Glycosylation is a highly sophisticated post-translational modification of proteins that occurs in the endoplasmic reticulum (ER) and the Golgi apparatus. The vital biological functions served by protein glycosylation can be separated into two types. The first is the role of glycosylation in the extracellular environment, where a variety of sugar chains participate in protein secretion and stability, cell–cell adhesion and signaling, innate immunity, embryogenesis, and morphogenesis. Another role is in quality control of glycoproteins in the ER, a topic that has received recent attention. Quality control of glycoproteins is categorized into three kinds of reactions; the first is folding of newly synthesized glycoproteins, the second is ER-associated degradation of terminally misfolded or unassembled glycoproteins, and the last is sorting and transport of glycoproteins between organelles. In all three processes, N-glycans on the glycoproteins are used as tags to initiate the reactions. The process of glycosylation is conserved in yeast, plants, invertebrates and vertebrates, including mammals. Here, I focus on the intracellular lectins that participate in these processes and discuss the role of glycosylation based on the structural differences of N-glycans.

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Lymphocyte homing is mediated by a cascade of adhesive interactions between circulating lymphocytes and specialized endothelial cells comprising high endothelial venules (HEVs). Sulfated O-glycans expressed on HEVs, collectively called peripheral lymph node addressin (PNAd), interact with L-selectin expressed on lymphocytes, contributing to the initial step of the lymphocyte homing. In chronic inflammatory states, PNAd is induced on HEV-like vessels but absent in non-lymphoid tissues under normal conditions. Such HEV-like vessels have been observed in various chronic inflammatory diseases including rheumatoid arthritis, lymphocytic thyroiditis, Helicobacter pylori-associated chronic gastritis, and inflammatory bowel disease (IBD), and implicated in lymphocyte recruitment in those diseases. In H. pylori-associated chronic gastritis, PNAd-expressing HEV-like vessels are induced, and the progression of chronic inflammation is highly correlated with appearance of these vessels. Furthermore, eradication of H. pylori by antibiotics resulted in disappearance of PNAd. These results indicate that inhibition of PNAd formation could have therapeutic effect by attenuating lymphocyte recruitment. In ulcerative colitis (UC), PNAd-expressing HEV-like vessels are induced, preferentially in the active phase, and T cells, particularly CD4⁺ T cells, are closely associated with these vessels, suggesting that T cell recruitment via PNAd-expressing HEV-like vessels plays at least a partial role in UC pathogenesis. Additionally, N-acetylglucosamine-6-O-sulfotransferase 1 (GlcNAc6ST-1) is suggested to be a candidate to regulate PNAd induction on HEV-like vessels in UC. These results provide a potential therapeutic strategy to treat UC by blocking T cell adhesion to PNAd-expressing HEV-like vessels. Inhibition or down-regulation of GlcNAc6ST-1 may be an alternative.

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N-Glycosylation of proteins is conserved in eukaryotes, which is one of the most abundant post-translational modification reactions, and nearly half of all known proteins in eukaryotes are glycosylated. In fact, changes in oligosaccharide structure (glycan) are associated with many physiological and pathological events, including cell adhesion, migration, cell growth, cell differentiation and tumor invasion. Glycosylation reactions are catalyzed by the action of glycosyltransferases, which add sugar chains to various glycans on glycoproteins, glycolipids and proteoglycans. Here, we focus mainly on the modification of N-glycans with N-acetylglucosaminyltransferase III (GnT-III), N-acetylglucosaminyltransferase V (GnT-V) and α2,6 sialyltransferase (ST6GalI) to address the important roles of N-glycans in integrin-meditaed cell adhesion and migration. In addition, we also discuss the potential roles of N-glycosylation sites on integrin α5 subunit.

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Protein therapeutics, such as antibodies and cytokines, is the largest class of new drug candidates being developed by pharmaceutical companies. Although most of these glycoproteins are produced in mammalian cells, there is concern that their large-scale production could be affected by an inadequate supply of bovine serum. There is also the risk of viral infection spreading through the use of contaminated protein therapeutics. Consequently, protein expression systems in yeast have been established because protein manufacturing costs are cheaper than in mammalian cells, and yeast systems are virus-free. However, yeasts cannot generate human-type glycans, and thus cannot produce therapeutic glycoproteins for human use. There has therefore been considerable interest in glycan remodeling, from yeast-type to human-type. ‘Humanized’ glycoproteins can now be generated in yeast by disrupting yeast-specific glycosyltransferases and introducing genes responsible for sugar-nucleotide synthesis, its transported from the cytosol to Golgi lumen, as well as their transfer and hydrolysis. A compound that inhibits yeast O-mannosyltransferase suppresses yeast-specific O-mannosyl modification, and can produce mucin-type glycoproteins. These systems are just being developed to the stage where the production in glycoengineered yeast of biopharmaceutical glycoproteins such as cytokines, antibodies for therapeutics, and enzymes for replacement therapy for lysosomal diseases are being evaluated for clinical applications. Yeast glycoprotein expression systems are expected to become the dominant approach for the production of human glycoproteins in the near future.

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Many glycoproteins and glycosaminoglycans are approved for clinical use. Carbohydrate moieties in biopharmaceuticals affect not only their physicochemical properties and thermal stability, but also their reactivity with their receptors and circulating half-life. Modification of glycans is one target of drug design for enhancement of efficacy. Meanwhile, there have been reports of serious adverse events caused by some carbohydrates. It is crucial to maintain the constancy of carbohydrate moieties for the efficient and safe use of glycosylated biopharmaceuticals. On the other hand, for scientific, safety-related, and economic reasons, changes in the manufacturing process are frequently made either during the development or after the approval of new biopharmaceuticals. Furthermore, the development of biosimilar glycoprotein products has been attempted by different manufacturers. Changes in pharmaceutical manufacturing processes possibly cause alteration of glycosylation and raise concerns about alteration of their quality, safety, and efficacy. In this review we provide some current topics of glycosylated biopharmaceuticals from the viewpoints of efficacy, safety, and the manufacturing process and discuss the significance of glycosylation analysis for development of biopharmaceuticals.

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This study was designed to determine the antioxidant levels in the urinary bladder wall layers as well as urothelium derived from different bladder regions. Samples of the urothelium, lamina propria, muscularis, and serosa were prepared from the pig's urinary bladder body, while samples used for regional mapping of the urothelium were prepared from trigone, ventral and dorsal middle bladder body, and apex region. Activities of superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase were determined. Concentrations of ascorbic acid and glutathione were also measured. Antioxidant activities, i.e. concentrations of superoxide dismutase, glutathione peroxidase, glutathione reductase and glutathione, were shown to be highest in the urothelium and progressively lower towards the serosa. Regional mapping of the urothelium singled out apex as the region with the lowest antioxidant activities, i.e. concentrations of glutathione peroxidase, ascorbic acid, and glutathione. The fact that antioxidants are concentrated in the urothelium implies that urothelium functions as a barrier against reactive species. The urothelium derived from the apex is the region with the lowest antioxidant levels and is therefore probably the region most liable to development of oxidative damage.

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We have developed the Rabbit Corneal Epithelial (RCE) Model to evaluate the in vitro eye irritation potential of chemicals including pharmaceuticals, cosmetics and their raw ingredients. In the model, a stratified culture of rabbit corneal epithelial cells is grown at the air–liquid interface on an amnion acting as a parabasal membrane. The alkaline exposure was restored each day in the presence of no irritants, although with the addition of sodium lauryl sulfate (SLS), which is a major irritant, the restoration of deficit was inhibited on the RCE model in a dose-dependent manner. The results of this test were comparable with those of the Draize test, and thus, this method using the RCE model may prove to be a useful and sensitive in vitro eye irritation test. The in vitro eye irritation potential of polyoxyethylene alkyl derivatives, polyoxyethylene lauryl ether (PLE), polyoxyethylene cetyl ether (PCE), polyoxyethylene stearyl ether (PSE), polyoxyethylene oleyl ether (POE), and polyoxyethylene behenyl ether (PBE) were evaluated using the RCE model containing an alkaline exposure. POE inhibited 90.2% of the restoration of deficit at a concentration of 0.5% on the 4th day after addition. Depending on the structure, an activity relationship was defined. The polyoxyethylene alkyl derivatives had distinctly different inhibitory potencies against the restoration of deficit, according to their substitution patterns. POE inhibited the restoration of deficit greater than other polyoxyethylene alkyl derivatives on the RCE model. These results indicated that the oleyl chain of POE is an important factor for inhibiting the restoration of deficit on the RCE model.

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Primary human hepatocytes are extensively used to study the potential of drugs to induce cytochrome P450 (CYP). However, the activities of these enzymes decrease rapidly during culture. Previously we reported that in a layered co-culture system with HepG2 and bovine endothelial cells, the expression levels of various CYP genes were significantly increased compared with the monolayer cultured HepG2 cells. Here, we examined the induction of CYP gene expression by an inducer by examining the effect of phenobarbital treatment on CYP gene expression in the co-culture system. In the layered co-cultured HepG2, expression of the CYP2C and CYP3A family genes was induced by phenobarbital treatment. We also detected CYP3A4 enzyme induction using this co-culture system. Moreover, the induction of hepatic drug transporters by phenobarbital was detected. These results suggest that functional regulation of the CYP and transporter gene pathway is retained in these layered co-cultured cells. Thus, this system may serve as a useful model for in vitro pharmacological studies on the coordinated regulation of transport and metabolism.

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Phytosterols and triterpenes are synthesized by oxidosqualene cyclases (OSCs) via the isoprenoid pathway. Here, GsAS1—a full-length β-amyrin synthase cDNA isolated from Gentiana straminea MAXIM.—was characterized. Its open reading frame consists of 2268 bp, predicted to encode a 756 residue protein containing four QW and one Asp-Cys-Thr-Ala-Glu (DCTAE) motifs, which are both well conserved among known triterpene synthases. The deduced GsAS1 peptide sequence shares 76.2% homology with that of Panax ginseng β-amyrin synthase. A phylogenetic analysis showed that GsAS1 is closely related to other plant OSCs, and particularly to the β-amyrin synthases. When the GsAS1 sequence was heterologously expressed in Escherichia coli, an 88 kDa gene product was produced, and this reacted with the appropriate antibody. The sequence was also heterologously expressed in the Pichia pastoris yeast. GsAS1 is expressed in a tissue-specific manner, with its expression in the leaf being ca. 4.5-fold than that in the root, and nearly three-fold than that in the stem. GsAS1 expression was up-regulated by treatment with methyl jasmonate (MeJA) over a period from 6 h to 10 d post treatment. The accumulation oleanolic acid increased after induction by MeJA.

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Alteration in the expression of claudins, consisting of tight junctions (TJs), has been reported in various clinically isolated tumors. Claudins play an important role not only in the intercellular barrier function of TJs but also in migration and invasiveness of cancer cells. However, the use of different types of cells and different claudins in these studies has complicated the picture. In this study, we systematically examined the effect of claudin (claudin-1, -2, -3, -4 and -15) overexpression on the paracellular permeability, migration and invasiveness of Caco-2 colonic cancer cells. Overexpression of claudin-4 or claudin-2 increased or decreased, respectively, paracellular permeability. Overexpression of claudin-4 specifically stimulated the invasive activity of the Caco-2 cells. Furthermore, activation of matrix metalloproteinase (MMP)-2 and MMP-9 were observed in the claudin-4-overexpressing cells, suggesting that the invasive activity was stimulated through an increase in MMP activity. Overexpression of claudin-2 or claudin-3 and -4 stimulated or inhibited, respectively, the migration activity of the Caco-2 cells. Immunostaining analysis revealed that each of the overexpressed claudins localized at TJs under the conditions used to evaluate paracellular permeability. In contrast, they localized mainly in intracellular compartments under experimental conditions designed to assess cell invasion and migration. Overall, the results of this study show that the effect exerted by the claudins on the intercellular barrier function of TJs, as well as on cell migration and invasive activity, differs depending on the particular claudin species. Furthermore, the subcellular localization of the claudins varies according to the culture conditions.

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2-Aryl-3-bromoquinolin-4(1H)-ones and 2-aryl-3-bromo-1-methylquinolin-4(1H)-one were evaluated for their antiangiogenic effects. Results showed that the 2-aryl-3-bromoquinolin-4(1H)-ones (QNHFBr and QNHClBr), and the 3-bromo-2-(4-chlorophenyl)-1-methylquinolin-4(1H)-one (QNMeClBr) reduced endothelial cell numbers in an assay of cell proliferation, showing increased cytotoxicity at higher doses. The compounds also inhibited neovessel growth in an ex vivo assay of angiogenesis. Furthermore, low levels of proangiogenic factors, namely, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor/placental growth factor (VEGF/PlGF) were obtained in cultures treated with these compounds. Thus, the tested compounds, QNHFBr, QNHClBr, and QNMeClBr exhibit antiangiogenic activity.

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We examined the biological activities present in Streptomyces strains preserved at the National Institute of Technology and Evaluation Biological Resource Center, and found a metabolite of Streptomyces roseolilacinus NBRC 12815 that showed a potent anti-tyrosinase activity. The compounds with anti-tyrosinase activity were purified by several chromatographic procedures. Final HPLC analysis revealed at least two anti-tyrosinase compounds with different retention times (12815A and B). The identification of two anti-tyrosinase compounds was performed with instrumental analysis and database search. The results obtained suggest that the active compounds are SF 2583A and B. Compound 12815A (IC₅₀ values; about 9 μM) showed more potent tyrosinase inhibition than compound 12815B (IC₅₀ values; about 1086 μM). The only structural difference between 12815A and B is the presence of an additional chloric atom. In addition, the activity of 12815A was markedly decreased under acidic conditions, resulting in irreversible inactivation. However, the inactivated 12815A still exhibited residual activity when exposed to detergent, Tween 80. These results suggest that the chlorine and the hydration water are very important in the exertion of anti-tyrosinase activity by 12815A.

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Ultraviolet (UV)-induced DNA damage is a crucial molecular trigger for sunburn cell formation and skin cancer. Nucleotide excision repair (NER) is the main mechanism in repairing UVB-induced DNA damage to mammalian cells. The purpose of this study was to investigate the functional role of ginsenoside Rb1 in UV-induced DNA damage and apoptosis in HaCaT (keratinocyte cell line) cells, and Xpc⁻ knockout mouse keratinocytes. Flow cytometry and Hoechst 33258 staining were performed in analyzing UV-induced apoptosis in keratinocytes treated with ginsenoside Rb1. The ImmunoDotBlot assay was used to detect cyclobutane pyrimidine dimers, the main sign of DNA damage. Western blot analysis was applied for analyzing Xeroderma pigmentosum-C (XPC) and excision repair cross-complementing 1 (ERCC1), two of the NER proteins. Ginsenoside Rb1 inhibited UV-induced apoptosis of keratinocytes and caused a notable reduction in UV-specific DNA lesions which was due to induction of DNA repair. This reduction was not observed in Xpc⁻ knockout keratinocytes. Ginsenoside Rb1 induced the expression of specific components of the NER complex, such as XPC and ERCC1. Our results demonstrate that ginsenoside Rb1 can protect cells from apoptosis induced by UV radiation by inducing DNA repair.

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Curcumin reportedly has anti-allergic effects and can inhibit the release of histamine from mast cells. In the present study, fourteen benzylidenecyclopentanone analogues of curcumin were studied for their effects on histamine release from rat basophilic leukemia (RBL-2H3) cells. After screening, four selected compounds: 2,5-bis(4-hydroxybenzylidene)cyclopentanone; 2,5-bis(4-hydroxy-3-methoxybenzylidene)cyclopentanone; 2,5-bis(4-hydroxy-3,5-dimethylbenzylidene) cyclopentanone; and 2,5-bis(4-hydroxy-3,5-diethylbenzylidene)cyclopentanone were studied for their concentration-dependent effects on histamine release and Ca²⁺ uptake. In RBL-2H3 cells and rat peritoneal mast cells stimulated with antigen or compound 48/80, respectively, the methoxy-hydroxy analogue was more potent than curcumin in inhibiting histamine release. In contrast, the inhibitory effects of methyl/ethyl analogues were less potent than those of curcumin. Moreover, these compounds abrogated histamine release induced by increased intracellular Ca²⁺ concentrations in response to stimulants such as thapsigargin and ionomycin. These compounds also showed potent inhibitory effects on ⁴⁵Ca²⁺ uptake in RBL-2H3 cells. The mechanism of the inhibitory effects of these curcumin analogues on histamine release appeared to be related to blockade of Ca²⁺ signaling events. These results provide useful information to guide the development of new synthetic compounds for the treatment of allergic and inflammatory diseases related to histamine or mast cells.

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Memantine, a non-competitive antagonist of NMDA receptors, has recently been used in Alzheimer's disease. The influences of memantine on behavioral changes, monoamine oxidase (MAO) activity and reuptake of both serotonin (5-HT) and dopamine in mice were examined in the present study. Memantine dose-dependently increased locomotor activity. This effect was inhibited by intraperitoneal (i.p.) administration of haloperidol. Furthermore, administration [intracerebroventricular (i.c.v.)] of memantine did not induce the head-twitch response (HTR). However, the 5-HT-induced HTR was potentiated by the combined administration of memantine. The enhanced HTR was inhibited by i.p. administration of haloperidol or 5-HT_2A antagonist ketanserin. Memantine at 1 mM inhibited both MAO-A and MAO-B activities in mouse forebrain homogenates to 37% and 64% of controls, respectively. Lineweaver–Burk plots analysis revealed competitive inhibition with both MAO-A and MAO-B. The inhibitions were also reversible. Memantine inhibited the reuptake of both 5-HT and dopamine into mouse forebrain synaptosomes. 5-HT and dopamine reuptakes were inhibited to 2% and 16% of controls, respectively, with 1 mM memantine. These findings suggest that the increased locomotor activity and enhanced 5-HT-induced HTR by memantine may result from the reuptake and turnover inhibitions of 5-HT and dopamine.

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Kami-shoyo-san (Jia-Wei-Xiao-Yao-San), Toki-shakuyaku-san (Dang-Gui-Shao-Yao-San) and Toki-shigyaku-ka-goshuyu-shokyo-to (Dang-Gui-Si-Ni-Jia-Wu-Zhu-Yu-Sheng-Jiang-Tang) are Kampo (traditional Chinese) medicines which are traditionally and effectively used for the treatment of chilly sensation (Hiesho) in Japan, but the active components and their detailed mechanisms have not yet been clarified. Etiologies of Hiesho include poor peripheral blood circulation and platelet aggregability contributes to peripheral blood circulation; therefore, we investigated the effect of Kampo medicines on platelet aggregation using rabbit platelets in vitro. Collagen and U46619, a thromboxane A₂ receptor agonist, caused rabbit platelet aggregation, which was potently inhibited by pretreatment of platelets with Kami-shoyo-san and Toki-shakuyaku-san in vitro. Toki-shigyaku-ka-goshuyu-shokyo-to, however, did not significantly inhibit collagen- or U46619-induced platelet aggregation. Therefore, we examined the effect on platelet aggregation of two herbal medicines, Atractylodis Lanceae Rhizoma and Poria, both of which are contained in Kami-shoyo-san and Toki-shakuyaku-san but not in Toki-shigyaku-ka-goshuyu-shokyo-to. As the results indicate, Atractylodis Lanceae Rhizoma inhibited platelet aggregation induced by collagen but not by U46619. Poria effectively inhibited U46619-induced platelet aggregation and it partially inhibited collagen-induced platelet aggregation. On the other hand, Atractylodis Lanceae Rhizoma and Poria did not inhibit adrenaline/adenosine diphosphate- or adrenaline/serotonin-induced platelet aggregation. These results suggest the possibility that the inhibition of platelet aggregation by two Kampo medicines, Kami-shoyo-san and Toki-shakuyaku-san, is one of the mechanisms underlying the improvement of Hiesho. Furthermore, Atractylodis Lanceae Rhizoma and Poria are possible herbal medicines for the inhibition of platelet aggregation.

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Previous reports have shown that diltiazem and TMB, calcium channel antagonists, inhibit 5-hydroxytryptamine type 3A (5-HT_3A) receptor-mediated currents (I_5-HT) in cell lines and in heterologously expressed Xenopus oocytes. In the present study, we sought to elucidate the molecular mechanisms underlying diltiazem- and TMB-induced 5-HT_3A receptor regulations. We used the two-microelectrode voltage clamp technique to investigate the effect of diltiazem and TMB on 5-HT-mediated ion currents in Xenopus oocytes expressing wild-type or 5-HT_3A receptors harboring mutations in the gating pore region of transmembrane domain 2 (TM2). In oocytes expressing wild-type 5-HT_3A receptors, diltiazem and TMB dose-dependently inhibited peak I_5-HT with an IC₅₀ of 71.4±4.9 and 4.5±0.3 μM, respectively. Among various mutants of TM2, mutation V291A greatly attenuated and abolished the TMB- and diltiazem-induced inhibition of peak I_5-HT, respectively. Mutation V291A also induced constitutively active ion currents in the absence of 5-HT. Diltiazem and TMB inhibited constitutively active ion currents in a dose-dependent manner. The IC₅₀ values of constitutively active ion currents in V291A receptors were 165.3±11.1 and 6.6±0.5 μM for diltiazem and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), respectively. Results of site-directed mutagenesis experiments suggest that the Val291 residue could be a candidate for common interaction site for diltiazem- and TMB-8-mediated 5-HT_3A receptor regulations.

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Silibinin is known to have hepatoprotective, anti-carcinogenic and anti-inflammatory effects. However, roles of silibinin in the immediate-type allergic reactions (anaphylaxis) have not fully been investigated. In the present study, we have demonstrated that silibinin attenuated mast cell-mediated anaphylaxis-like reactions involved in allergic diseases. Oral administration of silibinin inhibited compound 48/80-induced passive cutaneous anaphylaxis-like reaction in mice. Silibinin also attenuated anti-dinitrophenyl (DNP) immunoglobulin (Ig) E-mediated passive systemic and cutaneous anaphylaxis. Silibinin had no cytotoxicity on rat peritoneal mast cells (RPMC). Silibinin dose-dependently reduced histamine release from RPMC activated by compound 48/80 or anti-DNP IgE. Moreover, silibinin inhibited the secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-6 in RPMC. Pretreatment of silibinin suppressed the antigen-stimulated calcium uptake and activation of nuclear factor-kappa B (NF-κB) in RPMC. Furthermore, silibinin increased the intracellular cAMP level. Increased cAMP, decreased calcium uptake and suppressed NF-κB activity might be involved in the inhibitory effect of silibinin on the secretory response. Our findings provide possibility that silibinin may serve as an effective therapeutic agent for allergic diseases.

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The activation of aryl hydrocarbon receptor with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to be antagonized by co-treatment with resveratrol. However, such a protective effect has been suggested from studies using subcutaneous injection of this polyphenol. To evaluate the practical usefulness of resveratrol, this study examined the protective effect of oral resveratrol on the sub-acute toxic effects of TCDD in C57BL/6J mice. A TCDD-induced wasting syndrome was not alleviated by treating mice for 28 d with oral resveratrol. However, subcutaneous injection of resveratrol for 5 d significantly improved the symptoms. Neither oral nor subcutaneous administration of resveratrol alleviated TCDD-induced hepatomegaly and thymic atrophy. Steatosis produced by TCDD was markedly counteracted by co-treatment with oral resveratrol, whereas resveratrol injected subcutaneously had no effect. The reason for the lack of protective effect via the latter dosing route was assumed to be due to the minor accumulation of hepatic lipids 5 d after TCDD treatment. To clarify the mechanisms, the activity of ethoxyresorufin O-deethylase and the content of thiobarbituric acid-reactive substances in the liver were measured. Both indices increased by TCDD treatment were significantly suppressed by subcutaneous injection of resveratrol. In contrast, oral resveratrol failed to rescue them. In agreement with the greater protective effects of subcutaneously-injected resveratrol, pharmacokinetic studies indicated that the area under the curve extrapolated to infinity (AUC_∞) was 8.2-times greater following subcutaneous injection compared with oral administration. These data suggest that 1) oral resveratrol is attractive candidate as an agent capable of combating dioxin toxicity and 2) increasing the bioavailability of this polyphenol enhances its protective effect.

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Epithelial-mesenchymal transition (EMT) is an important mechanism in kidney fibrosis. While Salvianolic acid-B (Sal B) has been well appreciated to show a protective effect of tissue fibrosis, the objective of this study was to investigate the influence of Sal B on the transdifferentiation of renal tubular epithelial cells. Human kidney proximal tubular cell line (HK-2) was used as the proximal tubular cell model and EMT was induced with 5 ng/ml of human tranforming growth factor (TGF)-β1. The effects of the Sal B on cell morphology were observed by phase contrast microscopy, and the possible mechanisms were studied by immunocytochemistry and real-time reverse transcription-polymerase chain reaction. Our results revealed that Sal B could inhibit TGF-β1-induced myofibroblast phenotype and restored the epithelial morphology in a dose-dependent manner. It was partially through modulating α-smooth muscle actin (SMA) increase and E-cadherin reduction. These observations strongly suggest that Sal B is a potent inhibitor of TGF-β1-induced EMT and might be a promising agent for treating tubulointerstitial fibrosis.

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We have developed a convenient method for the detection of Lophophora williamsii using loop-mediated isothermal amplification (LAMP). We designed six species-specific primers for L. williamsii, including two loop primers. This L. williamsii-specific primer set was used for LAMP of total DNA extracted from L. williamsii and from L. diffusa. Real-time monitoring of LAMP was achieved by measuring turbidity due to the formation of magnesium pyrophosphate. Amplification occurred in samples mixed with total DNA from L. williamsii, but not in those mixed with total DNA from L. diffusa. We could also visually observe L. williamsii by adding fluorescent detection reagent to the reaction tube and exposing it to UV light. This new method amplified L. williamsii selectively and is expected to be applicable to the detection of peyote.

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A sulfated galactan (FG) was isolated from the green alga, Codium fragile. Chemical analysis revealed that FG mainly consisted of D-galactose with pyruvic acid (12.3%) and sulfate (11.0%). Methylation and NMR analyses showed that FG was composed of →3)-β-D-Galp-(1→, β-D-Galp-(1→ and →3,6)-β-D-Galp-(1→ residue. In addition, pyruvic acid was suggested to be present as (1′-carboxy)-ethylidene cyclic ketal at O-3 and O-4 of non-reducing terminal galactose residues, whereas sulfate was substituted at O-4 of other galactose residues. When the antiviral potency of FG was evaluated, it inhibited the replication of herpes simplex virus type 2 (HSV-2), and the mechanism of action was suggested to be interference in the early steps such as virus adsorption to and penetration into host cells. Furthermore, it was shown that FG directly reduced virus infection rates. In a genital infection model using HSV-2-infected mice, FG improved mortality and lesion scores and reduced virus yields by intravaginal administration. These results suggest that FG might be a potent candidate as a prophylactic agent for HSV-2 infection.

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Migration/invasion is involved in the multiple steps of metastasis, resulting in a poor prognosis of breast cancer. (−)-Epigallocatechin-3-gallate (EGCG) in green tea inhibits the metastasis of some cancer cell lines. Cell migration/invasion assays using Boyden chambers demonstrated that (−)-epigallocatechin (EGC), another green tea catechin, inhibited heregulin-β1 (HRG)-induced migration/invasion of MCF-7 human breast carcinoma cells to approximately the same extent as EGCG. Assays of cytoskeletal reorganization, Western blotting and immunoprecipitation suggested that EGCG inhibited this migration/invasion by suppressing the HRG-stimulated activation of epidermal growth factor receptor-related protein B2 (ErbB2)/ErbB3/protein kinase B (Akt), whereas EGC did so through pathways including the disruption of the HRG-stimulated activation of ErbB2/ErbB3 but not Akt. EGC, as well as EGCG, could play an important role against the promotion of metastasis of breast cancer cells.

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We performed an in vitro assay for seven compounds from methanolic extract of Sappan Lignum (CSE) that inhibit the chemical mediators of inflammation using the J774.1 cell line: brazilin (1), sappanchalcone (2), protosappanin A (3), protosappanin B (4), protosappanin C (5), protosappanin D (6), and protosappanin E (7). Those compounds were evaluated for their inhibitory effects on nitric oxide (NO) and prostaglandin E₂ (PGE₂) production and their suppressive effects on tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) mRNA expression. As a result, we clarified that 1 inhibited NO production, and almost no inhibition in PGE₂. On the contrary, 2, 6, and 7 inhibited both NO and PGE₂ production and suppressed TNF-α, IL-6, COX-2, and iNOS mRNA expression. An examination of carrageenin-induced mouse paw edema suggested that the CSE contained active compounds other than 1, the main constituent in CSE. It was thus revealed that several compounds and mechanisms contributed to the anti-inflammatory effect of CSE.

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The bioassay-guided fractionation of a MeOH extract of the heartwood of Caesalpinia sappan L. provided two neuroprotective compounds, sappanchalcone (2) and 4-O-methylepisappanol (3), together with a methoxychalcone, isoliquiritigenin 2′-methyl ether (1), and three aromatic compounds, 4-O-methylsappanol (4), caesalpine J (5), pluchoic acid (6). At concentrations of 20—40 μm, compound 2 showed significant cytoprotective effects against glutamate-induced oxidative stress through the induction of heme oxygenase (HO)-1 in HT22-immortalized hippocampal cells. Compound 3 also showed moderate neuroprotective effect at 40 μm, but compounds 1, 4—6 did not show any protective effects against glutamate-induced cytotoxicity in HT22 cells.

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The mechanism of skin penetration enhancement by ultrasound under sonophoresis (US) or by an electrical field under iontophoresis (IP) was investigated using hairless mouse skin in vitro. The seven model chemicals with different molecular weights (122—1485) were dissolved in a hydrophilic gel. Donor gel with the chemicals was loaded on the skin surface and then the skin was treated with US (300 kHz, 5.2 W/cm², 5.4% duty-cycle) and IP (0.32±0.03 mA/cm²) individually or with US and IP in combination (US+IP). The penetration profiles of the chemicals with a molecular weight of less than 500 were influenced by the presence of an electric charge, the profiles of ionized chemicals for US+IP were the same as profiles for IP, while the penetration flux of a non-ionized chemical synergistically increased with US+IP compared with the individual flux of US and IP. The chemicals with molecular weight of more than 1000 showed synergistic effects with US+IP. The mathematical simulation assuming a bilayer skin model revealed that the synergistic effects were mainly influenced by electroosmosis in the stratum corneum (SC). Therefore the synergistic effects of US+IP was mainly caused by the SC diffusivity of chemicals increased by US and the electroosmotic water flow by IP application.

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The interferon (IFN) is a paradigm of secretory protein. However, it has been poorly understood how its secretion is regulated in polarized epithelial cells. Recently, we had shown that exogenous IFNs transiently expressed in polarized monolayers were predominantly secreted to the side on which gene transfection had been performed, while stably expressed IFNs were secreted almost equally to the both cell sides. Since those modes of secretion did not affect each other, epithelial cell layers seemed to have at least two protein sorting/secretion pathways, one for transient expression and the other for stable expression, for identical secretory proteins. Furthermore, this dual secretion profile seemed to be mediated by distinct post-trans Golgi network vesicles, suggesting the involvement of lipid rafts in the sorting multiplicity. To address this issue, here we studied the effects of cholesterol depletion with methyl-β-cyclodextrin (MβCD) on the secretion profile of IFN-β exogenously expressed in Madin–Darby canine kidney (MDCK) cells. The MβCD-treatment, however, did not affect the profile in either transient or stable expression, although the architecture of zonula occuludin-1, which links to the tight junction, was substantially disrupted by the treatment. Further analysis of Triton X-100-insoluble cell extracts by sucrose density centrifugation demonstrated that IFN-β was not apparently associated with lipid rafts in either transient or stable expression. These results suggest that lipid rafts may not be crucially involved in the regulation of secretion polarity of IFN-β in the epithelial cells.

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We investigated the role of acoustic cavitation on sonophoretic skin permeation of calcein, a model permeant, across excised hairless rat skin. Three different frequencies (41, 158, 445 kHz) and various intensities (60 to 300 mW/cm²) of ultrasound were applied. Cavitation generation in degassed and undegassed (normal) water was monitored using a commercially available cavitation meter, then compared with skin permeability from calcein solution consistent of them. In addition, the penetration of a fluorescent dye, rhodamine B, into gelatin gel as a skin alternative was observed to estimate the role of cavitation collapse in the solution at or near the skin surface. Cavitation generation in the undegassed water was dependent on the ultrasound frequency, and the rank order of the cavitation was 41 kHz>158 kHz>445 kHz. At 41 kHz, cavitation generation in degassed water was clearly lower than that in undegassed water. Calcein permeability during ultrasound application correlated well with the cavitation generation in the medium, suggesting the important role of the indirect actions of cavitation collapse which occurred in the applied solution rather than the direct action in the skin. When ultrasound (41 or 158 kHz) was applied to the gelatin gels covered with rhodamine B solution, alteration in the surface configuration, like spots, and the coincident penetration of the dye were observed only at 41 kHz, while no alteration in the surface configuration was evident at 158 kHz. These results suggest that cavitation collapses in the vicinity of the skin surface might be more important for solute penetration in addition to skin permeabilization.

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The use of urinary and/or biliary excretion data was considered as an alternative approach if the bioanalytical method lacked the appropriate sensitivity to adequately characterize the serum or plasma concentration–time profile. This approach is used for the analysis of plasma concentration–time profile under the lower limit of quantification (LLOQ) of various analytical instruments. The objective of this study was to develop a pharmacokinetic (PK) model that describes the plasma concentration–time profiles under LLOQ of HPLC using urinary and biliary excretion data. As model compounds, pyrrole (Py)-imidazole (Im) polyamides 1035 (MW, 1035.12) and 1666 (MW, 1665.78) were used. The cumulative urinary excretions of Py-Im polyamides 1035 and 1666 were 72.4±11.6 and 4.8±0.5% of the administered dose, respectively. The cumulative biliary excretion of Py-Im polyamide 1035 was 4.3±0.4% of the administered dose, and Py-Im polyamide 1666 was not detected. The plasma concentration–time profiles of Py-Im polyamide 1035 were adequately described using linear and non-linear output compartments. The developed PK model could be used to describe the plasma concentration profiles using the linear output compartment interpreted as the urine compartment and the non-linear output compartment interpreted as the bile compartment. This PK model will be able to provide a more accurate prediction of the plasma concentration profiles under LLOQ.

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We recently developed a multifunctional envelope-type nano device (MEND) for efficient nucleic acid delivery. Here, we report on the development of an octaarigine (R8)-modified MEND encapsulating small interfering RNA (siRNA) with a tumor-specific, cleavable, polyethylene glycol (PEG)-lipid (PPD). We first determined the optimal concentration of R8 and pH-sensitive fusogenic peptide (GALA) on the lipid envelope of MEND (R8/GALA-MEND). Then, we examined the combination of optimized R8/GALA-MEND with a PEG-lipid. When a conventional PEG-lipid was used, the R8/GALA-MEND failed to knockdown expression of the target gene. On the other hand, PPD-modified R8/GALA-MEND exhibited efficient silencing activity to the level of the PEG-unmodified R8/GALA-MEND. In addition, we compared a R8/GALA-MEND with a MEND composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) that is a conventional cationic lipid used as a lipoplex component. The knockdown ability of the R8/GALA-MEND was much higher than that of the DOTAP-based MEND at the dose that is commonly employed in in vitro siRNA transfection. These results demonstrate that the R8/GALA-MEND is a promising delivery system for the transfer of siRNA to tumor cells.

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The protein sericin is the main constituent of silk. We demonstrate the effects of sericin on corneal wound healing in rat debrided corneal epithelium. We also determined the effects of sericin on cell adhesion and proliferation in a human cornea epithelial cell line (HCE-T). Epithelium was removed from the corneas of rats with a BD Micro-Sharp^TM, and wounded corneas were dyed with a 1% fluorescein solution. The corneal wounds were monitored using a fundus camera TRC-50X equipped with a digital camera. The corneal wound of rats instilled with saline was approximately 10% healing at 12 h, and approximately 65% healing at 24 h after corneal epithelial abrasion. The corneal wounds of rats instilled with saline showed almost complete healing by 36 h after corneal epithelial abrasion. On the other hand, the corneal healing rate of rats instilled with sericin solution was higher than that of rats instilled with saline, and the corneal healing rate constant increased with increasing sericin concentration. In addition, the adhesion and proliferation of HCE-T cells treated with 0.01—0.5% sericin solutions were enhanced, reaching a maximum at treatments with 0.2 and 0.1% sericin solutions, respectively. The present study demonstrates that the instillation of sericin solution has a potent effect in promoting wound healing and wound-size reduction in rats, probably caused by increasing cell movement and proliferation.

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D-Psicose inhibits the growth of L1 stage Caenorhabditis elegans. Sugars, involved in the pentose phosphate pathway, were examined for their ability to reverse the inhibition. Among these sugars, D-ribose specifically exerted reversing activity in a competitive manner. The ingested sugars are probably phosphorylated, although it remains to be seen whether D-psicose is phosphorylated. The structural similarity of D-psicofuranose 6-phosphate (Pf6P) or D-psicofuranose (Pf) to D-ribofuranose 5-phosphate (Rf5P) suggests that Pf6P or Pf is reversibly docked in the active site(s) of ribose-5-phosphate isomerase(s) to act as an antimetabolite to Rf5P, leading to inhibition of the biosynthesis of nucleic acids. d-Psicose was much less potent against the L4 stage than against the L1 stage. This is probably because in the L4 stage the somatic cell lineages come to an end and the number of germ-line nuclei increases to about 1000.

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- The mixed material was Each of the materials was
- the extract was approximately 2.5%. A voucher specimen
- The extract power was stored

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Table 1

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