Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Volume 27, Issue 12
Displaying 1-33 of 33 articles from this issue
Analytical Biochemistry
Regular Articles
  • Akio Suzuki, Ryo Suzuki, Tadahide Furuno, Reiko Teshima, Mamoru Nakani ...
    2004 Volume 27 Issue 12 Pages 1891-1894
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    Communication between nerves and mast cells is a prototypic demonstration of neuro-immune interaction. Numerous studies have shown that the stimulation of nerves (or addition of neurotransmitters) can evoke activation of mast cells, and that mast cell-derived mediators can influence neuronal activity. However, the molecules involved in the membrane-membrane contacts between nerves and mast cells are still unknown. Here, we used an in vitro co-culture approach comprising interaction between immune (bone marrow-derived mast cell, BMMC) and nerve cells (superior cervical ganglia, SCG). The experiments showed clearly that the nerve-mast cell communication was supported by synapse-like structure and that N-cadherin, not E-cadherin, played an essential role in the synapse-like structure. In addition, we found that the synapse-like structure was assisted by clustering of β-catenin to N-cadherin.
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Biochemistry/Molecular Biology
Regular Articles
  • Takamasa Yokoyama, Yayoi Kamata, Kenzo Ohtsuki
    2004 Volume 27 Issue 12 Pages 1895-1899
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    The physiological correlation between casein kinase 2 (CK2) and two Src family tyrosine kinases (Src-TKs, Fyn and Src) was mainly investigated in vitro. It was found that (i) Thr-residues of these two Src-TKs were preferentially phosphorylated by CK2 using [γ-32P]GTP as a phosphate donor; (ii) this phosphorylation was highly stimulated in the presence of poly-Arg; (iii) full phosphorylation of two Src-TKs by CK2 resulted in significant reduction of their TK activities; and (iv) quercetin (a CK2 inhibitor) inhibited the CK2-mediated reduction of their Src-TK activities in vitro. Under the same experimental conditions, similar results were obtained with Yes. These results suggest that CK2 may be a protein kinase responsible for the suppression of at least three Src-TKs (Fyn, Src and Yes) through the specific phosphorylation of their Thr-residues at the cellular level.
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  • Toshifumi Shiraga, Toshiro Niwa, Yasuo Ohno, Akira Kagayama
    2004 Volume 27 Issue 12 Pages 1900-1906
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    In the current study, we investigated interindividual variability of the 2-hydroxylation, 3-glucuronidation, and 3-sulfation of ethynylestradiol (EE2) using human liver microsomes and cytosol. Km values for the 2-hydroxylation and 3-glucuronidation in pooled liver microsomes and for the 3-sulfation in pooled liver cytosol were 3.34, 23.3, and 2.85 μM, respectively. Vmax/Km (ml/min/g liver) was highest for the 3-sulfation, followed by 2-hydroxylation, suggesting that 3-sulfation is the major metabolic pathway of EE2 in human liver. All further studies were performed at a substrate concentration of 0.1 μM. Microsomal 2-hydroxylation and 3-glucuronidation activities ranged from 0.21 to 5.02 (2.04±1.34, mean±S.D., n=35) and 0.20 to 4.84 (1.20±1.00, n=35) pmol/min/mg protein, respectively. Cytosolic 3-sulfation activity ranged from 4.2 to 24.3 (11.8±4.4, n=21) pmol/min/mg protein. All the measured enzyme activities were neither gender-related nor age-dependent, except that 2-hydroxylation was significantly higher in females than in males (p<0.05). The relative contribution of CYP3A to the 2-hydroxylation in liver microsomes was estimated from the degree of inhibition by 1 μM ketoconazole. The degrees of inhibition were between 17.8 and 78.0% (51.6±16.0%, n=27). These results indicate that there are large interindividual differences in the enzyme activities towards the respective metabolic pathways of EE2 and the relative contribution of CYP3A to the 2-hydroxylation of EE2 in human liver.
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  • Taro Uchiyama, Tetsunori Matsumoto, Yoshiharu Suzuki, Masami Ishida, T ...
    2004 Volume 27 Issue 12 Pages 1907-1912
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    The effects of endogenous hyaluronan on cervical ripening during pregnancy were examined in rabbits. Hyaluronan of approximately 620 kilodalton (kDa) was detected in the uterine cervix on the 25th and 29th days of pregnancy, while it was not detected in cervix of non-pregnant animals. In addition, low-molecular-weight (less than 191 kDa) hyaluronan was present in cervix at the 29th day of pregnancy. Hyaluronan level in the cervix was lower on the 29th day than on the 25th day of pregnancy, whereas that in serum was significantly higher on the 29th day than on the 25th day of pregnancy. To clarify the physiological functions of endogenous hyaluronan, the effects of hyaluronan (HA600—700), which had approximately equal to endogenous hyaluronan, on uterine cervical tissues and uterine cervical fibroblasts were examined. Rabbits at the 23rd day of pregnancy were administered a vaginal suppository of HA600—700 (20 mg) daily for three days. Promotion of cervical ripening was observed, as well as detachment of collagen fiber bundles, and a reduced density of collagen fiber distribution. Total collagenolytic activity was increased significantly by HA600—700 (1.0 mg/ml) treatment in cultured uterine cervical explants of pregnant rabbits as compared with the untreated group. Moreover, very similar effects of HA600—700 treatment (0.1, 1.0 mg/ml) were observed in cultured uterine cervical fibroblasts. Further, in tissue cultures, but not cell cultures, of pregnant rabbit cervix, prostaglandin E2 (PGE2) production was enhanced by HA600—700 treatment. Therefore, it appears that endogenous hyaluronan is closely concerned with cervical ripening and dilatation in uterine cervix of pregnant rabbits.
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  • Shinya Abe, Satoko Suyama, Hitomi Shishido, Masayoshi Sato, Yosuke Oht ...
    2004 Volume 27 Issue 12 Pages 1913-1915
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    Gallium-67 (67Ga) has been used as a tumor or inflammation-imaging agent in nuclear medicine. We recently reported that the peak of serum alanine aminotransferase (ALT) level was 1 d after carbon tetrachloride (CCl4) treatment in rats. However, the peak of 67Ga uptake by the liver tissue and hepatocytes was 2 d after the treatment. If 67Ga is taken in the hepatic disorder phase, the pattern of 67Ga uptake by the hepatocytes should be consistent with that of the ALT level. In order to answer why it was not, we carried out a detailed examination. The lipid peroxidation of hepatocytes from CCl4-treated mice was greatly increased 1 d after CCl4 treatment; serum ALT was also increased 1 d after the treatment. The uptake of 67Ga by the liver tissue reached a maximum 1 and 2 d after the treatment, while maximum by the hepatocytes was achieved after 2 d. The incorporation of bromodeoxyuridine into the hepatocytes also reached maximum 2 d after the treatment. These results suggest that the uptake of 67Ga by the hepatocytes is carried out during liver regeneration rather than during hepatic disorder by CCl4 treatment.
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  • Ayako Kobayashi, Tatsuo Maeda, Masatomo Maeda
    2004 Volume 27 Issue 12 Pages 1916-1922
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    Transporter associated with antigen processing (TAP)-like (TAPL, ABCB9) is a half-type ATP binding cassette (ABC) protein belonging to subfamily B highly homologous to the TAP, a hetero-dimeric complex consisting of a TAP1 and a TAP2 subunit. Human TAPL, to which was tagged with green fluorescence protein (GFP) at its carboxyl terminus (TAPL-GFP), showed fluorescence on intracellular membranes similar to TAP1-GFP. A truncated form of TAPL-L-GFP (M1—S275 was followed by GFP) showed a similar cellular fluorescence pattern to TAPL-GFP. However, the fluorescence of TAPL-S-GFP (M1—G75) was distributed over all the cellular membranes including plasma membrane, indicating that the amino terminal region of TAPL (M1—S275) is essential for its localization to the intracellular membranes. A co-expression study demonstrated that TAPL-S-GFP was co-localized with TAPL-DR (DsRed-tagged TAPL) or TAP1-DR, suggesting that TAPL is able to interact with not only itself but also with TAP1 through the M1—G75 region of TAPL. It is also proposed that a further downstream sequence of TAPL would confine the distribution of TAPL-S-GFP to the intracellular membranes. Similarly, the distribution of TAP2-S-GFP (M1—R88) was restricted to the intracellular membranes by TAPL-DR or TAP1-DR, indicating that the M1—R88 region of TAP2 is able to interact with TAPL as well as TAP1. Therefore, TAPL would form a homo-dimer with itself, and a hetero-dimer with TAP1 and TAP2. TAPL-GFP was co-localized with the fluorescence endoplasmic reticulum (ER) marker, suggesting that TAPL is mainly localized to the ER in the intracellular membranes.
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  • Akira Kamei, Shinsuke Takamura, Makoto Nagai, Noriko Takeuchi
    2004 Volume 27 Issue 12 Pages 1923-1931
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    We reported previously that C-terminal truncated α-crystallins were found in lenses of hereditary cataractous rat ICR/f. In this study, we examined the phosphorylation of the crystalline lens proteins, αB-crystallin and αA-crystallin, in cataractous and normal rats of different ages and have found an increase in the phosphorylation of serine residues of truncated α-crystallin in cataractous lens. Phosphorylation and C-terminal truncation of α-crystallins could, both, reduce their chaperone-like activity and lead to cataract formation.
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  • Tohru Higaki, Taro Watanabe, Ichiro Tamatomi, Hidetoshi Tahara, Masano ...
    2004 Volume 27 Issue 12 Pages 1932-1938
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    Telomerase-negative immortal human cells maintained telomere length by a mechanism called alternative lengthening of telomeres (ALT mechanism). These cells (ALT cells) have two prominent characteristics of long telomere repeats at each chromosome end revealed by Southern blotting (terminal restriction fragments: TRF) and the presence extrachromosomal telomere repeat (ECTR) DNA. We report here that the TRF length of ALT cells revealed by the conventional unidirectional (UD) current or pulse-field (PF) current electrophoresis appeared to be over estimated. The TRF length determined by the pulse inverse-field (PIF) current electrophoresis (2—9 kbp depending upon cell lines) was much smaller than that (ca. 23 kbp) by UD or PF current electrophoresis. These results were in consistent with very weak telomere staining in situ at chromosome ends in ALT cells. When a mixture of HinfI-digested genomic DNA of human diploid fibroblasts and synthetic telomere repeat DNA with similar size of ECTR DNA was electrophoresed using a UD current, the apparent TRF size shifted to larger molecular weight, while the size shift did not occur by PIF current electrophoresis. These results together with other data indicate that the unusually long TRF of ALT cells determined by using conventional electrophoresis is an artifact produced by a complex formed by short TRF and short ECTR DNA.
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  • Syuhei Ishikura, Noriyuki Usami, Satoko Nakajima, Akiko Kameyama, Hiro ...
    2004 Volume 27 Issue 12 Pages 1939-1945
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    Mouse kidney contains two 3(17)α-hydroxysteroid dehydrogenases (HSDs) that show essentially the same properties except for their isoelectric points. However, the structural differences and physiological roles of the two enzymes remain unknown. In this study, we have isolated cDNAs for the two 3(17)α-HSDs from a total RNA sample of mouse kidney by reverse transcription-PCR. The identity of the cDNAs was confirmed by characterization of the recombinant enzymes that showed the same molecular weights, pI values, pH optima, substrate specificity and inhibitor sensitivity as those of the enzymes from mouse kidney. We also found that the recombinant enzymes reduce precursors of neuroactive progesterone derivatives, 5α-dihydrotestoserone, deoxycorticosterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate and estrone at low Km values of 0.3—2 μM. The two enzymes belonged to the aldo-keto reductase (AKR) family, and their 323-amino acid sequences differed only by five amino acids. The sequences of the two isoforms are identical to those of proteins that are predicted to be encoded in a gene for AKR1C21 in the database of the mouse genome. However, the mRNAs for the two isoforms were expressed in mouse kidney and other tissues, in which their expression levels were different. The results indicate an important role of 3(17)α-HSD in controlling the concentrations of various steroid hormones in the mouse tissues, and suggest the existence of two genes for the two isoforms of the enzyme.
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Pharmacology
Regular Articles
  • Hee Kang, Kyoo-Seok Ahn, Chongwoon Cho, Hyun-Su Bae
    2004 Volume 27 Issue 12 Pages 1946-1950
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    Astragali Radix (AR), is a popular herbal medicine used to treat allergic diseases in Korea, Japan and China. Our study examined the effect of an AR ethanol extract on both in vitro and in vivo murine CD4 T cells' differentiation into Th1 and Th2 subsets. CD4 T cells from Balb/c mice were activated with anti-CD3/anti-CD28 mAb in the presence of AR for 2 d. AR treated cells showed an elevated level of IL-4 but a reduced level of IFN-γ secretion. In addition, in vitro Th1/Th2 polarization experiments revealed that AR enhanced the levels of IL-4 in Th2 cells but reduced the levels of IFN-γ in Th1 cells. To elucidate the effects of AR in Th1/Th2 lineage development during the in vivo condition, AR was administrated orally to BALB/c mice. The results demonstrated that AR administration significantly increased IL-4 production in both the serum and supernatant of splenocyte culture, while IFN-γ secretion was diminished upon in vivo activation with anti-CD3 antibody. Our data clearly indicates that AR selectively alters Th1/Th2 cytokine secretion patterns and provides the pharmacological basis for AR's clinical applications.
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  • Hiroshi Tsuneki, Kana Ito, Naoto Sekizaki, En-Long Ma, Yueren You, Jun ...
    2004 Volume 27 Issue 12 Pages 1951-1956
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    Nicotinic acetylcholine receptors are found in microvascular endothelial cells. To reveal the functional role in cerebral angiogenic processes, we studied the nicotinic modulation of proliferation activity in cultured bovine and porcine cerebral microvascular endothelial cells. The proliferation activity was determined by an increase in the number of cells present in culture dishes. When the bovine cerebral endothelial cells at different passages were cultured in the presence of nicotine (10 nM), the proliferation activities were significantly increased in the cells at passage 1 and passage 3, but not at passage 4. Reverse transcriptase–polymerase chain reaction studies demonstrated that the expression of mRNAs coding for α3 nicotinic receptor subunit was significantly reduced in the bovine cerebral endothelial cells at passage 4, compared with that at passage 1. The proliferation of porcine cerebral endothelial cells (passage 1) was enhanced by acetylcholine (10 nM—100 μM) in the presence of atropine, a muscarinic antagonist, and this enhancing effect was inhibited by hexamethonium (100 μM, a nicotinic antagonist). The stimulation by acetylcholine (1 μM, with atropine) or nicotine (10 nM) induced the phosphorylation of a mitogen-activated protein (MAP) kinase (extracellular-signal regulated kinase: ERK) in the serum-starved endothelial cells. In the presence of PD98059 (2 μM, a MAP kinase kinase inhibitor) and atropine, acetylcholine (1 μM) failed to enhance the proliferation of porcine cerebral endothelial cells. These results demonstrate that nicotinic stimulation promotes the proliferation of bovine and porcine cerebral microvascular endothelial cells, at least in part, through the MAP kinase activation.
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  • Soichiro Akamatsu, Aiko Watanabe, Makoto Tamesada, Risa Nakamura, Shin ...
    2004 Volume 27 Issue 12 Pages 1957-1960
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    Mycelia of the edible mushroom Lentinus edodes (shiitake) were cultivated in a solid medium, and two fractions were obtained by hot-water extraction (L.E.M.) and then ethanol extraction followed by Sephadex LH-20 column chromatography (ESMe). The L.E.M. and ESMe were then examined for their hepatoprotective effect on dimethylnitrosamine-injured mice. Both fractions decreased the blood aspartate aminotransferase and alanine aminotransferase levels, partially inhibited the overaccumulation of collagen fibrils, and suppressed the overexpression of genes for α-smooth muscle actin and/or heat-shock protein 47 in the mice. Both fractions also inhibited the morphologic change and proliferation of isolated rat hepatic stellate cells (HSCs), which play a central role in liver fibrosis, in a dose-dependent manner and without cytotoxicity. The direct interaction between the extracts and HSCs appears to be important for the hepatoprotective activity. Polyphenols contained in both fractions are considered to be potential candidates for expressing the hepatoprotective effects. The finding of antifibrotic activity in extracts from an edible mushroom is expected to be helpful in the development of hepatoprotective agents with few side effects.
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Notes
  • Michihisa Tohda, Prawpan Suwanakitch, Rattima Jeenapongsa, Hisae Hayas ...
    2004 Volume 27 Issue 12 Pages 2021-2023
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    The rat with permanent occlusion of the bilateral common carotid arteries (2VO) is useful model for the study of dementia. The expression changes of amyloid precursor protein (APP), secretase, α7 nicotinic acetylcholine receptor (α7NicR) and acetylcholine esterase (AChE), which are involved in Alzheimer's disease, were examined by quantitative RT/PCR in this model rat brain. The expression of APP, α7NicR and secretase were increased 4 d after 2VO. The α7NicR level at 2 d after operation already tended to increase. These result suggest that α7NicR expression was enhanced at early stage of brain ischemia. Using this model to find drugs which regulate the α7NicR expression will be useful to assay the materials with anti-dementive effect.
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  • Ji-Jing Yan, Jun-Sub Jung, Young-Jin Hong, Yoo-Sun Moon, Hong-Won Suh, ...
    2004 Volume 27 Issue 12 Pages 2024-2027
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    Antioxidants have been shown to be effective in murine models of sepsis. Protocatechuic acid has antioxidant activity. In the present study, the protective effects of protocatechuic acid and its derivatives were investigated in a mouse model of septic shock induced by lipopolysaccharide (LPS)/D-galactosamine (GalN). Pretreatment of animals with protocatechuic acid effectively suppressed LPS/GalN-induced lethality; protocatechuic acid isopropyl ester was the most effective among the various derivatives of protocatechuic acid. Protocatechuic acid isopropyl ester was also effective in protection against the high-dose LPS-induced shock. Pretreatment with protocatechuic acid isopropyl ester effectively suppressed the LPS/GalN-induced increase in plasma tumor necrosis factor (TNF)-α alanine aminotransferase (ALT), nitrite/nitrate levels, and hepatic malondialdehyde levels. In contrast, it markedly enhanced the LPS/GalN-induced increase in plasma interleukin (IL)-10 levels, without any changes in IL-6 plasma levels. These results suggest that protocatechuic acid isopropyl ester could be useful for the prevention of sepsis.
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  • Vanu Ramkumar Ramprasath, Palanivelu Shanthi, Panchanatham Sachdananda ...
    2004 Volume 27 Issue 12 Pages 2028-2031
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    The article relates to investigation of the anti-inflammatory effects of Semecarpus anacardium LINN. nut extract (SA), and also an anti-inflammatory drug, indomethacin, on carrageenan-induced paw edema and cotton pellet granuloma tests for their effects on acute and chronic phases of inflammation, respectively. The effect of SA on developing and developed adjuvant arthritis was also evaluated. SA significantly decreased the carrageenan-induced paw edema and cotton pellet granuloma. Indomethacin also decreased the acute and chronic phases of inflammation. SA decreased the adjuvant induced (arthritis) paw edema after the treatment, in both developing and developed adjuvant arthritis. These results indicate that the potent anti-inflammatory effect and therapeutic efficacy of Semecarpus anacardium LINN. nut extract against all phases of inflammation, is comparable to that of indomethacin.
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  • Yuhki Sato, Fumihiko Katagiri, Shin Inoue, Hiroki Itoh, Masaharu Takey ...
    2004 Volume 27 Issue 12 Pages 2032-2034
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    The herbal medicine Ninjin-to has been used for the treatment of gastroenteritis, esogastritis, gastric atony, gastrectasis, vomiting, and anorexia. One of the mechanisms of the empirical effects is assumed to be due to local changes in neuropeptide levels. Sensory afferent neurons in the gastrointestinal mucosa regulate neuropeptides [calcitonin gene-related peptide (CGRP), substance P, etc.], which play various physiologic roles. To determine whether the pharmacologic effects of Ninjin-to on the gastrointestine are due to changes in gastrointestinal mucosa regulatory peptide levels, we examined the effects of Ninjin-to 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 6.0 g of Ninjin-to caused significant increases in plasma CGRP-IS at 40 min and 60 min, and in substance P-IS levels at 90 min, compared with a placebo group. These results may indicate that the pharmacologic actions of Ninjin-to are closely related to changes in CGRP-IS and substance P-IS levels.
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Pharmacognosy
Regular Articles
  • Atsuhide Hamada, Saburo Yoshioka, Daisuke Takuma, Junko Yokota, Tailin ...
    2004 Volume 27 Issue 12 Pages 1961-1964
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    Eriobotrya japonica has been used as a medicinal plant for a long time, and its leaves are known to have many physiological actions such as anti-inflammatory, antitussive, and expectoran. In contrast, Eriobotrya japonica seeds are only known to contain amygdalin, and almost no investigations of its pharmacological action have been performed. Moreover, some anticancer agents such as adriamycin cause renal disorders as an adverse effect, and the mechanism of the adverse effect is considered to involve oxidative stress. We have reported that Eriobotrya japonica seed extract has an inhibitory effect on liver disorders. In this study, we prepared a 70% ethanol extract of Eriobotrya japonica seeds and administered the extract to rats with renal disorder induced by a single administration of 7 mg/kg body weight adriamycin, and investigated the usefulness of the extract. Increases in indices of renal function, plasma urea nitrogen, were significantly inhibited in rats treated with the Eriobotrya japonica extract compared to rats treated with tap water. In addition, the renal tissue level of reduced glutathione was significantly high in rats that ingested the extract, while the lipid peroxide levels in plasma and renal tissue were significantly low. However, no effect on renal tissue antioxidative enzymes was observed, suggesting that Eriobotrya japonica seed extract has direct antioxidative action. Based on these findings, Eriobotrya japonica seed extract may be effective in reducing the oxidative stress of adriamycin-induced renal disorder. Therefore, ingestion of Eriobotrya japonica seed extract may contribute to a reduction of the adverse effects of adriamycin.
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  • Toshitsugu Taguri, Takashi Tanaka, Isao Kouno
    2004 Volume 27 Issue 12 Pages 1965-1969
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    The antibacterial activities of 10 different plant polyphenols were evaluated by comparing their minimum inhibitory concentrations (MICs) against several food-borne pathogenic bacteria, Staphylococcus aureus (20 strains), some serotypes of the genus Salmonella (26 strains), Escherichia coli (23 strains), and some species of the genus Vibrio (27 strains). The polyphenols examined were epigallocatechin (1), epigallocatechin-3-O-gallate (2), punicalagin (3), tannic acid (4), castalagin (5), prodelphinidin (6), geraniin (7), procyanidins (8), a theaflavin mixture of black tea (9), and green tea polyphenols treated with loquat polyphenol oxidase (10). The average MICs of all polyphenols against S. aureus and the genus Vibrio (192±91 and 162±165 μg/ml, respectively) were much lower than the values against the genus Salmonella and E. coli (795±590 and 1519±949 μg/ml, respectively) (p<0.01). The coefficient of variation of the MICs of all polyphenols against S. aureus was the least and that against the genus Vibrio was the greatest. The mean MICs of each plant polyphenol against S. aureus (98—389 μg/ml) and the genus Vibrio (68—488 μg/ml) were similar. The relatively lower mean MIC values of 1, 2, 5, and 6 suggest the importance of 3,4,5-trihydroxyphenyl groups in antibacterial activity.
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  • Katarina Hostanska, Thomas Nisslein, Johannes Freudenstein, Juergen Re ...
    2004 Volume 27 Issue 12 Pages 1970-1975
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    We previously reported that the antiproliferative effect of an isopropanolic-aqueous extract of black cohosh (iCR) on MCF-7 estrogen-responsive breast cancer cell line was due to the induction of apoptosis. Here we address the question to what extent apoptosis induction can be ascribed to one of the two major fractions of iCR, the triterpene glycosides (TTG) or the cinnamic acid esters (CAE). Furthermore, as black cohosh is routinely administered orally, we studied whether its pharmacological effects would withstand simulated liver metabolism. The antiproliferative activity of TTG and CAE as well as of rat liver microsomal S9 fraction-pretreated iCR on MCF-7 cells were investigated by WST-1 assay. The features of cell death induced were tested for apoptosis by flow cytometry (light scatter characteristics, Annexin V binding). Irrespective of S9-pretreatment, 72 h iCR treatment induced a dose-dependent down regulation of cell proliferation with the same IC50 of 55.3 μg/ml dry residue which corresponds to 19.3 μg/ml TTG and 2.7 μg/ml CAE. The degree of apoptotic MCF-7 cells was also comparable. Both, isolated TTG and CAE fractions inhibited cell growth, the IC50 being 59.3 μg/ml and 26.1 μg/ml, respectively. Interestingly, whereas IC50 and apoptosis induction correspond well for the whole extract, TTG and CAE fractions induced apoptosis at concentrations (25 and 5 μg/ml) well below those required for significant growth inhibition. Observation of this study firstly showed that the cell death induced by iCR withstood a metabolic activation system. In addition, TTG and CAE compounds significantly contributed to its apoptotic effect, CAE being the more potent inhibitor of proliferation and apoptosis inducer.
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  • Jung Seop Roh, Ji Young Han, Jung Han Kim, Jae Kwan Hwang
    2004 Volume 27 Issue 12 Pages 1976-1978
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    In order to develop a new skin whitening agent, safflower (Carthamus tinctorius L.) seeds were evaluated for melanogenesis inhibitory activity and its active principles were identified following activity-guided isolation. The 80% aqueous methanol extract and ethyl acetate fraction from safflower seeds showed a significant inhibition for mushroom tyrosinase. Three active compounds, N-feruloylserotonin, N-(p-coumaroyl)serotonin, and acacetin, were isolated from the ethyl acetate fraction as the active principles. Compared with arbutin (IC50=0.223 mM), the IC50 values of these compounds were 0.023, 0.074, and 0.779 mM for N-feruloylserotonin, N-(p-coumaroyl)serotonin, and acacetin, respectively. It was also found that N-feruloylserotonin and N-(p-coumaroyl)serotonin strongly inhibited the melanin production of Streptomyces bikiniensis and B16 melanoma cells in comparison with a known melanogenesis inhibitor, arbutin.
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  • Madoka Hosoi, Michiho Ito, Toru Yagura, Robert Phillip Adams, Gisho Ho ...
    2004 Volume 27 Issue 12 Pages 1979-1985
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    cDNA cloning of a monoterpene synthase from Perilla frutescens whose steam-distilled oil contains 92.9% perillaketone, was performed by the PCR method using primers designed based on limonene synthase. The full-length nucleotide sequence of this cDNA consisted of 1978 bp including a 1827-bp translational region encoding a deduced protein of 608 amino acids, which was similar to that of limonene synthase from P. frutescens (85% identity). Functional expression of this clone in Escherichia coli yielded an active monoterpene synthase enzyme, which converted geranyl diphosphate into 53.8% myrcene, 20.9% sabinene, 19.8% linalool and 5.5% limonene. As for the extraction of reaction products, we performed SPME (solid phase micro extraction) as well as conventional solvent extraction, and compared these two extraction methods.
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Notes
  • Hyuncheol Oh, Dae-Gill Kang, Ji-Woong Kwon, Tae-Oh Kwon, Seung-Yeob Le ...
    2004 Volume 27 Issue 12 Pages 2035-2037
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    Bioassay-guided fractionation of the EtOAc-soluble extract of Sedum sarmentosum afforded a new flavonoid, quercetin-3-O-α-(6′′′-caffeoylglucosyl-β-1,2-rhamnoside) (1), along with four known flavonoids, quercetin 3-O-α-(6′′′-p-coumaroylglucosyl-β-1,2-rhamnoside) (2), isorhamnetin-3-β-glucopyranoside (3), quercetin-3-β-glucopyranoside (4), and kaempferol-3-α-arabinopyranoside (5). Purification of these compounds was conducted with the application of various chromatographic methods. Compounds 1—5 inhibited angiotensin I converting enzyme (ACE) activity in a concentration-dependent manner. Compounds 1—5 had 50% inhibitory concentration values of 158.9±11.1 μM, 351.6±3.9 μM, 408.9±4.6 μM, 708.8±23.1 μM, and 392.8±13.4 μM.
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  • Yoshiki Mino, Masaki Shirakawa, Yuichi Harada, Kazumichi Kuroda
    2004 Volume 27 Issue 12 Pages 2038-2041
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    The nucleotide sequences of partial ferredoxin (Fd)-cDNAs (corresponding to the amino acid sequence of 22—87 in the total 97 amino acids of ferredoxin) were determined for Datura arborea, D. stramonium, D. metel, and related Datura plants. Non-synonymous substitutions were noted at 4 positions and a synonymous substitution was seen at position 82 (Gln [CAA] (arborea) vs. Gln [CAG] (stramonium and metel)). The nucleotide sequence of Fd-cDNA may provide more detailed information regarding the relative taxonomic positions of plants than the amino acid sequence. However, Datura plants in the same section (metel, fastuosa, and innoxia) and of different varieties (stramonium var. stramonium and stramonium var. tatula) showed identical Fd-cDNA nucleotide sequences. This result suggests that there are very close relationships among the plants in each group.
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Biopharmacy
Regular Articles
  • Georges Da Violante, Naima Zerrouk, Isabelle Richard, Jean-Louis Frend ...
    2004 Volume 27 Issue 12 Pages 1986-1992
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    The Caco-2 cell model is a valuable tool for studying intestinal biotransformation of xenobiotics and to evaluate the potential of human intestinal absorption of new compounds. These properties were evaluated with Caco-2/TC7 cells in accelerated conditions to reduce maturation lag time from 21-d to 3-d in order to increase time and labor efficiency. Transmission electron and fluorescent microscopy were used for morphological characterization. Alkaline phosphatase and lactate dehydrogenase activities were assessed within time. Cytochrome P450 expression was studied by RT-PCR. Apparent permeabilities of a set of passively absorbed molecules across Caco-2/TC7 cell monolayers were determined to evaluate potential of both systems for prediction of human intestinal absorption. Microscopic images revealed that cells under both conditions differentiated as enterocyte-like cells but did so heterogeneously in the 3-d model. TEER values have shown that the 3-d model is a leakier cell system with higher mannitol Papp (cm/s). Biochemical characterization (hydrolase activities, CYP450 expression) suggested that the 3-d model was at a lower maturation level than the 21-d model. Carrier-mediated uptake of L-Phe was lower in the 3-d model suggesting that this model has limited application for mechanistic studies. Reasonable correlation was obtained between the two models (r2=0.88, p>0.01) for 11 passively absorbed compounds with high potential of rank ordering of compounds. Although results suggested that the 3-d cells are under-differentiated, they could be usable to estimate the oral absorption of passively absorbed compounds.
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  • Natesan Subramanian, Subhabrata Ray, Saroj Kumar Ghosal, Ranjan Bhadra ...
    2004 Volume 27 Issue 12 Pages 1993-1999
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    Celecoxib is a hydrophobic and highly permeable drug belonging to class II of biopharmaceutics classification system. Low aqueous solubility of celecoxib leads to high variability in absorption after oral administration. Cohesiveness, low bulk density and compressibility, and poor flow properties of celecoxib impart complications in it's processing into solid dosage forms. To improve the solubility and bioavailability and to get faster onset of action of celecoxib, the self-microemulsifying drug delivery system (SMEDDS) was developed. Composition of SMEDDS was optimized using simplex lattice mixture design. Dissolution efficiency, t85%, absorbance of diluted SMEDDS formulation and solubility of celecoxib in diluted formulation were chosen as response variables. The SMEDDS formulation optimized via mixture design consisted of 49.5% PEG-8 caprylic/capric glycerides, 40.5% mixture of Tween20 and Propylene glycol monocaprylic ester (3 : 1) and 10% celecoxib, which showed significantly higher rate and extent of absorption than conventional capsule. The relative bioavailability of the SMEDDS formulation to the conventional capsule was 132%. The present study demonstrated the suitability of mixture design to optimize the compositions for SMEDDS. The developed SMEDDS formulations have the potential to minimize the variability in absorption and to provide rapid onset of action of celecoxib.
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  • Noriyasu Fukuoka, Toyohisa Tsukamoto, Junji Uno, Michio Kimura, Shushi ...
    2004 Volume 27 Issue 12 Pages 2000-2005
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    This study investigated most suitable transforming factor related to the daily Phenobarbital dose (D) providing a steady-state serum concentration (Ct) and analyzed the influences of concomitant antiepileptic drugs on Ct quantitatively. Data obtained by routine therapeutic drug monitoring from a total of 326 epileptic patients treated with multiple oral administrations of phenobarbital (PB) as a powder, were used for the analysis. A total of 156 patients were administered PB alone, and 92, 57, and 21 patients were coadministered one, two, and three different antiepileptic drugs, respectively. Valproic acid (VPA), carbamazepine (CBZ), phenytoin (PHT), zonisamide (ZNS), clonazepam, and ethosuximide were coadministered with PB. For administration of PB alone, four types of transforming factor corresponding to clearance, i.e., total body weight, total body water volume, body surface area and extracellular water volume (VECW) were proposed. With VECW as a transforming factor, the level/dose (L/D) ratio (:Ct/(D/VECW)) was independent of the patient's age and gender. Ct was dependent on only one variable regarding D/VECW and expressed as Ct=0.989×(D/VECW). The coadministration of one drug caused a difference in the gradient of the regression line of PB alone, and the influence of each drug was detected by dividing each mean L/D ratio of PB plus one other drug by that of PB alone. VPA, CBZ, and PHT significantly increased (p<0.01) the L/D ratio to 1.48, 1.35, and 1.23 of the value for PB alone, respectively. With coadministration of multiple drugs, the L/D ratio rose significantly (p<0.05) as the number (≦2) of drugs coadministered increased regardless of the type, and also increased with the concomitant use of 3 drugs compared with 2 drugs. For a more detailed analysis, we defined the parameter ηi (i=1, 2, …, 6) and an alteration ratio Ri, representing the influence of each antiepileptic drug on the L/D ratio of PB alone. A model based on the assumption that each coadministered drug competitively inhibited PB-metabolizing enzyme, was adopted. The analysis clarified that VPA, CBZ, and PHT significantly increased (p<0.05) the L/D ratio of PB to 1.466, 1.177, and 1.186, respectively. In the case of the addition or discontinuance of concomitant treatment with antiepileptic drugs in the same patient, the estimated L/D ratios were calculated using the value of each Ri and compared with the measured value. The mean of prediction error was calculated as 23.1%. Our results appear valid and Ri should be available for clinical use.
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  • Mutsunobu Yoshioka, Noriaki Ohnishi, Tomokazu Koishi, Yukihisa Obata, ...
    2004 Volume 27 Issue 12 Pages 2006-2009
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    The effects of Ginkgo biloba leaf extract (GBE), a widely used herbal dietary supplement in Japan, on the pharmacokinetics and pharmacodynamics of nifedipine (NFP), a calcium-channel blocker, were studied using 8 healthy volunteers. Simultaneous oral ingestion of GBE (240 mg) did not significantly affect any of the mean pharmacokinetic parameters of either NFP or dehydronifedipine, a major metabolite of NFP, after oral administration of NFP (10 mg). However, the maximal plasma NFP concentrations in 2 subjects were approximately doubled by GBE, and they had severer and longer-lasting headaches with GBE than without GBE, with dizziness or hot flushes in combination with GBE. The mean heart rate after oral administration of NFP with GBE tended to be faster than that without GBE at every time point. Accordingly, it was concluded that GBE and NFP should not be simultaneously ingested as much as possible, and careful monitoring is needed when administering NFP concomitantly with GBE to humans.
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Notes
  • Mutsunobu Yoshioka, Noriaki Ohnishi, Noriko Sone, Suguru Egami, Koji T ...
    2004 Volume 27 Issue 12 Pages 2042-2045
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    The effects of Ginkgo biloba leaf extract (GBE), one of the most widely used herbal dietary supplements in Japan and the United States, on the pharmacokinetics of nifedipine (NFP), a typical probe of P450 (CYP) 3A, but not a substrate of the multidrug transporter P-glycoprotein (P-gp), were studied using rats. Simultaneous oral treatment with GBE (20 mg/kg) did not affect the pharmacokinetics after intravenous administration of NFP (2.5 mg/kg). However, the maximal plasma NFP concentration, the area under the concentration–time curve and absolute bioavailability after oral administration of NFP (5 mg/kg) were significantly increased by simultaneous oral treatment with GBE, approximately 1.6-fold, 1.6-fold and 2.1-fold, respectively. These results suggest that the concomitant oral use of GBE appeared to reduce the first-pass metabolism of orally administered NFP, by inhibiting CYP3A, possibly but not P-gp, in rats.
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  • Naoya Koizumi, Hiroyuki Mizuguchi, Masuo Kondoh, Makiko Fujii, Takao H ...
    2004 Volume 27 Issue 12 Pages 2046-2048
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    Recombinant adenovirus (Ad) vectors based on Ad type 5 have been widely used for gene transfer experiments. Conventional Ad type 5 vectors have a narrow range of tropism and are limited by the size of the transgene that can be packaged. To overcome these limitations, we previously developed an Ad vector (Ad5/35 vector) containing a chimeric Ad type 5 and 35 fiber protein. In the current study, we evaluated the ability of the Ad5/35 vector to transfer genes into human trophoblast cell lines (JAR, JEG-3 and BeWo cells), which are used as in vitro models of human placenta. We compared the gene transfer efficiency of Ad5/35 to that of conventional Ad vector. We found that expression of CD46, which are receptors for Ad5/35 vector, are higher than that of coxsackievirus and adenovirus receptor in all 3 trophoblast cell lines, as determined by flow cytometry. Next, we compared the transducing activity of Ad5 vector and Ad5/35 vector that each expressed luciferase as a reporter gene. Ad5/35 vector had greater gene transfer activity than the conventional Ad vector in all 3 trophoblast cell lines (1.82-fold in JAR cells, 5.37-fold in BeWo cells, 6.11-fold in JEG-3 cells). Thus, Ad vector that contains chimeric type 5 and 35 fiber protein can be a powerful tool for gene transfer experiments in human trophoblast cell lines.
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Miscellaneous
Regular Articles
  • Prithviraj Karaka, Kuppusamy Asok Kumar, Laxmi Rani Basu, Asish Dasgup ...
    2004 Volume 27 Issue 12 Pages 2010-2013
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    Dicyclomine hydrochloride is an antispasmodic agent. The MIC of dicyclomine against standard strains of Gram positive and Gram negative bacteria were performed by NCCLS broth dilution technique. These drugs showed a rapid killing action on Gram positive bacteria, Staphylococcus aureus NCTC 6571, 8530 and several other reference strains. The killing effect against Gram negative bacteria, Shigella boydii 8 NCTC 254/66 and Salmonella typhimurium NCTC 74 showed that the drug was bacteriostatic with respect to these strains. High rate of killing was achieved for most strains of Gram positive bacteria within 2 h. When administered to Swiss strain of white mice at doses of 30 and 60 μg/g of mouse, the drug could significantly protect the animals challenged with 50 MLD of Salmonella typhimurium NCTC 74. According to χ2 test, the in vivo data were highly significant (p< 0.001). Since dicyclomine showed a remarkable inhibitory action against several pathogenic bacteria, in the course of time, it may be developed as a potent antimicrobial agent for many bacterial infections.
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  • Naoki Sakane, Yoko Asano, Tomoko Kawamura, Tomohiro Takatani, Yasuhiro ...
    2004 Volume 27 Issue 12 Pages 2014-2020
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    Fetal liver (FL) hematopoiesis is thought to be important for expanding the cell number during ontogeny. In order to investigate the cellular interaction molecules among FL stromal and hematopoietic cells, we established a monoclonal antibody, Ndk-10, that reacts with FL stromal cells but not with dish non-adherent cells. When Ndk-10 was added to an FL stromal and hematopoietic cell-coculture, it inhibited the survival of c-kit+ cells. The inhibitory activity of Ndk-10 was also observed in the fetal liver organ culture. The Ndk-10 recognized a 150 kD molecule in the adherent cells of FL and kidney, and the N-terminal amino acid sequence was identical to that of mouse aminopeptidase N/CD13. The peptidase activity of CD13 was inhibited by Ndk-10, and addition of its specific inhibitor resulted in the same inhibitory activity as Ndk-10. We propose that aminopeptidase N/CD13 is a critical molecule that regulates the survival of c-kit+ cells in the FL microenvironment.
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Notes
  • Masaya Kawase, Yuka Hayashi, Fumie Kinoshita, Eiji Yamato, Jun-ichi Mi ...
    2004 Volume 27 Issue 12 Pages 2049-2051
    Published: 2004
    Released on J-STAGE: December 01, 2004
    JOURNAL FREE ACCESS
    Montmorillonite, a bioinert clay mineral, was examined as a novel vector for an oral gene-delivery system. The complex of montmorillonite and plasmid DNA encoding the enhanced green fluorescent protein (EGFP) gene was prepared at various weight ratios, and then transfected into cultured intestinal epithelial cells (IEC-6) in vitro. The EGFP gene was clearly transcribed when the transfection was performed using the montmorillonite–plasmid complex at a weight ratio of 0.05:1. In contrast, no gene expression was detected by reverse transcription-polymerase chain reaction (RT-PCR) analysis when the transfection was performed with naked plasmid. Various plasmid preparations were given orally to mice, and the gene expression in the stomach and small intestine was examined by RT-PCR. Although no gene expression was detected in the mice receiving an oral administration of naked plasmid or polyethyleneimine–plasmid complex, the EGFP gene complexed with montmorillonite was expressed in the small intestine. These results indicate that montmorillonite protected the plasmid DNA from the acidic environment in the stomach and DNA-degrading enzymes in the intestine, and successfully delivered it into cells of the small intestine.
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