Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Volume 33 , Issue 8
Showing 1-33 articles out of 33 articles from the selected issue
Analytical Biochemistry
Regular Articles
  • Xiao-Guang Gong, Yun-Fu Lv, Xin-Qiu Li, Fu-Guo Xu, Qing-Yong Ma
    Type: Regular Article
    Subject area: Analytical Biochemistry
    2010 Volume 33 Issue 8 Pages 1261-1267
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    Pancreatic cancer is the fourth leading cause of cancer-related death in the western countries and it is resistant to almost all cytotoxic drugs. In the current study, we explored the gemcitabine resistance induced by the interaction between Annexin A2 (ANXA2) and alternatively spliced segment of tenascin-C (TNfnA-D). In the pancreatic cancer cell culture system in vitro, it was proved that exogenous recombinant TNfnA-D combined with the cell surface ANXA2 specifically and their interaction suppressed gemcitabine-induced cytotoxicity on pancreatic cancer cells in a dose-dependent manner. Meanwhile, the TNfnA-D/ANXA2 interaction increased the phosphorylation of phosphatidylinositol 3-kinase (PI3K), Akt, inhibitory κB (IκB) kinase α/β (IKKα/β), IκBα, and p65 nuclear factor-κB (NF-κB) significantly. Inhibition of Akt and PI3K with their specific inhibitors partially reversed the suppression of gemcitabine-induced cytotoxicity elicited by TNfnA-D/ANXA2 interaction. Activation of p65 NF-κB was dependent on the phosphorylation of PI3K/Akt. The phosphorylated IKKα/β induced the phosphorylation and degradation of IκBα, the sequential phosphorylation, nuclear translocation and activation of p65 NF-κB. Pyrrolidine dithiocarbamate (PDTC) effectively blocked the activity of p65 NF-κB in response to TNfnA-D. Down-regulation of p65 NF-κB with its specific small interfering RNA (siRNA) restored the gemcitabine-induced cytotoxicity suppressed by TNfnA-D/ANXA2 interaction. For the first time, this study show that ANXA2/TNfnA-D interaction induced gemcitabine resistance via the canonical PI3K/Akt/NF-κB signaling pathways in pancreatic cancer cells. Therefore, therapy targeting ANXA2/TNfnA-D and/or p65 NF-κB may have potential clinical application for patients with pancreatic cancers.
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  • A-Rang Im, Youmie Park, Yeong Shik Kim
    Type: Regular Article
    Subject area: Analytical Biochemistry
    2010 Volume 33 Issue 8 Pages 1268-1273
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    Chondroitin sulfate (CS) is a glycosaminoglycan that composed of hexosamine (D-galactosamine) and hexuronic acid (D-glucuronic acid) unit arranged in an alternating unbranched sequence. CS is an essential component of the extracellular matrix (ECM) of connective tissue. It is mainly covalently attached to core proteins in the form of proteoglycans so that it exhibits specific interactions with proteins for cell growth, differentiation, division and migration. In this study, CSs were purified from the cartilage and backbone of sturgeon (Acipenser sinensis). To characterize their biochemical properties, we performed disaccharide compositional analysis after chondroitinase ABC digestion, high performance size exclusion chromatography (HPSEC) and 1H-NMR spectroscopy. We also investigated the effects of CSs on fibroblast proliferation and adhesion to determine whether wound healing was accelerated in vitro and proliferation of different mitogen-activated protein kinases (MAPK) signaling pathways was facilitated. The CS purified from sturgeon cartilage was primarily composed of 4-sulfated CS (88.8%) and sturgeon backbone CS contains more than 60% 6-sulfated CS. The average molecular weights of CSs obtained from sturgeon cartilage and backbone were found to be 8 and 43 kDa, respectively. Our results showed that both CSs are able to increase cell adhesion, induce proliferation and migration on fibroblasts and may accelerate wound healing by inducing MAPK signaling pathways.
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Note
  • Atsushi Hirano, Masaaki Takahashi, Eri Kinoshita, Masaaki Shibata, Tos ...
    Type: Note
    Subject area: Analytical Biochemistry
    2010 Volume 33 Issue 8 Pages 1426-1429
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    Etravirine (TMC-125, ETV) is a second-generation non-nucleoside reverse transcriptase inhibitor (NNRTI) that demonstrates potent activity against NNRTI-resistant strains of human immunodeficiency virus type-1 (HIV-1). Thus, ETV has been used in combination with ritonavir-boosted protease inhibitor (PI) and integrase inhibitor for therapy-experienced HIV-1-infected patients. On the other hand, as ETV is a substrate and inducer of cytochrome P450 3A4 (CYP3A4), ETV may induce metabolism of PI and alter the concentrations of co-administered PIs. In order to ensure optimal drug efficacy and prevention of resistance, it is essential to monitor plasma concentrations of ETV and PIs. Here we describe the application of HPLC with UV detection for the simulataneous assay of ETV and 4 PIs, darunavir (DRV), atazanavir (ATV), ritonavir (RTV) and lopinavir (LPV). In this study, the calibration curve of each drug was linear with the average accuracy ranging from 93.6 to 110.9%. Both intra- and interday coefficients of variation for each drug were less than 11.6%. The mean recovery of all drugs ranged from 88.0 to 97.5%. The limit of quantification was 0.04, 0.04, 0.04, 0.05 and 0.07 μg/ml for ETV, DRV, ATV, RTV and LPV, respectively. These results demonstrate that our HPLC-UV method can be used for routine determination of plasma concentrations of ETV and 4 PIs in clinical settings.
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Biochemistry
Regular Articles
  • Qi Zhao, Xiaohong Cao, Bin Zeng, Chunling Wang, Le Yan, Chengjian Xu
    Type: Regular Article
    Subject area: Biochemistry
    2010 Volume 33 Issue 8 Pages 1274-1278
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    A lectin from Musca domestica larva was purified by affinity chromatography on a glactose-Sepharose 4B column. Musca domestica larva lectin (MLL) inhibited the growth of BEL-7402 cells in a time and concentration-dependent way. The results of Hoechst 33258 staining indicated that MLL induce BEL-7402 cells apoptosis based on the typical apoptotic morphological changes. Subsequently, we found that MLL treatment abrogated glutathione antioxidant system and induced mitochondria to generate reactive oxygen species (ROS) accumulation, resulting in reduction of mitochondrial transmembrane potential. The induction of cell apoptosis was caused by the upregulation of Bax, the downregulation of Bcl-2, the cytochrome c release and the activation of the caspases pathways.
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  • Jeong Hwan Kim, Soo-Jin Jeong, Hee-Young Kwon, Sang Yoon Park, Hyo-Jun ...
    Type: Regular Article
    Subject area: Biochemistry
    2010 Volume 33 Issue 8 Pages 1279-1284
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    Adverse effects, nephrotoxicity and hepatotoxicity, of anticancer drugs such as cisplatin have limited the usage for cancer therapy. Therefore, development or identification of supplement agents in anticancer drugs is attractive to reduce side effects and enhance antitumor activity. Here, we found that decursin isolated from Angelica gigas showed protective effects of cisplatin-induced damage in normal human primary renal epithelial cells (HRCs). We found that decursin significantly blocked cisplatin-induced cytotoxicity by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay in HRCs. Further, we found that decursin inhibited sub-G1 and cell death by suppression of cleavage of caspase-3, -9 and poly(ADP-ribose) polymerase (PARP) induced by cisplatin treatment in HRCs. Importantly, decursin effectively restored the activities of Cu/Zn superoxide dismutase (SOD), catalase and glutathione peroxidase in cisplatin-treated HRCs. Taken together, our findings demonstrate that decurcin prevents cisplatin-induced cytotoxicity and apoptosis through the activation of antioxidant enzymes in HRCs and suggest further that combination of decursin might suppressed adverse effects of anticancer drugs in cancer patients.
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Note
  • Naoko Nishimura, Masayuki Takai, Eisaku Yamamoto, Keiji Hasumi
    Type: Note
    Subject area: Biochemistry
    2010 Volume 33 Issue 8 Pages 1430-1433
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    Plasma hyaluronan-binding protein (PHBP), a serine protease that can activate coagulation factor VII and prourokinase, circulates as a single-chain form (pro-PHBP), and is autoproteolytically converted to an active two-chain form with the aid of an effector such as spermidine and heparin. In this study, we screened natural sources for inhibitors of spermidine-induced pro-PHBP autoactivation. As an active agent, we purified bikaverin from a culture of a fungus. Bikaverin inhibited spermidine-induced autoactivation with an IC50 of 0.45 μM, while it also inhibited the active form of PHBP (IC50=0.8 μM). Additional screening of related compounds led to the identification of purpurin, a plant anthraquinone, as a specific inhibitor: IC50=6.6 μM for spermidine-induced autoactivation; no inhibition of heparin-induced autoactivation and active PHBP. Alizarin and emodin, which structurally differed from purpurin in the position or the number of the hydroxyl groups, were less active and nonspecific. Thus, the position and/or the number of the hydroxyl group affect both the potency and selectivity of the anthraquinone inhibitors.
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Molecular and Cell Biology
Regular Articles
  • Shujun Li, Hai Shi, Fuyun Ji, Biaoluo Wang, Quanxin Feng, Xiangying Fe ...
    Type: Regular Article
    Subject area: Molecular and Cell Biology
    2010 Volume 33 Issue 8 Pages 1285-1290
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    This study aimed to investigate the mechanism by which the human lung cancer drug resistance-related gene BC006151 regulates chemosensitivity by down-regulating BC006151 expression via antisense gene transfer in H446/CDDP cells. A retroviral vector containing the antisense BC006151 sequence was constructed and transfected into H446/CDDP cells. Transfection of the empty vector served as a negative control. The two groups of transfected cells were treated with various chemotherapeutic agents, after which morphological changes in cell ultrastructure were compared by transmission electron microscopy, cell proliferation and chemosensitivity to particular chemotherapeutic agents were compared by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method, the effects of chemotherapy on cell cycle and apoptosis were compared by flow cytometry, and Bcl-2 was evaluated by immunohistochemistry and Western blot analysis. Results showed that apoptotic body-like structures were identified by transmission electron microscopy in the antisense gene-transfected cells. MTT founded that these cells exhibited a significantly lower level of proliferation than the control cells (p<0.01), together with a markedly increased sensitivity to various chemotherapeutic agents (p<0.01). Flow cytometry analysis revealed that a G1 phase arrest accounted for the reduction in proliferation in the antisense gene-transfected cells; increased apoptosis was also detected (p<0.01). Both immunohistochemistry and western blot analysis confirmed that Bcl-2 expression was significantly down-regulated in the antisense gene-transfected cells compared to controls. In a word, down-regulation of BC006151 can significantly inhibit proliferation and increase apoptosis of H446/CDDP cells after chemotherapy, and this gene may play an important role in the development of multidrug resistance in lung cancer.
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  • Achinto Saha, Takashi Kuzuhara, Noriko Echigo, Atsuko Fujii, Masami Su ...
    Type: Regular Article
    Subject area: Molecular and Cell Biology
    2010 Volume 33 Issue 8 Pages 1291-1299
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    The expression of “growth arrest and DNA damage inducible genes 45 and 153” is related to apoptotic induction of cells. GADD45 is an effector gene of the tumor suppressor p53, and GADD153 is associated with cellular function of cancer prevention. Curcumin, isolated from the plant Curcuma longa (LINN), has been investigated as a promising cancer preventive in food because curcumin, a phenolic and coloring compound, is widely ingested in the Indian subcontinent. However, the exact mechanisms of action of curcumin have not yet been clearly elucidated. Based on our successful results with green tea catechins as cancer preventive, we studied the relationship between the expression of GADD45 and 153 and apoptotic induction in human lung cancer cell line PC-9. In our study curcumin increased the expression of GADD45 and 153 in a p53-independent manner. Curcumin also inhibited the growth of PC-9 cells and induced G1/S arrest of the cell-cycle followed by strong induction of apoptosis. Treatment with GADD45 and 153 small interfering RNAs (siRNAs) inhibited the apoptotic induction in PC-9 cells by curcumin. Moreover, curcumin induced the expression of cyclin dependent kinase inhibitor genes p21 and p27, while it inhibited the expression of numerous genes, including Bcl-2, cyclin D1, CDK2, CDK4 and CDK6. All the results with PC-9 cells suggest that the up-regulation of GADD45 and 153 by curcumin is a prime mechanism in the anticancer activity of curcumin.
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Microbiology
Regular Article
  • Ya-li Lv, Fu-sheng Zhang, Juan Chen, Jin-long Cui, Yong-mei Xing, Xian ...
    Type: Regular Article
    Subject area: Microbiology
    2010 Volume 33 Issue 8 Pages 1300-1306
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    Endophytic fungi are rich in species diversity and may play an important role in the fitness of their host plants. This study investigated the diversity and antimicrobial potential of endophytic fungi obtained from Saussurea involucrata KAR. et KIR. A total of 49 endophytic fungi were isolated from S. involucrata and identified using morphological and molecular techniques. Extracts of fermentation broth from the 49 fungi were tested for antimicrobial activity against pathogenic microorganisms using the agar diffusion method. Forty-eight out of the 49 endophytic fungi were identified and grouped into 14 taxa. Cylindrocarpon sp. was the dominant species isolated from S. involucrata, followed by Phoma sp. and Fusarium sp. Among the 49 endophytic fungi, 9 root isolates having darkly pigmented, septate hyphae were identified as dark septate endophytic (DSE) fungus, and 12 fungi inhibited at least one test microorganism. Moreover, 5 strains showed a broader spectrum of antimicrobial activity and 4 strains displayed strong inhibition (+++) against pathogenic fungi. The results indicate that endophytic fungi isolated from S. involucrata are diverse in species and a potential source of antimicrobial agents.
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Pharmacology
Regular Articles
  • Shao-Rui Chen, Xiang-Zhen Xu, Yu-Hua Wang, Jian-Wen Chen, Suo-Wen Xu, ...
    Type: Regular Article
    Subject area: Pharmacology
    2010 Volume 33 Issue 8 Pages 1307-1313
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    In this study we investigated the anti-inflammatory effects of an icariin derivative (3,5-dihydroxy-4′-methoxy-6″,6″-dimethy1-4″,5″-dihydropyrano[2″,3″:7,8]-flavone). We found that this icariin derivative inhibits tumor necrosis factor-α (TNF-α) production, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expression, and protein expression in lipopolysaccharide (LPS) stimulated RAW264.7 macrophages. It also alleviates paw edema induced by carrageenan in mice. To clarify the molecular mechanisms underlying these anti-inflammatory effects, we examined the effects of this compound on the phosphorylation of mitogen-activated protein kinase (MAPK), phosphorylation of inhibitory kappaBalpha (IκBα), and nuclear translocation of p65 subunit of nuclear factor (NF)-κB, and found it suppresses the activation of p38 MAPK and inhibits translocation of NF-κB p65 to the nucleus through decreasing the phosphorylation of IκBα. As a result of these properties, this icariin derivative can be considered as a potential drug for inflammatory diseases.
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  • Daisuke Shii, Shizu Nakagawa, Miwa Yoshimi, Osamu Katsuta, Tomoko Oda, ...
    Type: Regular Article
    Subject area: Pharmacology
    2010 Volume 33 Issue 8 Pages 1314-1318
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    We investigated the efficacy of cyclosporine A (CyA) eye drops on ocular symptoms in late phase and delayed-type reactions in guinea pig allergic conjunctivitis models. An emulsion of ovalbumin (OVA) and Freund's complete adjuvant (FCA) was intraperitoneally injected into guinea pigs, and 15% OVA solution was applied topically to the eyes to elicit late phase reactions. Following the early phase reaction, increased scores for hyperemia, swelling, edema, and discharge were detected 6 h after antigen challenge, and CyA eye drops significantly inhibited the increase in scores for edema and discharge, the increase in the number of infiltrating inflammatory cells, and the percentage of eosinophils among polymorphonuclear leukocytes in conjunctival tissue. To induce delayed-type reactions, guinea pigs were sensitized by injecting FCA into the footpad, followed by injections of purified protein derivative into palpebral conjunctivae 24 d later. Increased scores for hyperemia, swelling, and discharge were detected 6 h after the induction of delayed-type allergy, and CyA eye drops significantly inhibited the increase in scores for hyperemia and swelling. In contrast, betamethasone sodium phosphate eye drops showed a tendency to inhibit the symptoms in both late phase and delayed-type reactions, or inflammatory cell infiltration in the late phase reaction, but the inhibition was not significant. These results suggest that CyA eye drops are useful for suppressing ocular symptoms in both late phase and delayed-type reactions in allergic conjunctivitis models.
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  • Romina Nassini, Eunice Andrè, David Gazzieri, Gaetano De Siena, ...
    Type: Regular Article
    Subject area: Pharmacology
    2010 Volume 33 Issue 8 Pages 1319-1323
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    Ingestion of elevated amounts of ethanol in humans and rodents induces hemorrhagic gastric lesions, at least in part by increasing oxidative stress. The present study was undertaken in order to evaluate the influence of a bicarbonate-alkaline mineral water (Uliveto®) on ethanol-induced hemorrhagic gastric lesions in mice. Lesions were evaluated by both macroscopic and microscopic analysis. In a first set of experiments, mice were allowed to drink Uliveto® or reference water ad libitum until 3 h prior to intragastric (i.g.) ethanol (23 ml/kg) administration. Neither Uliveto® nor reference water did afford any protection. In a second set of experiments, acute exposure to reference water (35 ml/kg, i.g.), given 30 min before ethanol, did not inhibit gastric lesions. However, administration of the same amount of Uliveto® caused a remarkable reduction in ethanol-evoked gastric lesions. Ethanol administration increased 4-hydroxy-2-nonenal levels, a byproduct of oxidative stress, in the luminal part of the gastric mucosa. This response was substantially reduced by about 70% by Uliveto®, but not by reference water. Reference water, added with the bicarbonate content, present in the Uliveto® water, protected against ethanol-induced lesions. Thus, acute pre-exposure to bicarbonate-alkaline mineral water (Uliveto®) protects from both oxidative stress and hemorrhagic gastric lesions caused by ethanol. The elevated bicarbonate content of Uliveto® likely accounts for the protection against ethanol-induced gastric injury.
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  • Ya Liu, Hai-Gang Zhang, Yi Jia, Xiao-Hui Li
    Type: Regular Article
    Subject area: Pharmacology
    2010 Volume 33 Issue 8 Pages 1324-1330
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    Panax notoginseng saponins (PNS) are highly valued traditional Chinese medicine. The effects of PNS (120 mg/kg, once daily administrated intragastrically (i.g.)) on atherosclerosis induced by a high-cholesterol diet and chronic inflammation, which was derived through zymosan (10 mg/kg, once every 2 d) administration intraperitoneally, were evaluated in rabbits for 8 weeks. A normal group, a simple high-fat diet group, and a zymosan plus high-cholesterol diet group (Zym) were used as controls. Typical pathologic changes associated with atherosclerosis in rabbits following induction by zymosan were alleviated by PNS treatment. After 2, 4, 6, and 8 weeks of treatment, PNS decreased the serum levels of total cholesterol, triglyceride, low-density lipoprotein cholesterol, interleukin-6 and C-reactive protein as well as increased high-density lipoprotein cholesterol level significantly in comparison with those in the Zym group, except for triglycerides at week 2. In addition, PNS treatment significantly decreased the mRNA expression levels of monocyte chemoattactant protein-1 and nuclear factor-κB/p65 in the aorta wall after 8 weeks of treatment compared with the Zym group. In conclusion, PNS attenuates atherogenesis through an antiinflammatory action and regulation of the blood lipid profile.
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  • Gurpreet Kaur, Amteshwar Singh Jaggi, Nirmal Singh
    Type: Regular Article
    Subject area: Pharmacology
    2010 Volume 33 Issue 8 Pages 1331-1336
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    The present study was designed to investigate the ameliorative potential of pralidoxime in tibial and sural nerve transection-induced neuropathy in rats. Tibial and sural nerve transection was performed by sectioning tibial and sural nerve portions (2 mm) of the sciatic nerve, and leaving the common peroneal nerve intact. The pinprick, acetone, hot and cold tail immersion tests were performed to assess the degree of motor functions, mechanical hyperalgesia, cold allodynia, heat and cold hyperalgesia respectively. Biochemically, the tissue thio-barbituric acid reactive species (TBARS), super-oxide anion contents (the markers of oxidative stress) and total calcium levels were measured. Tibial sural nerve transection resulted in the development of mechanical hyperalgesia, cold allodynia, heat and cold hyperalgesia along with the rise in oxidative stress and calcium levels. However, administration of pralidoxime (10, 20 mg/kg intraperitoneally (i.p.)) for 14 d attenuated tibial and sural nerve transection-induced cold allodynia, mechanical, hot and cold hyperalgesia. Furthermore, pralidoxime also attenuated tibial and sural nerve transection induced increase in oxidative stress and calcium levels. It may be concluded that pralidoxime has ameliorative potential in attenuating the painful neuropathic state associated with tibial and sural nerve transection, which may possibly be attributed to decrease in oxidative stress and calcium levels.
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  • Guang Zeng, Tao Tang, Han-Jun Wu, Wan-Hui You, Jie-Kun Luo, Yuan Lin, ...
    Type: Regular Article
    Subject area: Pharmacology
    2010 Volume 33 Issue 8 Pages 1337-1342
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    Parkinson's disease (PD) is associated with mitochondrial dysfunction, oxidative stress, and activation of the apoptotic cascade. In the study, we investigated the effects of salvianolic acid B (Sal B) on 1-methyl-4-phenylpyridinium (MPP+)-treated SH-SY5Y cells, a classic in vitro model for PD. We found Sal B inhibited the loss of cell viability by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The underlying mechanisms of Sal B action were further studied. Treatment of SH-SY5Y cells with MPP+ caused a loss of cell viability and mitochondrial membrane potential, condensation of nuclei, elevation in the level of reactive oxygen species (which was associated with cytochrome c release), an increase in the Bax/Bcl-2 mRNA ratio, and activation of caspase-3. Sal B ameliorated the MPP+-altered phenotypes. These results indicate that the Sal B protected SH-SY5Y cells against MPP+-induced apoptosis by relieving oxidative stress and modulating the apoptotic process. Our findings suggest that salvianolic acid B may be a promising agent to prevent PD.
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  • Houli Cao, Qian Feng, Wei Xu, Xiaorong Li, Zhuang Kang, Yanqin Ren, Li ...
    Type: Regular Article
    Subject area: Pharmacology
    2010 Volume 33 Issue 8 Pages 1343-1348
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    Genipin is a metabolite of geniposide isolated from an extract of Gardenia fructus. Some observations suggested that genipin could induce cell apoptosis in hepatoma cells and PC3 human prostate cancer cells. However, the effects of genipin on HeLa human cervical carcinoma cells are still unknown. In this study, we provided evidences that genipin induced the death of HeLa cells through apoptotic pathway in a dose-dependent manner. Genipin could remarkably induce cytotoxicity in HeLa cells and inhibit its proliferation. Induction of the apoptosis by genipin was confirmed by analysis of DNA fragmentation and induction of sub-G1 peak through flow cytometry. The results also showed that genipin-treated HeLa cells cycle was arrested at G1 phase. Western blot analysis revealed that the phosphorylated c-Jun NH2-terminal kinase (JNK) protein, phospho-Jun protein, p53 protein and bax protein significantly increased in a dose-dependent manner after treatment of genipin for 24 h, and to our knowledge, the activation of JNK maybe result in the increase of the p53 protein level, and the increase of the p53 protein led to the accumulation of bax protein, bax protein further induced cell apoptotic death eventually. Taken all these together, it is possible to develop genipin as an anti-cancer drug.
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Notes
  • Susumu Ohkawara, Toshiko Tanaka-Kagawa, Yoko Furukawa, Tetsuji Nishimu ...
    Type: Note
    Subject area: Pharmacology
    2010 Volume 33 Issue 8 Pages 1434-1437
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    Transient receptor potential vanilloid subtype 1 (TRPV1) is a non-selective cation channel activated by capsaicin. TRPV1 is expressed not only on human sensory neurons but also on human epidermal and hair follicle keratinocytes. Therefore, TRPV1 could have the potential to be a therapeutic target for skin disorders. To search for novel TRPV1 agonists, we screened 31 essential oils by using human TRPV1-expressing HEK293 cells. TRPV1 was activated by 4 essential oils: rose, thyme geraniol, palmarosa, and tolu balsam. The dose–response curves for TRPV1 activation by the essential oils revealed a rank order potency [the half-maximal effective concentration (EC50)] of rose>palmarosa>thyme geraniol>tolu balsam, and rank order efficiency (% activity in response to 1 μM capsaicin) of tolu balsam>rose>palmarosa>thyme geraniol. Moreover, the dose–response curves for TRPV1 activation by citronellol (main constituent of rose oil) and geraniol (main constituent of thyme geraniol and palmarosa oils) were consistent with the potency and efficiency of each essential oil. In contrast, benzyl cinnamate and benzyl benzoate (main constituent of tolu balsam oil) and geranyl acetate (main constituent of thyme geraniol oil) did not show TRPV1 activity. In this first-of-its-kind study, we successfully investigated the role of some essential oils in promoting human TRPV1 activation, and also identified two monoterpenes, citronellol and geraniol, as new human TRPV1 agonists.
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  • Chang-qing Yang, Peng-yuan Sun, Da-zhi Ding, Hiroshi Moriuchi, Yoichi ...
    Type: Note
    Subject area: Pharmacology
    2010 Volume 33 Issue 8 Pages 1438-1442
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    The effects of Sho-seiryu-to (TJ-19), an ethical Kampo formulation, on bleomycin (BLM)-induced pulmonary fibrosis in rats was examined. Pulmonary fibrosis was induced by intratracheal instillation of a single dose of BLM (5 mg/kg). The TJ-19 used consisted of at least 21 constituents, as determined by three-dimensional HPLC analysis, and was administered orally twice a day at a dose of 1.5 g/kg until the end of the study period. Changes in general appearance and body weight were monitored. Twenty-eight days after BLM instillation, the animals were sacrificed and the study parameters were measured. TJ-19 attenuated the loss in body weight, increase in lung/body weight ratio and concentration of hydroxyproline and malondialdehyde in the lung tissues induced by BLM administration. TJ-19 also prevented BLM-induced fibrotic changes in the lung histology. These protective effects of TJ-19 were observed when administration was started 1 week before and simultaneously with the instillation of BLM. These results suggest that TJ-19 has prophylactic potential against BLM-induced pulmonary fibrosis, and may therefore be a promising drug candidate and medicinal resource for preventing BLM-induced and idiopathic pulmonary fibrosis.
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Medicinal Chemistry
Regular Article
  • Yayoi Saegusa, Chiharu Sadakane, Junichi Koseki, Yoshihiro Hasegawa, S ...
    Type: Regular Article
    Subject area: Medicinal Chemistry
    2010 Volume 33 Issue 8 Pages 1349-1354
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    Transforming growth factor-β1 (TGF-β1) plays an important role in the development of glomerulonephritis. The study of experimental glomerulonephritis in rats was performed to examine the antinephritic effects of TJN-331, a new herbally-derived chemical compound. To clarify the action of TJN-331 ((E)-N-(3,4-dimethoxyphenethyl)-N-methyl-3-(3-pyridyl)-2-propenamide) on TGF-β1 production, glomeruli were isolated from rats with antiglomerular basement membrane (GBM) nephritis and incubated for 48 h with test drugs in vitro. Next, we examined the effects of TJN-331 on rat anti-GBM nephritis induced by injection with anti-GBM serum. TJN-331 dose-dependently inhibited the increase in total and mature TGF-β1 production from nephritic glomeruli, although it did not inhibit TGF-β1 production from normal glomeruli. Administration of TJN-331, at a dose of 2 mg/kg/d, per os (p.o.), prevented proteinuria and increased crescent formation and adhesion of capillary walls to Bowman's capsule. The increases in mature TGF-β1 protein production and TGF-β1 staining score in nephritic rats were reversed by TJN-331 treatment. These results suggest that TJN-331 inhibits proteinuria and histopathological changes in glomeruli via suppression of TGF-β1 production from inflamed glomeruli.
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Pharmacognosy
Regular Articles
  • Kazuko Yoshikawa, Naoki Kokudo, Toshihiro Hashimoto, Kyosuke Yamamoto, ...
    Type: Regular Article
    Subject area: Pharmacognosy
    2010 Volume 33 Issue 8 Pages 1355-1359
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    Sparassis crispa (SC), known as Hanabiratake in Japanese, is an edible mushroom with various medicinal properties. We isolated 3 novel phthalides, designated hanabiratakelide A (1), B (2), and C (3), from the SC fruit body. In this investigation, 3 known phthalides (4—6), ubiquinone-9, and 2 known unsaturated fatty acids were also isolated. Their structures were elucidated primarily through extensive NMR experiments. The isolated compounds 1—6 were tested for their anti-oxidant activity. The in vitro superoxide dismutase-like activity of the 3 hanabiratakelides was stronger than that of vitamin C. The compounds also exerted inhibitory effects on lipopolysaccharide-stimulated nitric oxide and prostaglandin E2 production by a murine macrophage cell line, RAW264. In addition, the growth of the colon cancer cell lines Caco-2 and colon-26 was significantly inhibited by treatment with the 3 hanabiratakelides. In vivo, the frequency of azoxymethane-induced aberrant crypt foci was reduced in SC-fed F344/N rats compared to rats fed a standard diet. In conclusion, 3 novel phthalides, hanabiratakelides, derived from SC were shown to possess anti-oxidant, anti-inflammatory, and anti-tumor activity.
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  • Eun-Young Kim, Jung-Hyun Kim, Mee-Ra Rhyu
    Type: Regular Article
    Subject area: Pharmacognosy
    2010 Volume 33 Issue 8 Pages 1360-1363
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    Ligusticum wallichii is an herb widely used to treat vascular disorders in Asian countries, and tetramethylpyrazine (TMP) has been identified as one of its vasorelaxant active components. This study was performed to examine the endothelium-independent relaxation produced by the butanol-soluble fraction of L. wallichii extract (LwBt) and its possible mechanisms of action in isolated rat aortic rings. The effects were compared with those of TMP. LwBt produced vasorelaxation that increased gradually after 2—3 min of LwBt administration and reached a maximum within 30 min. LwBt-induced relaxation was significantly attenuated by pretreatment with 4-aminopyridine and apamin. Additionally, LwBt attenuated CaCl2-induced vasoconstriction in high-potassium depolarized medium. Thus, LwBt-induced vasorelaxation apparently involved inhibition of calcium influx, mediated by the opening of voltage-dependent and/or Ca2+-activated potassium channels. On the other hand, the effect of TMP was significantly attenuated by pretreatment with glibenclamide, and 4-aminopyridine had no effect. In conclusion, LwBt-induced endothelium-independent vasorelaxation was mediated by the opening of voltage-dependent potassium channels, while TMP-induced relaxation was mediated by the opening of ATP-dependent potassium channels. These effects of LwBt may be due to a substance other than TMP.
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Notes
Biopharmacy
Regular Articles
  • Yi-Dong Yan, Jong Soo Woo, Joon Heok Kang, Chul Soon Yong, Han-Gon Cho ...
    Type: Regular Article
    Subject area: Biopharmacy
    2010 Volume 33 Issue 8 Pages 1364-1370
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    The purpose of this research was to prepare and evaluate a non-bitter donepezil hydrochloride (DH) orally disintegrating tablet (ODT) for enhanced patient compliance. Taste masking was done by preparing microspheres with different ratios of drug and Eudragit® EPO using spray drying method. The entrapment of the drug into microspheres was confirmed by scanning electron microscope (SEM) and X-ray powder diffraction. It was found that microspheres with a drug–polymer ratio of 1 : 2 could mask the taste obviously by inhibiting the release of DH in simulated salivary fluid. Microspheres-loaded tablets containing Polyplasdone NF and Low substituted Hydroxypropyl Cellulose (L-HPC) both at a 10% level showed rapid disintegration, in vitro (15.5 s) and in vivo (19.8 s), which were faster than that of marketed tablets (36.7, 41.3 s, respectively). Results from taste evaluation in human volunteers revealed that the ODTs with taste-masked microspheres had significantly enhanced palatability. Dissolution in vitro and pharmacokinetics in rats were evaluated for the tested ODTs compared to the donepezil hydrochloride commercial product (ARICEPT®). Both tablets showed comparable dissolution patterns in vitro and similar area under curve from 0 to 24 h (AUC0—24), Cmax and Tmax of DH in vivo to each other, suggesting that the tested ODTs might give the similar drug efficacy in rats compared to that of ARICEPT®. Thus, it was concluded that DH ODTs with masked taste were obtained by Eudragit® EPO-based microspheres, drug loaded microspheres neither decreased the bioavailability nor delayed the release of DH.
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  • Wen-Jun Yao, Kao-Xiang Sun, Yi Liu, Na Liang, Hong-Jie Mu, Chen Yao, R ...
    Type: Regular Article
    Subject area: Biopharmacy
    2010 Volume 33 Issue 8 Pages 1371-1377
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    The aim of this study was to investigate the effect of Poly(amidoamine) (PAMAM) dendrimers on corneal permeation of puerarin (PUE). Permeation studies were performed using excised cornea of rabbits by a Valia–Chien diffusion apparatus. Drug-treatment studies were carried out by measuring the penetration of puerarin on cornea in PAMAM–PUE physical mixture or PAMAM–PUE complex, and cornea-treatment studies were carried out by measuring the penetration of puerarin on PAMAM dendrimer pretreated cornea in puerarin solution. The results showed that the permeability coefficient of puerarin in PAMAM–PUE physical mixture was enhanced by 2.48 (G3), 1.99 (G4) and 1.36 (G5) times on average, respectively compared to control. However, no significant permeability enhancement of puerarin in PAMAM–PUE complex was found compared to control. This may attribute to free drug concentration was lower in PAMAM–PUE complex which served as a depot and exhibited slow-released behavior of drug. Cornea-treatment studies showed that the lag time of puerarin was decreased, while the cumulative amount within 2.5 h (Q2.5) and the permeability coefficient of puerarin increased compared to control. The permeability coefficient of puerarin was linear correlated to the molecular weight of PAMAM dendrimer (r2=0.99). This indicates that higher generation of PAMAM dendrimer more easily interact with cornea or loosen the epithelium cell junctions than lower generation to increase the flux of puerarin. Overall, the study showed that PAMAM dendrimer increased the corneal permeation of puerarin mainly by altering the corneal barrier.
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  • Masako Saito, Junichi Kawana, Tetsuro Ohno, Kazuhiko Hanada, Masahiro ...
    Type: Regular Article
    Subject area: Biopharmacy
    2010 Volume 33 Issue 8 Pages 1378-1384
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    Carvedilol is a β-adrenoceptor antagonist used for treating chronic heart failure (CHF). Two clinical studies were conducted to evaluate the population pharmacokinetics and pharmacodynamics of R- and S-carvedilol, and associated covariates, in patients with CHF. Fifty-eight patients (male=45, female=13) with New York Heart Association class I—IV CHF were enrolled in two clinical studies. R- and S-carvedilol concentrations were measured using HPLC at steady-state after oral administration of carvedilol at 1.25—20 mg o.d. or b.i.d. The data from both studies were used to estimate the population pharmacokinetic parameters and covariates using the nonlinear mixed effects model program. For 40 patients evaluated in one clinical study, the cytochrome P450 (CYP)2D6 *1, *10, and *5 genotypes were determined using allele-specific primer PCR, and individual patients' oral clearance (CL/F) of both enantiomers were estimated by the empirical Bayes method. A one-compartment model with a first-order absorption rate was established, in which body weight and α1-acid glycoprotein were significant covariates. Individual CL/F values for carvedilol were significantly lower in Japanese CHF patients with the CYP2D6 *1/*5, *5/*10 and *10/*10 genotypes. Estimation of the population pharmacokinetic parameters and their covariates for each enantiomer in Japanese patients with CHF showed that the CL/F values for R- and S-carvedilol were dependent on body weight, α1-acid glycoprotein, and CYP2D6 genotype. Prediction of exposure to free plasma carvedilol is important for dosage adjustment of β-blocker therapy in patients with CHF.
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  • Shinji Sato, Keiji Fukumura, Ayae Nishiyama, Ikuo Kanamoto, Yutaka Ino ...
    Type: Regular Article
    Subject area: Biopharmacy
    2010 Volume 33 Issue 8 Pages 1385-1393
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    Glycemic index (GI) is an indicator of glucose absorption into the systemic circulation after ingestion of foods. However, the plasma glucose level is determined by not only the absorption of glucose, but also the disappearance of glucose which is regulated by the insulin response. The aim of this study was to estimate the values of GI and glucose utilization including the absorption and the disappearance of glucose (UGLU) in the rice vermicelli (RV) using the resulting glucose clearance (CLGLU). Fifteen healthy subjects participated in this study. Alterations in plasma glucose and insulin levels were determined after ingestion of a reference food (12.5, 25, 50, 75 g glucose) and the test foods (white rice, long grain rice and six RV products; 50 g available carbohydrate). Time–course changes in plasma glucose levels were analyzed using a simple kinetic model. The values of CLGLU were calculated using the resulting kinetic parameters. A standard curve in which the incremental area under the curve of plasma insulin levels (AUCINS(ref)) was plotted against the resulting CLGLU(ref) was generated using the reference food. The values of GI and UGLU in the RV products were calculated from the observed clearances (CLGLU), and the predicted clearances (CLGLU(predict)) respectively, which were estimated using the standard curve of AUCINS(ref) vs. CLGLU(ref). It was clarified that the values of UGLU in the RV products (20—57%) were lower than those of GI (35—62%).
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  • Hiroaki Todo, Eriko Kimura, Hirotaka Yasuno, Yoshihiro Tokudome, Fumie ...
    Type: Regular Article
    Subject area: Biopharmacy
    2010 Volume 33 Issue 8 Pages 1394-1399
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    The permeation pathway of macromolecules and nanospheres through skin was evaluated using fluorescent isothiocyanate (FITC)-dextran (average MW, 4 kDa) (FD-4) and nanospheres (500 nm in diameter) in hairless rat abdominal skin and porcine ear skin as well as a three-dimensional cultured human skin model (cultured skin model). A low molecular hydrophilic compound, sodium fluorescein (FL) (MW, 376 Da), was used for comparison. FL penetrated the stratum corneum and permeated the viable epidermis of hairless rat skin, whereas less permeation of FL was observed through the cultured skin model, suggesting that the primary permeation pathway for the hydrophilic material may be skin appendages through the rat skin. A macromolecular compound, FD-4, was distributed through the hair follicles of the rat skin. In addition, nanospheres were detected in the hair follicles of porcine skin, although no skin permeation was detected. These findings suggest that appendage routes such as hair follicles can be a penetration pathway of macromolecules and nanospheres through skin.
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  • Kei Higuchi, Hisashi Iizasa, Yoshimichi Sai, Satomi Horieya, Kyeong-Eu ...
    Type: Regular Article
    Subject area: Biopharmacy
    2010 Volume 33 Issue 8 Pages 1400-1406
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    The syncytiotrophoblast, which regulates maternal–fetal transfer of drugs, consists of a single layer in humans, but two layers, i.e., SynI and SynII, in rodents. Polar distribution of transporters in the apical and basal plasma membranes of syncytiotrophoblast is important for placental function in terms of vectorial transport of substrates, but the mechanisms that control protein distribution in the syncytiotrophoblast remain unclear. We have previously established rat syncytiotrophoblast cell lines, TR-TBT 18d-1 and TR-TBT 18d-2, which retain characteristics of SynI and SynII, respectively. In this study, we aimed to characterize the gene expression profiles in the two layers by using these cell lines. DNA microarray analysis indicated that more than 25 mRNAs, including cytoskeleton binding proteins, ezrin and CLP36, are differentially expressed between TR-TBT 18d-1 and TR-TBT 18d-2. Quantitative real time-polymerase chain reaction (PCR) analysis indicated that mRNA expression of ezrin, CLP36, CCN1, and CCN2 is higher in TR-TBT 18d-1 and mRNA expression of elf-1a, hsc70 and flot2 is higher in TR-TBT 18d-2, compared with their counterparts. Immunohistochemical analysis indicated that ezrin is expressed in rat placental villi in vivo, and is located on the apical membranes of TR-TBT 18d-1, while CLP36 is located in the apical and basal sides of TR-TBT 18d-1. The expression of ezrin was highest at gestational days 14 and 18 and was highest among the ezrin/radixin/moesin (ERM) family members. These results may help to clarify the mechanisms controlling polarization of the syncytiotrophoblast and the significance of the double epithelial layers in rat and mouse.
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  • Miki Masago, Mari Takaai, Jumpei Sakata, Asuka Horie, Toshikazu Ito, K ...
    Type: Regular Article
    Subject area: Biopharmacy
    2010 Volume 33 Issue 8 Pages 1407-1412
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    The aim of the present study was to compare the membrane transport mechanisms of procainamide with those of quinidine using renal epithelial LLC-PK1 and intestinal epithelial LS180 cells. In LLC-PK1 cells, the transcellular transport of 10 μM quinidine in the basolateral-to-apical direction was similar to that in the opposite direction, and 1 mM tetraethylammonium (TEA) did not affect the transcellular transport of the drug. On the other hand, the transcellular transport of 10 μM TEA and procainamide in LLC-PK1 cells was directional from the basolateral side to the apical side. In addition, this directional transcellular transport of procainamide was diminished in the presence of 1 mM TEA. In LS180 cells, the temperature-dependent cellular uptake of 100 μM quinidine and procainamide was markedly increased by alkalization of the apical medium, and was inhibited significantly by 1 mM several hydrophobic cationic drugs, but not by TEA. The rank order of the inhibitory effects of hydrophobic cationic drugs on the uptake of procainamide in LS180 cells was imipramine>quinidine>diphenhydramine≈pyrilamine>procainamide, which was consistent with that on the uptake of quinidine. These findings suggested that procainamide (but not quinidine) was transported by cation transport systems in renal epithelial cells, but that both procainamide and quinidine were taken up by another cation transport system in intestinal epithelial cells.
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Note
  • Yasuko Obata, Yuki Otake, Kozo Takayama
    Type: Note
    Subject area: Biopharmacy
    2010 Volume 33 Issue 8 Pages 1454-1457
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    Prochlorperazine (PCPZ) is used as a drug of first choice to treat opioid-induced nausea and vomiting. To examine the feasibility of the development of a transdermal drug delivery system for PCPZ, we performed an in vitro skin permeation study with hairless mouse skin. When the concentration of L-menthol in the hydrogel was 0—0.5%, the PCPZ flux was small; on the other hand, the flux was increased remarkably when the L-menthol concentration was higher than 1%. The optimal formulation of hydrogel would be contained 20% isopropanol (IPA), 10% N-methyl-2-pyrrolidone (NMP), 2% L-menthol and 1% PCPZ. The strong inhibitory effects to stereotyped behavior were observed at 4 h after administration of PCPZ hydrogel, and the efficacy was sustained for at least 8 h after the administration in mice in vivo. Thus, it was considered that PCPZ was delivered to brain via systemic circulation by the administration of PCPZ hydrogel.
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Miscellaneous
Regular Articles
  • Tomoyoshi Ishikawa, Takahiko Ito, Ryosuke Endo, Keiko Nakagawa, Eiji S ...
    Type: Regular Article
    Subject area: Miscellaneous
    2010 Volume 33 Issue 8 Pages 1413-1417
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    Monoclonal antibodies are widely used for the treatment of various diseases, and because therapeutic monoclonal antibodies are stored in an aqueous solution or in a lyophilized state, the preparation of a stabilizing formulation that prevents their deterioration (degradation and aggregation) is crucial. Given the structural similarities of the immunoglobulin G (IgG) framework regions and a diversity of only four subclasses, we aimed to find common conditions that stabilize many different antibodies. In this study, we analyzed the effect of pH (the most critical factor in establishing a stable formulation) on human monoclonal antibodies from subclasses IgG1, IgG2, and IgG4, all of which have been utilized in antibody therapeutics. We found that human IgGs are stable with minimal heat-induced degradation and aggregation at pH 5.0—5.5 irrespective of their subclass. We also found that IgG1 is more susceptible to fragmentation, whereas IgG4 is more susceptible to aggregation. This basic information emphasizing the influence of pH on IgG stability should facilitate the optimization of formulation conditions tailored to individual antibodies for specific uses.
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  • Rika Nakamura, Rie Satoh, Ryosuke Nakamura, Takayoshi Shimazaki, Mie K ...
    Type: Regular Article
    Subject area: Miscellaneous
    2010 Volume 33 Issue 8 Pages 1418-1425
    Published: August 01, 2010
    Released: August 01, 2010
    JOURNALS FREE ACCESS
    To produce crops that are more tolerant to stresses such as heat, cold, and salt, transgenic plants have been produced those express stress-associated proteins. In this study, we used immunoproteomic and two-dimensional difference gel electrophoresis (2D-DIGE) methods to investigate the allergenicity of transgenic potatoes expressing Arabidopsis DREB1A (dehydration responsive element-binding protein 1A), driven by the rd29A promoter or the 35S promoter. Immunoproteomic analysis using sera from potato-allergic patients revealed several immunoglobulin E (IgE)-binding protein spots. The patterns of protein binding were almost the same between transgenic and non-transgenic potatoes. The IgE-binding proteins in potato were identified as patatin precursors, a segment of serine protease inhibitor 2, and proteinase inhibitor II by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) MS/MS. 2D-DIGE analysis revealed several differences in protein expression between non-transgenic potato and transgenic potato; those showing increased expression in transgenic potatoes were identified as precursors of patatin, a major potato allergen, and those showing decreased expression in transgenic potatoes were identified as lipoxygenase and glycogen (starch) synthase. These results suggested that transgenic potatoes may express slightly higher levels of allergens, but their IgE-binding patterns were almost the same as those of control potatoes. Further research on changes in protein expressions in response to environmental factors is required to confirm whether the differences observed in this study are due to gene transfection, rather than environmental factors.
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