the levels of CGRP-like immunoreactive substances (IS) and substance P-IS in plasma taken from five healthy subjects. A single oral administration of DKCT 7.5 g caused significant increases in plasma CGRP-IS at 40 min, and in substance P-IS levels at 20 and 60 min, compared with a placebo group. The present study may indicate that the pharmacologic action of DKCT is closely related to changes in CGRP- and substance P-IS levels.
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Toxicology
Regular Articles
  • Ja Yong Lee, Da Woon Jung, Hyun Ah Park, Soo Jeong Kim, Jin Ho Chung, ...
    2004 Volume 27 Issue 11 Pages 1792-1796
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    The effect of taurine intake on the biliary disposition and toxicity of acetaminophen (APAP) was examined in male Golden-Syrian hamsters. Animals were provided with taurine (5 mM) in drinking water for 1 week followed by APAP treatment (250 mg/kg, i.p.). Biliary excretion and plasma concentrations of APAP and its major metabolites were determined for up to 360 min. Taurine increased the bile flow, whereas the concentration of APAP or the metabolites in bile was not altered significantly. Accordingly the total biliary excretion of APAP and the metabolites was increased in hamsters fed taurine. Taurine increased the plasma concentrations of APAP-glutathione (GSH) and APAP-mercapturate, but the APAP-glucuronide or APAP-sulfate concentration was not changed. The area under the curve of the plasma APAP concentration was reduced significantly, suggesting that the elimination of APAP was enhanced by taurine intake. However, the hepatotoxicity resulting from a dose of APAP (450 mg/kg, i.p.) was not altered by taurine intake as determined by the elevation of serum alanine aminotransferase, aspartate aminotransferase, and sorbitol dehydrogenase activities. The results suggest that taurine administration could affect the disposition of APAP by enhancing its metabolism through the GSH-dependent pathway and also by increasing the biliary excretion of this drug and its metabolites. The pharmacological significance of this finding remains to be examined.
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Medicinal Chemistry
Regular Articles
  • Toshiro Matsui, Sumi Ebuchi, Tomoko Fujise, Kanthi J. M. Abesundara, S ...
    2004 Volume 27 Issue 11 Pages 1797-1803
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    To clarify the suppression of postprandial blood glucose rise via α-glucosidase (AGH) inhibitory action by natural compounds, propolis was examined in this study. A single oral administration of propolis extract (50% methanol fraction on XAD-2 column chromatography) in Sprague–Dawley rats demonstrated a potent antihyperglycemic effect with the significant AUC0—120 min reduction of 38% at a dose of 20 mg/kg compared to that of controls. Among the active compounds isolated from the fraction, 3,4,5-tri-caffeoylquinic acid was found to be a prominent candidate that exerts the effect and shows a strong maltase-specific inhibition with an IC50 value of 24 μM. In addition, the noncompetitive inhibition power apparently increased with the number of caffeoyl groups bound to quinic acid.
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Notes
  • Mitsuteru Numazawa, Wakako Handa, Keiko Yamada
    2004 Volume 27 Issue 11 Pages 1878-1882
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    To gain insight into the mechanism for irreversible inactivation of aromatase by 6β-bromoandrostenedione (1), one of the earliest discovered suicide substrates, in relation to the catalytic function of the enzyme, the 2,2-dimethyl derivative of compound 1, steroid 4, and its 6α-isomer 5, as well as 2-methyl-1,4-diene steroid 8 and its 6α-bromide 10, were synthesized. All of the steroids inhibited aromatase activity in human placental microsomes with apparent Ki's ranging between 10 and 81 nM. The 2,2-dimethyl-6β- and 6α-bromo steroids 4 and 5 were extremely powerful inhibitors (Ki: 14 and 10 nM, respectively), but these two did not cause a time-dependent inactivation of aromatase in the presence of NADPH; in contrast, the 2-methyl-1,4-diene steroids 8 and 10 caused time-dependent inactivation with apparent kinact of 0.035 and 0.071 min−1, respectively, in a suicide manner. These results indicate that the 2,2-dimethyl function of the 6β-bromide 4 would prevent the inactivation of aromatase caused by inhibitor 1 in a suicide manner, probably through steric activity, whereas the 2-methyl group of steroid 8 did not significantly affect the suicidal inactivation by the parent 1,4-diene steroid, a typical suicide substrate.
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Pharmacognosy
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  • In Kyung Rhee, Natalie Appels, Bertil Hofte, Bahadir Karabatak, Cornel ...
    2004 Volume 27 Issue 11 Pages 1804-1809
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    In an attempt to isolate the active compound while detecting acetylcholinesterase inhibitory activity, we applied a fluorometric flow assay system to an on-line coupled preparative HPLC. The MeOH extract of Nerine bowdenii showed a strong inhibitory peak in the on-line assay, and the active compound was isolated by CPC and HPLC. It was identified as ungeremine by analysis of its 1H-NMR, 2D-NMR, and NOESY spectra. The assignment of the active N. bowdenii constituent was also confirmed by co-TLC, co-HPLC, and co-1H-NMR experiments using an authentic sample of synthetic ungeremine. The IC50 value of ungeremine was 0.35 μM, showing stronger activity than galanthamine (2.2 μM).
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  • Ki-Ho Cho, Young-Suk Kim, Hyung-Sup Bae, Sang-Kwan Moon, Woo Sang Jung ...
    2004 Volume 27 Issue 11 Pages 1810-1813
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    Chunghyuldan (Daio-Orengedokuto in Japanese) (CHD) has been used as an antihyperlipidemic and antiischemic agent in Korea. To evaluate in vitro the efficacy of Chunghyuldans (CHDs) metabolized with and without human intestinal microflora against brain ischemia, we investigated its anti-inflammatory effect on LPS-induced RAW264.7 cells. Both metabolized CHD (MCHD) and CHD showed antioxidant activities in vitro, and inhibited nitric oxide (NO) and prostaglandin E2 (PGE2) productions in lipopolysaccharide (LPS)-induced RAW264.7 cells. These also inhibited enzyme activities and protein expressions of inducible NO synthase and cyclooxygenase-2 in LPS-induced RAW264.7 cells. MCHD-inhibitory activity against NO and PGE2 productions in LPS-induced RAW264.7 cells was more potent than those of CHD. These results suggest that CHD may show potent anti-inflammatory activity in vivo and can improve brain ischemia.
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  • Joyce Govinden-Soulange, Naresh Magan, Ameenah Gurib-Fakim, Anne Gauvi ...
    2004 Volume 27 Issue 11 Pages 1814-1818
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    The composition of the leaf oils, obtained by hydrodistillation, of five endemic Psiadia species of the Asteraceae family were studied by GC/MS on both polar and non-polar columns. The analysis showed that the volatile components of the oils were made up essentially of monoterpenes, sesquiterpenes, aliphatics and other shikimic acid derivatives. With respect to the non-volatile components, great variations were observed: P. lithospermifolia contained (E)-isoasarone (51.5%); P. penninervia: eugenol (5.1%); P. terebinthina: eugenyl-acetate (4.0%); P. viscosa: pentyl-4-(1-methylethyl benzoate) (25.8%); P. arguta: isoeugenol (56.5%). In vitro antimicrobial assays using the agar-well diffusion method, revealed that most of the oils were not very active against the tested microorganisms except for that of P. lithospermifolia, which significantly inhibited the growth of Bacillus cereus, Staphylococcus aureus and Pseudomonas aureofaciens, Aspergillus ochraceus, Candida pseudotropicalis, Kluyveromyces lactis and Fusarium moniliforme. This activity has been attributed to the presence of δ-elemene, (E)-farnesene, α-curcumene, selina-4,7(11)-diene, (E,Z)-α-farnesene, β-bisabolene some of which have established antimicrobial profiles. Likewise, the fungi toxic action of the oil of P. arguta against Aspergillus ochraceus, Candida pseudotropicalis, and Fusarium moniliforme, may be attributed to the presence of isoeugenol, eugenol being known to be mycotoxic especially against Aspergillus species.
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Notes
  • Sang-Myung Lee, Jin-Gyu Park, You-Hee Lee, Cheal-Gyu Lee, Byung-Sun Mi ...
    2004 Volume 27 Issue 11 Pages 1883-1886
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    In order to determine on the anti-complement activity of triterpenes, following eleven triterpenoides were isolated from the fruits of the Zizyphus jujuba MILL: ceanothane-type triterpenes: colubrinic acid (1), zizyberenalic acid (11); lupane-type triterpenes: alphitolic acid (2), 3-O-cis-p-coumaroyl alphitolic acid (3), 3-O-trans-p-coumaroyl alphitolic acid (4), betulinic acid (7), betulonic acid (9); and oleanane-type triterpenes: 3-O-cis-p-coumaroyl maslinic acid (5), 3-O-trans-p-coumaroyl maslinic acid (6), oleanolic acid (8), oleanonic acid (10). These compounds were examined for their anti-complement activity against the classical pathway of the complement system. Among them, compounds 5, 6, and 8 exhibited significant anti-complement activity with IC50 values of 101.4, 143.9, and 163.4 μM, respectively, whereas the ceanothane-type and the lupane-type triterpenes were inactive. This suggests that the oleanane-structure plays an important role in inhibiting the hemolytic activity of human serum against erythrocytes.
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Biopharmacy
Regular Articles
  • Jia-You Fang, Yann-Lii Leu, Tsong-Long Hwang, Hsiao-Chin Cheng
    2004 Volume 27 Issue 11 Pages 1819-1825
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    The purpose of this study was to evaluate the essential oils from sweet basil (Ocimum basilicum, OB) as skin permeation enhancers to promote the percutaneous absorption of drugs. The in vitro and in vivo irritancy of the essential oils was also examined. Terpenes with various carbon numbers (mono-, sesqui-, di-, and tri-) were identified in both the lower-polarity fraction (OB-1) and higher-polarity fraction (OB-2). In vitro skin permeation and deposition of indomethacin were significantly enhanced after treatment with OB essential oils. The enhancing effect of OB-1 was greater than that of OB-2 in the in vitro permeation and in vivo cutaneous microdialysis analyses as well as in the plasma concentration of indomethacin. On the other hand, the in vivo study showed that OB-2 had a greater ability to retain the drug within the skin than did OB-1. Enhancement of the skin permeation of drugs by OB essential oils might be mainly due to improvement in the partitioning of the drugs to the stratum corneum. Both in vitro cell cultures (keratinocytes and skin fibroblasts) and in vivo transepidermal water loss showed no or only negligible irritation to skin by OB essential oils.
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  • Toyonori Kato, Yayoi Hayashi, Katsuhisa Inoue, Hiroaki Yuasa
    2004 Volume 27 Issue 11 Pages 1826-1830
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    The mechanism of intestinal glycerol transport was investigated by using the in vitro everted sac method involving the rat small intestine. The uptake of glycerol into everted sacs was saturable with a Michaelis constant (Km) of 0.77 mM and a maximum transport rate (Jmax) of 11.5 nmol/min/100 mg wet tissue weight (wtw), suggesting the involvement of carrier-mediated transport, and was accompanied by unsaturable transport (passive transport) with a membrane permeability clearance (CLm,d) of 4.9 μl/min/100 mg wtw. The carrier-mediated uptake of glycerol was inhibited by the removal of Na+ and also by the addition of 2,4-dinitrophenol (DNP) and sodium azide (NaN3), which are metabolic inhibitors. These results suggest that the carrier-mediated glycerol transport is Na+-dependent and secondary active. Since glycerol uptake was also inhibited by p-chloromercuribenzene sulfonate (pCMBS), a thiol-modifying reagent, cysteine residues, which have a thiol group, seem to play an important role in the function of the carrier. We further found that glycerol uptake was selectively inhibited by glycerol-3-phosphate, chloramphenicol and voglibose, which are alcohol-related compounds analogous to glycerol. Several other compounds that did not inhibit glycerol uptake included D-glucose and 5-fluorouracil, which are known to be transported by specific carriers, and none of the selective inhibitors of glycerol uptake inhibited the uptake of D-glucose and 5-fluorouracil. Therefore, the carriers for these two compounds do not seem to be involved in glycerol uptake. It is likely that the carrier-mediated transport system involved in glycerol uptake is specific to glycerol and, possibly, some analogous compounds with hydroxyl groups. It would be interesting to examine the possibility that the carrier-mediated glycerol transport system might be involved in drug absorption and also that it might be used for oral drug delivery.
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  • Takeshi Kimura, Mamiko Arai, Hiroya Masuda, Atsufumi Kawabata
    2004 Volume 27 Issue 11 Pages 1831-1833
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    Given the absence of standard guidelines for use of recombinant human erythropoietin in patients with end-stage renal disease in Japan, in the present study, pharmacists actively managed the erythropoietin therapy, and the therapeutic and pharmacoeconomic outcome was evaluated. We compiled in-hospital guidelines for proper use of erythropoietin for outpatients with renal anemia under hemodialysis, and made recommendations, particularly about changes in the doses of erythropoietin and administration of iron preparations, to physicians. The clinical test values and the dosages of erythropoietin were monitored for 9 months and analyzed. As results of our participation, the number of renal anemia patients with over 30% of the hematocrit value as a therapeutic target increased from 7 to 32 among 41 patients. Twenty three of the 41 patients could decrease the dose of erythropoietin, and 5 patients could cease receiving the drug. Monthly total units of erythropoietin used for the 41 patients could also be decreased from 915000 units to 642000 units, resulting in considerable improvement of cost performance. Thus, active participation of pharmacists in management of renal anemia had great therapeutic and pharmacoecomic impact in Japan, as in North America.
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Miscellaneous
Regular Articles
  • Denise Pimenta da Silva Leitão, Ademar Alves da Silva Filho, An ...
    2004 Volume 27 Issue 11 Pages 1834-1839
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    Streptococcus mutans triggers dental caries establishment by two major factors: synthesis of organic acids, which demineralize dental enamel, and synthesis of glucans, which mediate the attachment of bacteria to the tooth surface. Propolis is a natural product that may prevent dental caries. Baccharis dracunculifolia DC (Asteraceae), a native plant from Brazil, is the most important botanical origin for the production of green propolis (Brazilian propolis) by honeybees. However, whether B. dracunculifolia (Bd) has an anticariogenic effect, like green propolis, remains unknown. Herein, we have made a comparative evaluation of the effects of extracts from green propolis and Bd on the glucan synthesis and acidogenic potential of S. mutans. The inhibitory effects of the extracts on bacterial acid production were evaluated through the potentiometric measurement of pH from bacterial suspensions treated with serial concentrations of both extracts. Besides presenting close inhibitory values at the same concentration range, Bd leaf rinse and green propolis extracts had similar IC50 values (0.41 and 0.34 mg/ml, respectively). Both extracts produced a bacteriostatic effect on S. mutans cultures at a concentration of 0.40 mg/ml. Estimated inhibitory values of green propolis and Bd leaf rinse extracts on the synthesis of insoluble glucans (IC50=12.9 and 25.0 μg/ml, respectively) and soluble glucans (IC50=50.4 and 49.1 μg/ml, respectively) were not significantly different from each other at p<0.05. The results demonstrate that Bd leaf rinse and green propolis extracts have similar inhibitory effects on the S. mutans cariogenic factors evaluated herein, and allowed us to suggest that Bd leaves may be a potential source for pharmaceutical products employed for this purpose.
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  • Hiroyuki Morita, Ryoichi Hasunuma, Kiichiro Kawaguchi, Yoshiyuki Adach ...
    2004 Volume 27 Issue 11 Pages 1840-1843
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    The protective effects of an antibiotic polymyxin B (PLB), having lipopolysaccharide (LPS)-binding activity, on infection-induced endotoxin shock in mice were investigated. Infection with 108 colony forming units of an attenuated Salmonella typhimurium aroA strain caused lethal endotoxin shock to ddY mice. Treatment with PLB 1 h post infection (p.i.) resulted in significant reduction of mortality and bacterial numbers in livers. In addition, treatment with PLB 1 h p.i. resulted in a transient increase at the early stage and gradual decline in plasma LPS levels. Although plasma levels of sCD14 and high mobility group box chromosomal protein-1 (HMGB-1) increased according with progression of infection, increases in plasma levels of sCD14 and HMGB-1 were downregulated by treatment with PLB 1 h p.i. However, the lethal shock was not blocked by treatment with anti-CD14 monoclonal antibody at 3 h and 6 h p.i. Interestingly, administration of PLB 6 h p.i. did not show any protective activities, indicating that a time window for effective PLB action is present.
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  • Takashi Satoh, Yuki Tomikawa, Kaori Takanashi, Shinji Itoh, Shungo Ito ...
    2004 Volume 27 Issue 11 Pages 1844-1849
    Published: 2004
    Released: November 01, 2004
    JOURNALS FREE ACCESS
    To determine the inhibition effects of drugs on the glucuronidation of estradiol (E2), 29 drugs that have been reported to induce gynecomastia were examined in the presence of UDP-glucuronic acid using human hepatic microsomes (pooled) as the enzyme source. The percentage inhibition of the E2 glucuronidation was determined at drug concentrations of 1 μM (approximate therapeutic concentration) and 100 μM (non-clinical overdose concentration) based on the rate constants for the 3- and 17-glucuronidation of E2 (11.2 and 2.52 pmol/min/mg protein, respectively). The only drug that exhibited 50% or higher inhibition of the 3-glucuronidation at a concentration of 1 μM was manidipine (54.4%). When the concentration was 100 μM, manidipine exhibited 100% inhibition of the 3-glucuronidation, and other drugs that exhibited 50% or higher inhibition of the 3-glucuronidation were nicardipine (92%), nisoldipine (90%), nifedipine (84%), domperidone (81%), tacrolimus (80%), nitrendipine (77%) and ketoconazole (69%). Conversely, ipriflavone accelerated the formation of estradiol 3-glucuronide in the activity of 165% at the concentration of 100 μM. On the 17-glucuronidation, all of the drugs showed less than 50% inhibition at the concentration of 1 μM, but at the concentration of 100 μM, drugs that exhibited 50% or higher inhibition consisted of manidipine (79%), chlormadinone acetate (74%), nisoldipine (66%), nitrendipine (60%) and ketoconazole (55%). Although IC50 values of these drugs were all lower than the Km value (285 μM) for the 3-glucuronidation of E2, they were higher than the Km value for the 17-glucuronidation (18.8 μM). Thus, the effect of the drugs on the E2 glucuronidation should be greater for hydroxy group at the C-3 than that at the C-17 of E2 molecule. On the other hand, metabolic clearances (Vmax/Km) of the 3- and 17-glucuronidation were about 1/14th and 1/18th of that of the 2-hydroxylation of E2, respectively. The result implies that, when the contribution of the glucuronidation to enterohepatic circulation is taken into consideration, the effect of this metabolic inhibition in the estrogen pool cannot be ignored.
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