Acorbine, a Corbicula japonica-derived tripeptide containing non-proteinogenic amino acids, suppresses ethanol-induced liver injury.

Since ancient times, Corbicula extract has been believed in Japan to have hepatoprotective effects, but it remains unclear whether these claims are true, and if so, which component is responsible for hepatoprotection. In this study, we showed that Corbicula extract exerted a protective effect against liver damage. Recent work identified acorbine (ß-alanyl-ornithyl-ornithine), a novel tripeptide containing non-proteinogenic amino acids, in the extract of Corbicula japonica. Synthesized acorbine cured alcohol-induced liver damage in mice. In addition, acorbine purified from Corbicula extract exerted a protective effect against alcohol-induced hepatotoxicity in a culture liver model derived from mouse ES/iPS cells. Thus, acorbine is one of the components of Corbicula extract that protects hepatocytes against ethanol-induced death.

Curcumin and its derivatives inhibit 2,3,7,8,-tetrachloro-dibenzo-p-dioxin-induced expression of drug metabolizing enzymes through aryl hydrocarbon receptor-mediated pathway.

Certain dioxins, including 2,3,7,8,-tetrachloro-dibenzo-p-dioxin (TCDD), are exogenous ligands for an aryl hydrocarbon receptor (AhR) and induces various drug-metabolizing enzymes. In this study, we examined the effect of curcumin on expression of drug-metabolizing enzymes through the AhR and NF-E2 related factor 2 (Nrf2) pathways. Curcumin dose-dependently inhibited TCDD-induced expression of phase I enzyme cytochrome P450 1A1 (CYP1A1) and phase II enzymes NAD(P)H:quinone oxidoreductase-1 (NQO1) and heme oxygenase 1 (HO-1) but not tert-butyl hydroquinone-induced NQO1 and HO-1, suggesting that curcumin inhibited only AhR pathway, but not Nrf2 one directly. Furthermore, we used 14 curcumin derivatives and obtained the correlation between hydrophobicity of the compounds and suppressive effect against AhR transformation. Results from the quantitative structure active correlative analysis indicated that methoxy groups and ß-diketone structure possessing keto-enol tautomerism in curcumin were necessary to inhibit AhR transformation, and the addition of methyl and methoxy group(s) to the curcumin increased the inhibition effect.

Quality Characterization of Japanese Medicinal Paeoniae Radix by Metallomic Analysis.

Paeoniae Radix is one of the crude drugs frequently used in traditional Japanese medicine (Kampo medicine). It takes abundant labor and time to cultivate Paeonia lactiflora for medicinal use; high production cost is one of the main reasons why the domestic production of Paeoniae Radix is decreasing in Japan. To promote the production of Paeoniae Radix, we focused on Paeonia cultivars that produce commercially valuable flowers and investigated their possibility for medicinal use. We prepared 28 batches of peony roots derived from P. lactiflora, which were cultivated in Japan; 4 batches were crude drug samples, and 24 batches were cultivar roots. The elements contained in these samples were measured using inductively coupled plasma (ICP)-MS. The obtained data were then analyzed by principal component analysis (PCA) and back propagation artificial neural network (BPANN) analysis. No significant differences were found between the profiles of elements contained in crude drugs and cultivar roots. However, PCA results indicated a high similarity of the multielement fingerprints of crude drugs. Using the PCA results, we also assessed visible cluster trends and found that 5 batches of cultivars also showed fingerprints related to those of crude drugs. We certified this classification by BPANN. From the perspective of metallomics, our findings suggest that these 5 batches of Paeonia cultivars could be alternatives to crude drugs.

Substitution at the C-3 Position of Catechins Has an Influence on the Binding Affinities against Serum Albumin.

It is known that catechins interact with the tryptophan (Trp) residue at the drug-binding site of serum albumin. In this study, we used catechin derivatives to investigate which position of the catechin structure strongly influences the binding affinity against bovine serum albumin (BSA) and human serum albumin (HSA). A docking simulation showed that (-)-epigallocatechin gallate (EGCg) interacted with both Trp residues of BSA (one at drug-binding site I and the other on the molecular surface), mainly by π-π stacking. Fluorescence analysis showed that EGCg and substituted EGCg caused a red shift of the peak wavelength of Trp similarly to warfarin (a drug-binding site I-specific compound), while 3-O-acyl-catechins caused a blue shift. To evaluate the binding affinities, the quenching constants were determined by the Stern-Volmer equation. A gallate ester at the C-3 position increased the quenching constants of the catechins. Against BSA, acyl substitution increased the quenching constant proportionally to the carbon chain lengths of the acyl group, whereas methyl substitution decreased the quenching constant. Against HSA, neither acyl nor methyl substitution affected the quenching constant. In conclusion, substitution at the C-3 position of catechins has an important influence on the binding affinity against serum albumin.

Taurine as a marker for the identification of natural Calculus Bovis and its substitutes.

Calculus Bovis (C. Bovis) is a commonly used animal-derived therapeutic preparation. To meet the increasing clinical demand for the preparation, two artificial substitutes for Bos Taurus have been introduced in China: artificial C. Bovis and in vitro cultured C. Bovis. However, information on their efficacy and safety is inadequate. Therefore, we investigated the biological differences between the commonly used natural preparation and its two substitutes, with the aim of not only identifying the differences but also providing a procedure to distinguish between the different preparations.In the study, we prepared 9 natural C. Bovis, 2 artificial C. Bovis, and 2 in vitro cultured C. Bovis preparations for evaluation. Differences were noted between the three preparations relative to their effect on viability of cardiac fibroblasts from 1-day-old Wistar rats. Although natural C. Bovis had no effect on cell viability, 1-h treatment of the cells with 0.25 mg/ml of the substitutes significantly reduced cell viability, as detected by the MTS assay. Based on liquid chromatography and inductively coupled plasma mass spectrometry, the preparations also differed in composition. Indeed, the substitutes contained more taurine, cholic acid, iron, magnesium, and calcium than the natural preparations. They also differed spectroscopically.The present results reveal significant biological differences between natural C. Bovis and two of its substitutes. Since the substitutes appear to contain more taurine, cholic acid, and elements, these constituents may serve as markers to distinguish between natural C. Bovis and its substitutes.

Syngeneic implantation of mouse hepatic progenitor cell-derived three-dimensional liver tissue with dense collagen fibrils.

BACKGROUND: Liver transplantation is a therapy for irreversible liver failure; however, at present, donor organs are in short supply. Cell transplantation therapy for liver failure is still at the developmental stage and is critically limited by a shortage of human primary hepatocytes. AIM: To investigate the possibility that hepatic progenitor cells (HPCs) prepared from the portal branch-ligated hepatic lobe may be used in regenerative medicine, we attempted to enable the implantation of extracellular matrices containing organoids consisting of HPC-derived hepatocytes and non-parenchymal cells. METHODS: In vitro liver organoid tissue has been generated by accumulating collagen fibrils, fibroblasts, and HPCs on a mesh of polylactic acid fabric using a bioreactor; this was subsequently implanted into syngeneic wild-type mice. RESULTS: The in vitro liver organoid tissues generated transplantable tissues in the condensed collagen fibril matrix and were obtained from the mouse through partial hepatectomy. CONCLUSION: Liver organoid tissue was produced from expanded HPCs using an originally designed bioreactor system. This tissue was comparable to liver lobules, and with fibroblasts embedded in the network collagen fibrils of this artificial tissue, it is useful for reconstructing the hepatic interstitial structure.

Demonstration and partial characterization of a bacterial growth enhancer in sera.

During our research into the pathogenesis of Vibrio parahaemolyticus, we noticed that the concentration of serum added to the tissue culture medium (Dulbecco's modified Eagle's medium: DMEM) greatly affected its growth. Using gel filtration column chromatography, we clearly demonstrated that serum contains not only a bacterial growth inhibitor (BGI) but also a bacterial growth enhancer (BGE) for Vibrio parahaemolyticus. Our data indicate that the BGI is transferrin, whereas the BGE seems to be an undescribed small molecule (molecular weight of 1,000-3,000 Da) and is associated with magnesium and molybdenum ions. BGE activity was not decreased by heat treatment (at 60 or 100°C for 30 min) and affected the growth rate of a wide range of Gram-negative and Gram-positive bacteria. The addition of EDTA into DMEM lowered the growth rate, whereas the addition of BGE restored the growth activity. This study suggests that sera contain a previously undescribed small BGE molecule.

Quality and safety issues related to traditional animal medicine: role of taurine.

BACKGROUND: Calculus Bovis (:C.Bovis) is one of the most precious and commonly-used medicinal materials in Japan and China. As the natural occurrence is very rare, a source of supply for C. Bovis is far behind the actual need and great efforts have been taken for some substitutes of natural C. Bovis. Unfortunately, very little information is available on the quality and/or clinical efficacy of medication based on C. Bovis. To ensure sustainable use of traditional therapeutic agents derived from C. Bovis, we felt that several issues needed to be addressed: 1) the source of the C. Bovis materials and quality control; 2) the role of taurine in the efficacy of C. Bovis. METHODS: Nine samples of natural C. Bovis and its substitutes were collected. ICP-MS was used for elemental analysis and the characterization was performed by principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA) as multivariate approaches. The efficacy of C. Bovis was evaluated for morphology, viability and beating pattern on cultured cardiac myocytes and/or fibroblasts. RESULTS: PCA and multi-elemental focus was effective in discriminating C. Bovis samples derived from different habitats. A satisfactory classification using SIMCA was obtained among Australia C. Bovis, other habitats and the substitutes. Australian samples had better batch uniformity than other habitats and were composed of fewer elements. We have used Australian C. Bovis for assessment on its bioactive compounds. Rat cardiac cells incubated with C. Bovis extract (0.01-0.1 mg/ml) maintained normal morphology, viability and beating pattern. Cardiac myocytes and fibroblasts treated for 48 h with CA (0.5 mM) or DCA (0.1 mM) caused cell injury, as reflected by changes in appearance and a reduction of viability detected by the MTS assay. In cardiomyocytes, 0.5 h exposure of CA (0.5 mM) markedly decreased the velocity ratio of beating, whereas the simultaneous addition of 1 mM taurine largely prevented the decrease. CONCLUSIONS: The multi-elemental focus provided some references for the quality control and the efficacy of C. Bovis. Taurine partly attenuated the harmful actions of bile acids. It is plausible that the relationship between taurine and the bile acids contributes to therapeutic effect of C. Bovis.

Hepatoprotective effect of syringic acid and vanillic acid on CCl4-induced liver injury.

The mycelia of the edible mushroom Lentinula edodes can be cultured in solid medium containing lignin, and the hot-water extracts (L.E.M.) is commercially available as a nutritional supplement. During the cultivation, phenolic compounds, such as syringic acid and vanillic acid, were produced by lignin-degrading peroxidase secreted from L. edodes mycelia. Since these compounds have radical scavenging activity, we examined their protective effect on oxidative stress in mice with CCl(4)-induced liver injury. We examined the hepatoprotective effect of syringic acid and vanillic acid on CCl(4)-induced chronic liver injury in mice. The injection of CCl(4) into the peritoneal cavity caused an increase in the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. The intravenous administration of syringic acid and vanillic acid significantly decreased the levels of the transaminases. Four weeks of CCl(4) treatment caused a sufficiently excessive deposition of collagen fibrils. An examination of Azan-stained liver sections revealed that syringic acid and vanillic acid obviously suppressed collagen accumulation and significantly decreased the hepatic hydroxyproline content, which is the quantitative marker of fibrosis. Both of these compounds inhibited the activation of cultured hepatic stellate cells, which play a central role in liver fibrogenesis, and maintained hepatocyte viability. These data suggest that the administration of syringic acid and vanillic acid could suppress hepatic fibrosis in chronic liver injury.

[Development of novel culture system for regulation of hepatocyte function].

Cultured hepatocytes are expected to be used for drug screening and bioartificial liver. Since hepatocytes lose their functions very rapidly in vitro, many attempts have been made to maintain their viability and functions. First, we want to introduce the surface modification of culture substrate using a starburst dendrimer. Addition of fructose to the terminal of the dendrimer was shown to be effective in maintaining hepatocyte function. As the second topic, we will show results of the use of a three-dimensional carrier for hepatocyte cultivation. Hepatocytes and bone marrow stromal cells were cocultured in silane beads, and packed into a radial flow-type bioreactor. The perfusion culture showed the effectiveness of bone marrow stromal cells for the maintenance of hepatocyte function. The next topic will be the trial of adenoviral gene transfer into hepatocytes. Thioredoxin gene was chosen because the products play important roles in redox control and antiapoptosis. The introduction of the gene could inhibit apoptosis and maintain the hepatocyte viability. Finally, we want to introduce the results on differentiation of stem cells into hepatocytes, because it is very difficult to obtain sufficient number of human hepatocytes. Human mesenchymal stem cells were cultured in the presence of several protein factors and the hepatocyte-specific marker was expressed after 2 weeks of induction culture. The use of human stem cells could be an important strategy for the support of a drug development system.

Multipotent cells from the human third molar: feasibility of cell-based therapy for liver disease.

Adult stem cells have been reported to exist in various tissues. The isolation of high-quality human stem cells that can be used for regeneration of fatal deseases from accessible resources is an important advance in stem cell research. In the present study, we identified a novel stem cell, which we named tooth germ progenitor cells (TGPCs), from discarded third molar, commonly called as wisdom teeth. We demonstrated the characterization and distinctiveness of the TGPCs, and found that TGPCs showed high proliferation activity and capability to differentiate in vitro into cells of three germ layers including osteoblasts, neural cells, and hepatocytes. TGPCs were examined by the transplantation into a carbon tetrachloride (CCl4)-treated liver injured rat to determine whether this novel cell source might be useful for cell-based therapy to treat liver diseases. The successful engraftment of the TGPCs was demonstrated by PKH26 fluorescence in the recipient's rat as to liver at 4 weeks after transplantation. The TGPCs prevented the progression of liver fibrosis in the liver of CCl4-treated rats and contributed to the restoration of liver function, as assessed by the measurement of hepatic serum markers aspartate aminotransferase and alanine aminotransferase. Furthermore, the liver functions, observed by the levels of serum bilirubin and albumin, appeared to be improved following transplantation of TGPCs. These findings suggest that multipotent TGPCs are one of the candidates for cell-based therapy to treat liver diseases and offer unprecedented opportunities for developing therapies in treating tissue repair and regeneration.

Fucoidan partly prevents CCl4-induced liver fibrosis.

Fucoidan, a sulfated polysaccharide extracted from brown algae, has a wide range of biological activities, including anti-inflammatory, anti-viral, and anti-tumor activities. In the present study, we investigated the effects of fucoidan on CCl4-induced liver fibrosis. Administration of fucoidan reduced CCl4-induced acute and chronic liver failure. Hepatic fibrosis induced by CCl4 was also attenuated by injection of fucoidan. Damage to hepatocytes and activation of hepatic stellate cells are key events in liver fibrosis, and, interestingly, treatment of hepatocytes with fucoidan prevented CCl4-induced cell death and inhibited the proliferation hepatic stellate cells. These results indicate that fucoidan might be a promising anti-fibrotic agent possessing dual functions, namely, protection of hepatocytes and inhibition of hepatic stellate cell proliferation.

Glucose transporter mediation responsible for morphological changes of human epithelial cells on glucose-displayed surfaces.

Cellular morphology is one of the important factors for coordinating cell signaling. In this study, the morphological variation via glucose transporter (GLUT)-mediated anchoring was investigated in the cultures of human mammary epithelial cells in the presence or absence of insulin on culture surfaces with the changed ratios of d- and l-glucose displayed. With increasing ratio of d-glucose displayed on the surfaces, the cells showed a stretched shape in the culture with 10 mug/cm(3) insulin, reaching the highest extent of cell stretching at 100%d-glucose display, whereas round cells were predominant at 0%d-glucose display. In the absence of insulin, on the other hand, the extent of cell stretching showed a concave profile in terms of the ratio of d-glucose displayed, the extent being highest at 50%d-glucose display. Blocking of integrin alpha(5)beta(1) or GLUTs1 and 4 on the cells using corresponding antibodies revealed that the primary mechanism for cell attachment was based on integrin-mediated binding, and that GLUTs1 and 4 contributed largely to morphological changes of cells. Confocal microscopy further revealed that GLUT4 localization occurred in response to d-glucose display as well as insulin addition. In the absence of insulin, GLUT4 spots were extensively observed in the cell body regardless of whether d-glucose was displayed or not. However, in the presence of insulin, the broad distribution of GLUT4 appeared on the basal and apical sides of cells at 100%d-glucose display, in contrast with its localization only on the apical side of cells at 0%d-glucose display. These results suggest that the quantitative balance between GLUTs on the cytoplasmic membrane and d-glucose displayed on a culture surface determines the cell morphology, as explained by the receptor saturation model.

[Application of mesenchymal stem cells to liver regenerative medicine].

Stem cell-based therapy has received attention as a possible alternative to organ transplantation, owing to the ability of stem cells to repopulate and differentiate at the engrafted site. We transplanted bone marrow-derived mesenchymal stem cells (BMSCs) into liver-injured rats to test the therapeutic effect. Rat bone marrow cells were cultured in the presence of hepatocyte growth factor (HGF). RT-PCR and immunocytochemical analysis indicated that the BMSCs expressed the albumin mRNA and the production of protein after cultivation with HGF for 2 weeks. The BMSCs appeared to differentiate into hepatocyte-like cells in response to the culture with HGF. After labeling with a fluorescent marker, the BMSCs were transplanted into CCl(4)-injured rats by injection through the caudal vein. The liver was excised and blood samples were collected 4 weeks later. Engraftment of the transplanted BMSCs was seen with significant fluorescence in the injured liver. Transplantation of the BMSCs into liver-injured rats restored their serum albumin level and suppressed transaminase activity and liver fibrosis. Therefore, BMSCs were shown to have a therapeutic effect on liver injury. Recently, we have been trying to use mesenchymal stem cells isolated from dental papilla of discarded human wisdom teeth. Autologous transplantation of mesenchymal stem cells from bone marrow and dental papilla could be ethically and functionally promising for stem cell-based therapy.

Environmentally Friendly Measurement of Airborne Radon Using a Nonvolatile Liquid Scintillation Absorbent.

The practicality of using a liquid scintillation method with a nonvolatile liquid scintillation absorbent for the measurement of airborne Rn (radon) in a residence was examined. The relationship between the radioactivity absorbed by the liquid scintillation absorbent and the radon concentration in the air was investigated in a calibrated walk-in radon chamber. The equivalent radioactivity of radon was calculated for Po radioactivity immediately after radioactive equilibrium was attained using successive decay equations via alpha-particle spectrometry based on the 1 h, indirect, selective measurement of the Po alpha-particle spectrum generated after sampling radon. We confirmed that the amounts of radon absorbed in the liquid scintillation absorbent were proportional to the radon concentration in the air. The calibration curve that exhibited reliable quantitative linearity from 500 to 8,000 Bq m in air was extrapolated to the region between 0 and 500 Bq m using the least-squares method with data from 500 to 8,000 Bq m. The validity of the extrapolated curve at less than 500 Bq m was confirmed by comparison of the measured radon concentrations in the room and atmosphere with those determined using an existing ionization chamber. Variations in the absorption of radon were observed due to changes in temperature and humidity. The health and environmental safety of nonvolatile liquid scintillation absorbent was also considered.

Morphological regulation of rabbit chondrocytes on glucose-displayed surface.

A culture surface was designed to regulate morphology of rabbit chondrocytes by changing the ratio of D- and L-glucose isomers displayed on a glass plate. With increasing ratio of d-glucose displayed on the surfaces, the efficiency of cell attachment improved, meaning that the attachment exclusively occurred via mediation of an affinity between D-glucose displayed and glucose transporter on cell membrane. At 0% and 100% D-glucose display, the round-shaped cells appeared dominantly, and most of cells became stretched in shape at 50% d-glucose display, indicating that the frequency of round-shaped cells depicted a concave profile against the ratio of D-glucose displayed. From the cytoskeletal staining of F-actin and vinculin, the immature stress fibers with fewer focal contacts were recognized in both the round shaped cells and those stretched in shape on 100% D-glucose-displayed surface. The time-lapse observation revealed that the cells on 100% D-glucose-displayed surface conducted active migration and aggregation with formation of collagen type II. These results suggest that 100% D-glucose-displayed surface can offer culture environment to maintain the chondrocytic phenotype of cells, similarly to the conditions achieved in three-dimensional (3-D) culture.

Synergistic effect of D-glucose and epidermal growth factor display on dynamic behaviors of human epithelial cells.

We investigated the synergistic effect of D-glucose and epidermal growth factor (EGF) display on the dynamic cellular behaviors of morphology and migration in a culture of human epithelial cells. The time-lapse observation revealed that the cells on the D-glucose/EGF-displayed substrate were endowed with enhanced migration, accompanied with periodic changes in morphology between round and stretched shapes. Immunofluorescence staining of phosphotyrosine PY20 and vinculin was conducted to determine the intracellular localization of phosphorylated tyrosine expression and focal contact formation, respectively. On the substrate displaying D-glucose and EGF, the cells exhibited increases in the levels of the expression of phosphorylated tyrosine and the formation of focal contacts not only at the cellular periphery but also in the cell body. These findings supported the consideration that the displayed D-glucose causes the cells to be in close contact with the surface via grasping glucose transporters on the cytoplasmic membrane.

Response of human epithelial cells to culture surfaces with varied roughnesses prepared by immobilizing dendrimers with/without D-glucose display.

To investigate the response of human epithelial cells to substrates with nanoscale modifications, dendrimer-immobilized surfaces were prepared with or without D-glucose displayed as a terminal ligand, giving topographic structures with mean roughnesses (R(a)) of 1.8-11.0 nm. With an increase in the R(a) value up to 4.0 nm, the epithelial cells cultured on naked dendrimer surface without D-glucose display were somewhat stretched in their morphology compared with those on a nonmodified plain surface. However, for the R(a) values higher than 4.0 nm, such cell stretching was inhibited, resulting in the predominant existence of round-shaped cells. The change in cell morphology was appreciable on the surfaces with D-glucose-displayed dendrimers. When the R(a) value increased up to 4.5 nm on these surfaces, in particular, the enhancement of cell stretching was recognized, and fluorescence microscopic observation supported the hypothesis that the glucose-transporter-mediated adhesion of cells to the surface encouraged the development of filopodia and stress fibers, thereby improving focal contact with the surface. Our results suggest that the combination of displaying D-glucose and modulating roughness can promote cytoskeletal formation accompanied by marked cell elongation on culture surfaces.

Effects of Normothermic Conditioned Microwave Irradiation on Cultured Cells Using an Irradiation System with Semiconductor Oscillator and Thermo-regulatory Applicator.

We investigated the effects of microwave irradiation under normothermic conditions on cultured cells. For this study, we developed an irradiation system constituted with semiconductor microwave oscillator (2.45 GHz) and thermos-regulatory applicator, which could irradiate microwaves at varied output powers to maintain the temperature of cultured cells at 37 °C. Seven out of eight types of cultured cells were killed by microwave irradiation, where four were not affected by thermal treatment at 42.5 °C. Since the dielectric properties such as ε', ε" and tanδ showed similar values at 2.45 GHz among cell types and media, the degree of microwave energy absorbed by cells might be almost the same among cell types. Thus, the vulnerability of cells to microwave irradiation might be different among cell types. In HL-60 cells, which were the most sensitive to microwave irradiation, the viability decreased as irradiation time and irradiation output increased; accordingly, the decrease in viability was correlated to an increase in total joule. However, when a high or low amount of joules per minute was supplied, the correlation between cellular viability and total joules became relatively weak. It is hypothesized that kinds of cancer cells are efficiently killed by respective specific output of microwave under normothermic cellular conditions.

Chiral separation of acidic drug components by open tubular electrochromatography using avidin immobilized capillaries.

Avidin was immobilized covalently onto the inner surface of fused silica capillaries as a stationary phase in an open tubular capillary electrochromatography (OT-CEC) for chiral separations. The modification was attained by a combination of glutaraldehyde with both an amino-silylated fused silica surface and avidin using a Schiff base formation reaction. This method couples the advantage of high efficiency and small consumption of a chiral selector with the possibility of UV detection without limitations of protein absorption. In addition, the prepared capillary was stable for 50 days with over 100 runs. To evaluate the electrochromatographic performance of the prepared capillaries, the chiral separation of abscisic acid and arylpropionic acids were investigated. Effects of the modification condition of protein, pH of running buffer, and an organic modifier on the enantioseparation were also investigated. In addition, the avidin immobilized capillary was employed for the selective chiral analysis with an electrospray ionization mass spectrometry (ESI-MS) detection scheme.