Homoeriodictyol-7-O-β-D-glucopyranoside (HEDT-Glu) was isolated from Viscum coloratum and identified by MS, 1H- and 13C-NMR. A HPLC method was developed for determination of HEDT-Glu in rat plasma and tissues. All biological samples were pretreated by protein precipitation with acetone. Vanillin was selected as internal standard. The mobile phase consisted of methanol–water–glacial acetic acid (45 : 55 : 0.5, v/v/v). Good linearity were observed over the concentration ranges of 0.1—200.0 μg·ml−1 in rat plasma and 0.05—5.0 μg·ml−1 in tissues. Both intra- and inter-day precisions of HEDT-Glu, expressed as the relative standard deviation, were less than 13.1%. Accuracy, expressed as the relative error, ranged from −0.8 to 5.4% in plasma and from −5.6 to 9.4% in tissues. The mean extraction recovery of HEDT-Glu was above 73.17% in biological samples. The described assay method was successfully applied to the pre-clinical pharmacokinetic study of HEDT-Glu. After intravenous administration of HEDT-Glu to rat, AUC and CLtot were 16.04±3.19 μg·h·ml−1 and 0.85±0.17 l·kg−1·h−1, respectively. T1/2,α and t1/2,β were 0.06±0.01 h and 1.27±0.31 h, respectively. HEDT-Glu was cleared from the blood and mainly distributed to the liver and small intestine.
Lonicera japonica THUNB. is a commonly used anti-inflammatory herbal medicine. The therapeutic effectiveness of L. japonica depends significantly on its geographical origin. However, it is difficult to define criteria for confirming geographical authenticity using microscopic and chemical characteristics. In the present study, the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA loci of L. japonica from different origins and related species was sequenced. The mutation site found in the ITS region from geo-authentic L. japonica can be recognized by the restriction endonuclease EcoN I. Since PCR products from geo-authentic L. japonica cannot be digested completely, a quantitative restriction fragment length polymorphism analysis method was developed. The cleavage rate of PCR products by EcoN I was determined to be more than 70% in all geo-authentic L. japonica and less than 20% in non-geo-authentic L. japonica and other species from genus Lonicera. The rate correlated remarkably with the geographical origin of L. japonica. Therefore, this method can be used to classify geo-authentic L. japonica.
Macrophages play essential roles in the innate immune system. In this study, we show that macrophage functions such as phagocytosis and cytokine/chemokine expressions display a circadian rhythm that is regulated by a molecular clock. Phagocytosis, a crucial early reaction by which macrophages protect their host against foreign particles, exhibited a circadian variation that peaks during the light period and bottoms during the dark period. These diurnal changes of phagocytosis activity in macrophages were induced without exogenous stimulants such as bacterial infection. The expression of the clock genes including brain and muscle Arnt-like protein-1 (BMAL1) exhibited robust circadian rhythms in macrophages. The expression patterns of the clock genes in macrophages were similar to those in the suprachiasmatic nucleus and other peripheral tissues. Among inflammation factors examined, the level of monocyte chemoattractant protein-1 (MCP-1/JE) mRNA exhibited most robust circadian oscillation. Expression of other cytokines such as IL-1β, IL-6 and TNFα showed mild diurnal changes. Knockdown of the BMAL1 expression resulted in a decrease of the MCP-1/JE mRNA level in macrophages. BMAL1 increased significantly but weakly MCP-1/JE promoter activity. MCP-1/JE promoter activity is known to be regulated by nuclear factor-kappa B (NF-κB). NF-κB activity in BMAL1 knockdown macrophages was lower than that in control cells. Consequently, the circadian expression of MCP-1/JE in macrophages is regulated by BMAL1 through the activation of NF-κB. The results obtained in this study indicate that the innate immunoreactions involving macrophages are at least partly regulated by the autonomous clock machinery.
Peroxisome proliferators (PxPs) induce peroxisomal β-oxidation (Px-ox) in the liver of rodents and have a hypolipidemic function. To investigate hypolipidemic effect of PxPs, the relationship between TG fluctuation and Px-ox activity, as an indicator of the function of PxPs, was studied in primary cultured rat hepatocytes. Nafenopin (Nf) treatment of hepatocytes caused an increase in Px-ox activity in association with cellular TG accumulation in a time-dependent manner with a coefficient of r=0.918. This relationship between the activity and cellular TG were obtained using structurally diverse PxPs with a correlation coefficient of r=0.747. Treatment of the hypolipidemic drug, but non-PxP Pravastatin, decreased TG in the medium, but did not have the effects on cellular TG and Px-ox activity. The total amount of TG and diacylglycerol acyltransferase activity, the last enzyme in the TG de novo synthesis pathway, were not affected by Nf treatment. When hepatocytes were cultured with Brefeldin A, cellular TG was accumulated, the same as with Nf, however, Px-ox activity was not enhanced. Nf treatment markedly decreased the level of apolipoprotein B (apo B) in very low density lipoprotein (VLDL) fractions prepared from conditioned media and increased that of cellular apoB by Western blot analysis. Microsomal triglyceride transfer protein activity was not influenced by Nf. Together, with regards to TG lowering effect of PxPs, it is suggested that PxPs cause hepatocellular accumulation of TG without effects on TG biosynthesis and VLDL construction, and they might have inhibitory effect on VLDL secretion process.
Buckwheat is an ancient and specialty grain in China. Due to its unique chemical and bio-activity components, buckwheat has been found to have many uses in food products and medicine. However, very little is known about the toxicity of protease inhibitors from buckwheat. Here, the possible effects of a recombinant buckwheat trypsin inhibitor (rBTI) on the induction of apoptosis of the human K562 cell line were investigated by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays and flow cytometric analysis. MTT assay showed that rBTI could specifically inhibit the growth of K562 cells in a dose-dependent manner, but there were minimal effects on normal human peripheral blood mononuclear cells (PBMCs). Furthermore, comparison the effects of rBTI on K562 cells with those of negative control (BSA and the complex of BSA and rBTI) revealed that rBTI was highly toxic to K562 cells, and BSA hardly had any inhibition on proliferation in K562 cells. The analysis of flow cytometric indicated that the apoptosis of K562 cells were 31.0%, 32.8%, 35.3% and 52.1% after treated by rBTI in range of 12.5—100 μg/ml, respectively. The results suggested that rBTI can induce apoptosis of K562 cells and that it might be a potential protein drug of the trypsin inhibitor family.
Purpose: Vascular endothelial growth factor C (VEGF-C) is the most important molecule in lymphangiogenesis and its relationship with lymph node metastasis has attracted considerable interest. We investigated the relationship of VEGF-C or VEGF-A with clinicopathological factors in gastric cancer patients. Methods: Eighty gastric cancer patients who underwent gastric resection were analyzed immunohistochemically for expression of VEGF-C and VEGF-A protein. Results: Positive immunoreactivity of VEGF-C and VEGF-A was observed in 75 (93.8%) and 41 (51.3%) patients, respectively. VEGF-A expression was significantly correlated with tumor differentiation (p=0.0017) and vascular invasion (p=0.0004). And positive relationship of VEGF-C expression was only demonstrated with tumor differentiation (p=0.0168). Interestingly, however, the frequency of lymph node metastasis was significantly higher in the patients with expression of both VEGF-C and VEGF-A (strong positive expression, p=0.036). Furthermore, the expression of both was also significantly correlated with depth of tumor invasion, tumor differentiation, lymphatic invasion, and vascular invasion. Conclusion: The present results suggest that strong expression of VEGF-A in addition to VEGF-C expression is essential in lymph node metastasis, presumably because enhanced metastatic potential including lymphangiogenesis induced by both VEGF-A and VEGF-C is vital in lymph node metastasis of gastric cancer.
Neuroblastoma (NB) often causes spontaneously regression, and can mature to ganglioneuroma. The form with the most favorable prognosis expresses high levels of TrkA, a high-affinity receptor for nerve growth factor (NGF), whereas advanced NB and associated cell lines have abnormalities in the NGF/TrkA signaling pathway. A novel cyclophane, cyclophane pyridine (CPPy), was designed to conserve the tyrosine phosphorylation of TrkA, thereby enhancing NGF/TrkA signal transduction. We investigated whether this compound improved NGF-induced tyrosine phosphorylation of the Y490 domain of TrkA and conserved the expression of an early gene (c-fos) in human NB cell lines (IMR-32 and NB-39). As determined by Western blotting, TrkA (Y490) phosphorylation was enhanced by the combination of CPPy (10−8 M) and NGF (100 ng/ml) compared with NGF alone. CPPy also conserved NGF-induced c-fos mRNA expression. Moreover, CPPy induced the morphological differentiation of NB cells, leading to expression of the neuronal marker gene GAP-43. These data suggest that CPPy can induce the differentiation of NB cell lines by facilitating NGF-induced TrkA/Ras/MAPK signal transduction, and may therefore be an effective therapeutic agent for NB.
Many hypoglycemic and hyperglycemic episodes associated with clinical use of gatifloxacin (GFLX), a novel fluoroquinolone antimicrobial agent, have been reported in recent years. Some have reported hypoglycemia induced by fluoroquinolones, indicating that these agents may stimulate insulin secretion from pancreatic islet cells. In this study, we investigated the effect of GFLX on insulin homeostasis in islet cells using the insulin secreting cell line, HIT-T15. After 1 h incubation with over 100 μM of GFLX, insulin secretion from the cells was significantly augmented. However, the augmentation of insulin release induced by GFLX subsequently reached a plateau. Coincidentally, cellular insulin was decreased by 120 h incubation, and reactivity to re-stimulation by sulfonylurea was suppressed. The GFLX insulin depletion effect was stronger than the effects produced by such other fluoroquinolones as levofloxacin and ciprofloxacin. This study suggests that GFLX should induce insulin oversecretion from pancreatic islet cells in the short-term, and decrease insulin productivity or increase insulin disintegration in the long-term. These results are consistent with the clinical results of GFLX finding that hypoglycemic episodes were seen after a first single administration, and most hyperglycemic episodes were seen more than 2 d after the start of administration.
Mat-8 was fused with a Myc-tag or green fluorescent protein at its carboxyl terminus, and then expressed in Chinese hamster ovary K1 cells. Determination of the cellular localization of the tagged proteins suggested that they were localized on the intracellular membrane, being not only detected around the nuclear envelope but also partly overlapping with markers for endosomes and Golgi bodies. However, Mat-8 with the Myc-tag was detected on the plasma membrane as well as the intracellular membrane, when it was expressed in colorectal cancer cells. The membrane fraction of the cancer cells was solubilized and immuno-precipitated with an antibody for the Myc-tag. Western-blotting analysis demonstrated that the Na+/K+-ATPase α subunit was present in the precipitate. Furthermore, the immuno-precipitate obtained with an antibody for the Na+/K+-ATPase α subunit reacted with that for the Myc-tag. These results suggested that Mat-8 could be associated with Na+/K+-ATPase similar to other FXYD family members. The Gly41→Arg mutation in the transmembrane region of Mat-8 inhibited its association with the Na+/K+-ATPase α subunit and localization on the plasma membrane, whereas the Cys44→Ala or Cys49→Ala substitution did not. Thus the conserved Gly41 residue in the transmembrane domain could be indispensable for localization of Mat-8 on the cell surface.
Cytotoxic T cells and natural killer cells play key roles in cell-mediated cytotoxicity and can induce apoptosis in virus-infected and malignant cells by releasing cytotoxic granules. In the current study, apoptosis was induced in Jurkat cells, a human T cell line, by delivering granzyme B into the cells using BioPORTER®, a cationic lipid formulation. During granzyme B-induced apoptosis, there was an increase in the cell surface expression of Lewis X and Y antigens. To clarify the roles of initiator and executioner caspases in the expression of Lewis X and Y antigens, we treated Jurkat cells with granzyme B in the presence of caspase 3, 8, and 9 inhibitors. The results indicated that delivery of granzyme B into Jurkat cells induces apoptosis by activating caspase 3 and that caspase 3 but not caspase 8 and 9 plays a key role in enhancing the expression of Lewis X and Y antigens. Real-time PCR revealed that expression of the mRNAs for α1,3-fucosyltransferases FUT4 was increased at 3 h during granzyme B-induced apoptosis, while FUT9 mRNA expression gradually increased after 12 h. This increased expression of FUT4 mRNA occurred downstream of caspase 3 activation and resulted in the increased cell surface expression of Lewis X and Y antigens.
Nerve growth factor (NGF)-induced neurite-outgrowth of PC12 cells was enhanced by polyoxometalates such as Na9[SbW9O33]·19.5H2O (SbW9) and (NH3Pri)6[Mo7O24]·3H2O (Mo7). Western blotting analysis of polyoxometalate/NGF-treated PC12 cells showed a strong expression of growth-associated protein 43 (GAP-43), which is associated with the neurite outgrowth. Similar effects were observed for other polyoxometalates, K11H[(VO)3(SbW9O33)2]·27H2O ((VO)3(SbW9)2), K6[P2W18O62]·14H2O (P2W18), and K7[PTi2W10O40]·6H2O (PTi2W10), in contrast with little effect for monomeric tungstate and molybdate. Of the polyoxometalates tested in this study, SbW9 and Mo7 were the most potent.
In this study, we examined the effect of Cilostazol to induce metallothionein (MT) in vivo and in vitro. Intraperitoneal injection of Cilostazol increased the expression of both MT-1 and MT-2 mRNA and total MT protein in the mouse liver. Cilostazol also augmented MT-1 mRNA levels in the murine brain. In vitro exposure to Cilostazol significantly augmented intracellular MT protein levels in cultured human brain microvascular endothelial cells (HBMEC) and in the neuroblastoma cell line IMR32. Taken together, these findings suggest that Cilostazol is an inducer of MT in the murine liver and brain, and that it has the potential to directly induce MT in cells. The contribution of the anti-oxidative effect of MT to the anti-stroke effect of Cilostazol was discussed.
Benzastatin C, a 3-chloro-tetrahydroquinolone alkaloid from Streptomyces nitrosporeus, showed antiviral activity in a dose-dependant manner with EC50 values of 1.92, 0.53, and 1.99 μg/ml against herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and vesicular stomatitis virus (VSV), respectively. In contrast, benzastatin D, the corresponding dechlorinated derivative, did not exhibit any antiviral activity. These results indicate that the antiviral activity of benzastatin C is mediated, in part, due to the chlorine moiety in its molecular structure.
We cloned a gene smfY for multidrug efflux pump from chromosomal DNA of Serratia marcescens using drug-hypersensitive Escherichia coli KAM32 as the host, and characterized the pump. E. coli KAM32/pESM42 carrying the smfY showed significantly increased MICs of various drugs including DAPI, norfloxacin, benzalkonium chloride, acriflavine and ethidium bromide, compared with the control. We also detected energy-dependent ethidium and acriflavine efflux due to the SmfY. Sequence analysis revealed that the SmfY was a multidrug efflux pump of the MF (Major Facilitator) superfamily transporters. This is the first report of a multidrug efflux pump belonging to the MF superfamily in S. marcescens.
Phytochemicals contained in dietary plants provide a variety of health benefits and may reduce the risk of cardiovascular diseases. The aqueous extracts from three popular Thai dietary and herbal plants, Cratoxylum formosum, Syzygium gratum, and Limnophila aromatica, were investigated for the antioxidant and vascular protective activities in the in vitro and in vivo models. The free radical scavenging and antioxidant activities of plant extracts were evaluated in vitro by the 1,1-diphenyl-2-picrylhydrazyl assay, the ferric reducing antioxidant power assay, the intracellular antioxidant activity in rat peritoneal macrophages by dihydrofluorescein assay, and the inhibition of nitric oxide (NO) production in RAW 264.7 macrophages. In an animal model of oxidative stress and vascular dysfunction, male Sprague-Dawley rats were orally administered with aqueous plant extracts (1 g/kg/d) or N-acetylcysteine (NAC; 300 mg/kg/d) as a control for 6 d. On day four, all animals except the normal control group, were administered with phenylhydrazine (PHZ) intraperitoneally. It was demonstrated that the plant extracts possessed high free radical scavenging and antioxidant activities. PHZ induced severe hemolysis and hemodynamic disturbances and treatment with the extracts and NAC significantly improved the hemodynamic status. Vascular responsiveness to bradykinin, acetylcholine, and phenylephrine in PHZ-control rats was markedly impaired, and the plant extracts or NAC largely restored the vascular responses. Moreover, the plant extracts prevented loss of blood reduced glutathione and suppressed formation of plasma malondialdehyde, plasma NO metabolites and blood superoxide anion. It was concluded that the plant extracts possess antioxidants and have potential roles in protection of vascular dysfunction.
1H-NMR spectroscopy of urine has been proved to be a powerful tool in metabonomic investigations in the field of chemical toxicity evaluation and disease diagnosis. However, most studies on urinary metabolite profiling by 1H-NMR have been conducted using rat urine. In the course of experiment on 1H-NMR analysis of urine samples of mice administered selenium compound, we noticed a substantial variation of chemical shift of citrate among individual mice. To clarify the effect of urinary pH on chemical shift of citrate, we compared 1H-NMR spectra of urine samples obtained from selenium-treated mice and untreated mice as well as those from untreated rats. The results clearly showed a linear relationship between urinary pH and chemical shift of citrate in 1H-NMR spectra both in mice and rats. The urine of mice exhibited a wider variation of pH, resulting in a wider variation of chemical shift of citrate than that of rats. We also recognized a clear peak of trimethylamine in urine of mice, but not that of rats. These data indicate that more attention should be paid to the characteristics of mouse urine with special reference to pH and trimethylamine metabolism in the analysis of NMR spectroscopy.
Extracellular nucleotides have multiple biological actions in processes such as proliferation, differentiation, chemotaxis, and cytokine secretion through P2X receptors on the cell surface. To determine the biological activity of adenosine triphosphate (ATP) and the expression of P2 nucleotide receptors in murine bone marrow-derived hematopoietic cells and stem cells/progenitor cells, we investigated the effects of ATP in assays of cell proliferation and cell death in vitro. Our results demonstrated that several subtypes of P2X receptors were expressed on hematopoietic cells and that P2X7, in particular, was partially expressed in hematopoietic stem cells/progenitor cells. In addition, stimulation of hematopoietic cells with high concentrations of ATP caused severe inhibition of cell proliferation despite the presence of cytokine stimulation. We analyzed the apoptotic effects of stimulation with several different dosages of ATP and confirmed the enhanced apoptotic activity in hematopoietic cells and progenitor cells. Antagonists, against P2X receptors and ATP, suramin and oxidized ATP, inhibited the induction of cell death for murine hematopoietic cells. Our data suggest that extracellular nucleotides may provide a novel and powerful tool for regulating the cell fate of hematopoietic stem cells.
Amino acids, the building blocks of proteins, play significant roles in numerous physiological events in mammals. As the effects of amino acids on melanogenesis have yet to be demonstrated, the present study was conducted to identify whether amino acids, in particular alanine, glycine, isoleucine and leucine, influence melanogenesis in B16F0 melanoma cells. Glycine and L-isoleucine, but not D-isoleucine, reduced melanogenesis in a concentration-dependent manner without any morphological changes in B16F0 melanoma cells. L-Alanine and L-leucine, but not D-alanine and D-leucine, also reduced melanogenesis without any morphological changes in B16F0 melanoma cells. However these amino acids did not show a concentration-dependency. Combination of L-alanine and the other amino acids, particularly 4 amino acids combination, had an additive effect on the inhibition of melanogenesis compared with single treatment of L-alanine. None of the amino acids affected the activity of tyrosinase, a key enzyme in melanogenesis. These results suggest that L-alanine, glycine, L-isoleucine and L-leucine, but not the D-form amino acids, have a hypopigmenting effect in B16F0 melanoma cells, and that these effects are not due to the inhibition of tyrosinase activity. Combination of these 4 amino acids had the additive effect on hypopigmentation that was as similar as that of kojic acid.
Flavonoids have been reported to be potent antioxidants and beneficial in oxidative stress related diseases. Quercetin, a major flavonoid in food, deserves much attention because of its antioxidative activity. However, the actions of flavonoids including quercetin are complex and paradoxical. Quercetin caused apoptosis and/or cell death in various cells including cancer cells and normal cells. In this study, we investigated the effects of quercetin with or without hydrogen peroxide (H2O2) on cell death of PC12 cells, a neuronal cell line. We showed that quercetin at 10—30 μM alone caused cell death accompanied by caspase-mediated DNA fragmentation in undifferentiated PC12 cells. Quercetin did not inhibit and rather enhanced 0.1 mM H2O2-induced cell death. The toxic effect of quercetin was not inhibited by antioxidants such as N-acetylcysteine and GSH, although H2O2-induced cell death was inhibited by the antioxidants. Quercetin-induced cell death was reduced by 2 h treatment with nerve growth factor and serum. In addition, quercetin caused cell death in differentiated PC12 cells that were cultured with nerve growth factor for 6 d. Genistein, a soy isoflavone that has the pro-apoptotic activity, also caused cell death with DNA fragmentation. Further evaluation of the potential of dietary flavonoids as neuroprotective reagents is needed.
γ-Aminobutyric acid (GABA) was administered orally to rats for 60 d after excision of five-sixths of their kidney volume. A decrease in renal function parameters was observed in these nephrectomized rats. However, the administration of GABA ameliorated renal dysfunction, and a longer administration period of GABA increased its protective effect. In addition, tubular fibrosis was markedly increased at 10 and 60 d in nephrectomized control rats, while GABA administration for 10 d reduced tubular fibrosis to the normal level. Tubular atrophy was markedly induced by nephrectomy, and was significantly reduced by the administration of GABA at 60 d. Furthermore, the nephrectomized control rats exhibited an increased expression level of transforming growth factor-β1, where GABA significantly decreased it after administration for 10 d. The expression of fibronectin in the tubuli of rats administrated GABA for 60 d was completely and dose-dependently reduced as compared with nephrectomized control rats. However, the improvement effects in glomeruli were less. We also found that GABAA and GABAB receptors were specifically localized in tubuli. Specific agonists for GABAA and GABAB receptors improved renal function. These results suggest that GABA may have a beneficial effect on renal function in nephrectomized rats by inhibiting fibrosis and atrophy primarily in tubuli, and that it ameliorates losses of renal function in renal failure.
Reactive oxygen species (ROS) are involved in the deleterious effects of UV light on skin. The antioxidant defense system is considered to be crucial for protecting skin from ROS. Recently, we showed that fructose 1,6-diphosphate (FDP), a glycolytic metabolite, reduced oxidative stress in UVB-irradiated keratinocytes. This study set out to determine whether topically applied FDP could exert protective effects against UV-induced skin damage in hairless mice. An in vitro skin permeation study using Franz-type diffusion cells showed that the amount of [14C]-FDP that diffused through the skin increased in a time-dependent manner, and about 3.5% of the applied FDP penetrated the skin after 24 h. Topical application of FDP (1%) preserved the endogenous antioxidant capacity of skin such as catalase and glutathione, which were significantly reduced after UVB irradiation without FDP. FDP also reversed the loss of catalase protein and prevented the accumulation of carbonylated proteins induced by UVB irradiation. These results provide evidence that topically administered FDP could penetrate into the skin and attenuate UVB-induced oxidative skin damage in hairless mice.
In the present study, we examined the effects of nilvadipine and amlodipine, both dihydropyridine-derivative calcium antagonists, on the impairment of spatial memory induced by a combination of ischemia and β-amyloid (Aβ). Nilvadipine (3.2 mg/kg, i.p.) significantly prevented the impairment of spatial memory and neuronal apoptosis in this model. By contrast, amlodipine had no effect on this impairment of spatial memory. These findings suggest that nilvadipine may prevent impairment of spatial memory by inhibiting neuronal apoptosis; this drug might therefore be useful for the prevention of the progression to dementia in Alzheimer's disease (AD).
Triptolide is a potential anti-immune agent, and has shown multi-organic toxicity, however its toxic mechanism remained undiscovered. This paper aimed at characterizing the pharmacokinetic profiles of triptolide in rats to provide the clue to approach the toxic mechanism. The absorption, distribution, metabolism and excretion of triptolide were investigated in male Sprague-Dowley rats after single doses of oral and i.v. administration. After oral administration of 0.6, 1.2 and 2.4 mg/kg, the concentration of triptolide in plasma reached the maximum within 15 min, and declined rapidly with an elimination half-life from 16.81 to 21.70 min. The triptolide kinetics was fitted into one-compartment model after i.v. administration. Oral absolute bioavailability was 72.08% at the dose of 0.6 mg/kg. Triptolide was also rapidly distributed and eliminated in all selected tissues. Less than 1% triptolide of the dose was recovered from the bile, urine or feces as parent drug within 48 h. While triptolide could not be detected in tissues and plasma at 4 h post dose, rats in the group C (oral: 1.2 mg/kg) and D (oral: 2.4 mg/kg) showed obvious toxic response to triptolide and some of rats even died out. It was indicated that triptolide was metabolized extensively, eliminated rapidly, and also showed that the toxicity produced by the triptolide was lag behind the exposure concentration.
We investigated the neuritogenic effects of Tremella fuciformis (TF), which has been valued in traditional Chinese medicine as a remedy with nutritive and tonic actions, on PC12h cells. The cognitive improving effects of TF on scopolamine-induced (2 mg/kg, s.c.) amnesia in rats were also evaluated with using the Morris water maze task and by performing choline acetyltransferase (ChAT) immunohistochemistry. The water extract of TF (0.01—1 μg/ml) promoted neurite outgrowth of the PC12h cells in a dose dependent manner. TF was highly efficient at the concentration range of 0.1—1 μg/ml. Oral daily treatment with TF (100 or 400 mg/kg) for 14 consecutive days significantly reversed the scopolamine-induced deficit in learning and memory, and it alleviated decrease in cholinergic immunoreactivity induced by scopolamine in the medial septum and hippocampus. The results demonstrate that the promotion of neuritogenesis in neuronal culture cells by TF water extract is related with its activity for improving the performance of rats on a spatial learning and memory task. Moreover, the impairments of spatial learning and memory may be attributable to the decrease in activation of the septohippocampal cholinergic system and that TF ameliorated learning and memory deficits partly through its increasing the central cholinergic activity. Therefore, TF could represent a potentially useful agent that is able to improve the function of impaired cognitive processes.
Gastrodin is a component extracted from the rhizome of Gastrodia elata, and has been shown to possess protective effects against neuron damage induced by simulated cerebral ischemia in previous studies. But its neurochemical effects on the ischemic brain had not been well studied. The present study aimed at evaluating the effects of gastrodin on the changes of transmitter amino acids in rat hippocampus during cerebral ischemia/reperfusion. Microdialysis sampling was performed during ischemia and early reperfusion periods in rats, and the glutamate and gamma-aminobutyric acid (GABA) in the dialysate were measured using high-performance liquid chromatography (HPLC). Administration of gastrodin (100 mg/kg) before ischemia significantly reduced the ischemia-induced elevation of glutamate levels during the postischemic period, increased the rise of extracellular GABA during the reperfusion periods, thus decreased the glutamate/GABA ratios during ischemia and reperfusion. These results provide insights to explain the neurochemical effects of gastrodin when applied prior to an ischemic event.
Platelet derived growth factor (PDGF)-BB is one of the most potent vascular smooth muscle cell (VSMC) proliferative factors, and abnormal VSMC proliferation by PDGF-BB plays an important role in the development and progression of cardiovascular problems, including restenosis after coronary angioplasty and atherosclerosis. Previous phytochemical studies on the stems or root barks of Cudrania tricuspidata (Moraceae) resulted in the isolation of various isoprenylated xanthones and flavonoids, some of which have anti-cancer, hepatoprotective, anti-inflammatory and anti-oxidant activities. In the present study, we investigated the antiproliferative effect of cudratricusxanthone A isolated from the root bark of C. tricuspidata and its underlying mechanism in VSMCs. Antiproliferative effects of cudratricusxanthone A on VSMCs were examined by direct cell counting and [3H]-thymidine incorporation assays. Cudratricusxanthone A inhibited [3H]-thymidine incorporations into DNA in VSMCs that occurred in response to treatment with 50 ng/ml PDGF-BB. PDGF-BB-stimulated DNA synthesis was significantly reduced to 86.1, 80.2, 64.2 and 25.1% at concentrations of 0.1, 1, 2 and 3 μM, respectively. Moreover, pre-treatment with cudratricusxanthone A (0.1—3 μM) suppressed this PDGF-BB-stimulated cell number in a concentration-dependent manner. The inhibition percentages were 11.1, 22.7, 51.3 and 81.5% at the concentrations of 0.1, 1, 2 and 3 μM, respectively. We also investigated the mechanism of antiproliferative effects by cudratricusxanthone A in PDGF-BB-stimulated VSMCs. In Western blot analysis, 50 ng/ml PDGF-BB-stimulated phospholipase C (PLC)γ1, Ras, and extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylations were inhibited by cudratricusxanthone A (0.1—3 μM). Consisted with these findings, cudratricusxanthone A inhibited PDGF-receptor β chain (PDGF-Rβ) phosphorylation induced by PDGF-BB in a concentration-dependent manner. These findings suggest that the inhibitory effects of cudratricusxanthone A on DNA synthesis and proliferation by PDGF-BB-stimulated VSMCs are mediated by the suppressions of the PDGF-Rβ and its downstream signaling pathways. Our observation may explain in part mechanistic basis for the prevention of cardiovascular diseases, such as atherosclerosis and restenosis after coronary angioplasty by cudratricusxanthone A.
Human protein kinase CK2 is an ubiquitous serine/threonine kinase that is typically found in tetrameric complexes consisting of two catalytic (α and/or α′) and two regulatory β subunits. Although there is growing evidence that besides the participation of CK2 in a complex series of cellular functions, this protein kinase is involved in cell viability, cell proliferation, and neoplastic transformation. In the present study, a series of 3-(substituted-benzylidene)-1,3-dihydro-indolin-2-thione derivatives and the corresponding indolin-2-one congeners were tested for their inhibition of human recombinant protein kinase CK2 in vitro. The efficacy of these compounds was compared with their inhibitory results of p60c-Src tyrosine kinase. It was found that 3-(substituted-benzylidene)-1,3-dihydro-indolin-2-thione derivatives are more effective than indolin-2-one congeners for the inhibition of CK2 and p60c-Src tyrosine kinase.
To develop effective skin-lightening agents, we tested medicinal herbal extracts for their melanogenic-inhibitory activities. We isolated a sesquiterpenoid compound from the extract of Atractylodis Rhizoma Alba using the bioactivity-guided fractionation and identified it as selina-4(14),7(11)-dien-8-one (compound 1) with spectroscopic methods. Compound 1 dramatically reduced melanin synthesis of melan-a cells without any apparent cytotoxicity. Compound 1 did not inhibit cell-free tyrosinase activity but decreased tyrosinase activity in melanocytes. These effects were attributed to reduced expression of melanogenic enzymes such as tyrosinase, tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2). These results suggest that compound 1 may be an effective skin-lightening agent that regulates expression of melanogenic enzymes.
The free radical scavenging activities of Panax ginseng C.A. MEYER are known to increase by heat processing. Phenolic acids and Maillard reaction products (MRPs) have been suggested as active free radical scavenging components from our previous research, but heat processing-induced chemical and activity changes of ginsenosides considering the Maillard reaction have not yet been fully elucidated. In this study, we investigated the hydroxyl radical (·OH) scavenging activity changes of ginsengs and ginsenoside-Rb2 (Rb2) by heat processing using an electron spin resonance spectrometer. Especially, Rb2 was heat processed with the same amount of glycine, a frequently used amino acid in the Maillard reaction model system. As a result, the ·OH scavenging activities and brown compound levels of ginseng and glycine–Rb2 mixture were increased by heat processing. However, the increase in ·OH scavenging activities were not in accordance with the extents of browning. On the other hand, less-polar ginsenosides such as Rg3, Rg5, and Rk1 were generated from the glycine–Rb2 mixture by heat processing. The sugar moieties at carbon-20 of Rb2 were separated by the steaming process, less-polar ginsenosides were produced, and then the separated sugar moieties were thought to form MRPs with glycine. From the ·OH scavenging activity tests of Rb2, glycine, less-polar ginsenosides, and maltol, the increase in ·OH scavenging activity was thought to be more closely related to the generation of ·OH scavenging ginsenosides such as 20(S)-Rg3 and Rg5 by heat processing than MRPs.
Six diarylheptanoids (1—6) from the stem bark of Alnus hirsuta were investigated for their inhibitory activity against LPS-induced NF-kB activation and NO and TNF-α production. Among them, compounds 2, 3, and 6 displayed inhibitory activity against NF-kB activation and NO and TNF-α production with IC50 values of 9.2—9.9 μM, 18.2—19.3 μM, and 22.3—23.7 μM, respectively, in RAW264.7 cells. Three active compounds had no significant cytotoxicity in RAW264.7 cells at their effective concentrations. This is the first report of NF-kB-inhibitory activity of these compounds and supports the pharmacological use of A. hirsuta, which has been employed as a herbal medicine for the treatment of inflammatory diseases.
Phytoestrogens are naturally occurring compounds exerting estrogenic activity, and include isoflavonoids, flavonoids and lignans. In the present study, we evaluated the stimulating activity of six lignans, meso-dihydroguaiaretic acid, nordihydroguaiaretic acid, machilin A, guaiacin, isoguaiacin and isoguaiacin dimethylether, from Machilus thunbergii, on osteoblast differentiation employing primary cultures of mouse osteoblast as an in vitro assay system. Among the six lignans tested, arylnaphthalene type lignans such as guaiacin, isoguaiacin and isoguaiacin dimethylether significantly increased alkaline phosphatase activity, whereas bibenzylbutane type lignans such as meso-dihydroguaiaretic acid, nordihydroguaiaretic acid and machilin A showed little effects. Isoguaiacin and isoguaiacin dimethylether also increased collagen synthesis as well as calcium deposition. In addition, treatment of the mouse osteoblasts with tamoxifen markedly reduced ALP activity increased by isoguaiacin or isoguaiacin dimethylether, suggesting the involvement of estrogen receptor in the action of these lignans on osteoblast differentiation. Taken together, these results suggest that arylnaphthalene type lignans such as guaiacin, isoguaiacin and isoguaiacin dimethylether significantly increase osteoblast differentiation.
The purpose of the present study was to examine unilateral lung-selective gene transfer following the administration of naked plasmid DNA (pDNA) onto the pulmonary pleural surface in mice. Naked pDNA was administered intravenously, intraperitoneally, and instilled onto the right pulmonary pleural surface. Four hours later, right pulmonary pleural surface instillation of naked pDNA resulted in high gene expression in the right lung. On the contrary, intravenous and intraperitoneal administration of naked pDNA resulted in no detectable gene expression. After instilling naked pDNA onto the right or left pulmonary pleural surface, gene expressions in the applied lung were significantly higher than those in the other lung and tissues. In addition, gene expressions were detected only in the intrathoracic tissues, not in the intraperitoneal tissues. Four hours after instillation of naked pDNA onto the right pulmonary pleural surface, gene expression in the right lung was the highest, and thereafter gene expression in the right lung decreased gradually. This novel gene transfer method is expected to be a safe and effective treatment against serious lung diseases.
Atazanavir (ATV) is a low oral bioavailability (BA) compound and, clinically, is generally coadministrated with ritonavir (RTV), which boosts the oral BA of ATV by inhibiting cytochrome P450 (CYP) 3A, and P-glycoprotein (Pgp) via the same metabolic pathway. However, depending on pharmacokinetic interaction, RTV-boosted ATV has great potential for other comedication. In this study we demonstrated the pharmaceutical approach to BA improvement of ATV without RTV in rats, based on the solid dispersion system using sodium lauryl sulfate (SLS) as a carrier and Gelucire® 50/13 as an absorption enhancer. ATV solid dispersions in SLS were prepared by a conventional solvent method and, at ratios of ATV to SLS of 1 : 2 and 1 : 3, were demonstrated to form an amorphous state in powder X-ray diffraction (PXRD) analysis and exhibited 2.26- and 2.36-fold improvement in a dissolution test in comparison to bulk ATV, respectively. After oral administration to rats, ATV solid dispersion in SLS at a ratio of 1 : 2 showed a 3.5-fold increase in BA compared with bulk ATV. Moreover, the addition of Gelucire 50/13 to ATV solid dispersion, at a total ratio of Gelucire 50/13, ATV and SLS 1 : 1 : 2 gave 7.0- and 4.7-fold increase in Cmax and BA compared with bulk ATV, respectively, when the relative BA to RTV-boosted ATV reached 93%. The results in this study proved that a pharmaceutical approach could improve the bioavailability of ATV without pharmacokinetic interaction with RTV.
The non-steroidal antiandrogen flutamide is widely used for treatment of prostatic cancer, but causes side effects, including cholestatic hepatitis and fulminant hepatitis. We investigated the pathogenesis of flutamide-induced cholestatic hepatitis, focusing on the bile salt export pump (BSEP; ABCB11), which exports bile salts to the bile. We examined the inhibitory effects of flutamide and its active metabolite, hydroxyflutamide, on the transport of taurocholic acid (TCA) by membrane vesicles derived from hBSEP-expressing Sf9 cells. Flutamide inhibited the transport of TCA by hBSEP (IC50 value, about 50 μM), while hydroxyflutamide had no effect at up to 100 μM. When flutamide was administered to rats as a single oral dose of 100 mg/kg, the biliary excretion rate of bolus-injected [3H]TCA was decreased and the liver tissue concentration of flutamide exceeded 50 μM. Repeated doses of flutamide for 5 d (10 mg/kg/d) also decreased the biliary excretion rate of bolus-injected [3H]TCA. In this case, the liver tissue concentration of flutamide was below 0.1 μM. In both cases, no change in the mRNA level of rat Bsep was detected by RT-PCR. These results suggest that flutamide itself, but not its major metabolite, may cause cholestasis by inhibiting BSEP-mediated bile salt excretion.
Seizures have been reported in patients receiving fluoroquinolones, including levofloxacin (LVFX). In the present study, we investigated the effects of experimental renal failure and the concomitant treatment with ganciclovir on the pharmacodynamics of LVFX-induced seizures to identify whether these factors can alter the pharmacokinetics or the pharmacodynamics of LVFX. Male Wistar rats received an intravenous infusion of LVFX at 250, 500, or 1000 mg/h/rat until the onset of seizures, and samples of serum, brain, and cerebrospinal fluid (CSF) were obtained. The concentration of LVFX in CSF at the onset of seizures was not affected by the infusion rate, whereas that in serum and brain increased with increasing infusion rate. This suggests that the concentration of LVFX in CSF is an appropriate index of the drug concentration at the site of action. The concentration of LVFX in CSF at the onset of seizures was significantly lower in rats with renal failure than in the control rats. Pretreatment with methylguanidine, an uremic toxin, at 600 mg/h/rat for 8 min reduced the concentration of LVFX in CSF at the onset of seizures and the total body clearance of LVFX after the intravenous injection. In rats pretreated with ganciclovir at 500 mg/h/rat for 1 h, the concentration of LVFX in CSF at the onset of seizures was significantly lower than the control rats. These results suggest that renal failure and ganciclovir can be the risk factors for LVFX-induced seizures, and that they increase the sensitivity of the central nervous system to LVFX-induced seizures.
Cationic liposomes (CL) are one of the most widely studied non-viral vectors for gene delivery. It is well-known that CL induces cytotoxicity following lipofection. However, little is known regarding the mechanism involved in the cytotoxicity. In this study, the in vitro cytotoxicity of CL and its complex with pDNA (lipoplex) was investigated, and a part of the mechanism of induction as well. While free pDNA did not show any cytotoxicity, pDNA increased the cytotoxicity of CL via the formation of lipoplex. In addition, the lipoplex-induced cytotoxicity increased in a lipoplex dose-dependent manner, irrespective of the type of pDNA, cell line and the absence or presence of serum. An assay showed that apoptosis was largely induced by treatment with the lipoplex (lipofection), but not with CL alone, in the tested range of concentration of CL and pDNA. Furthermore, following treatment with lipoplexes, the cells exhibited the morphological features of apoptosis and DNA fragmentation. A cDNA microarray study showed that the lipofection up-regulated 45 genes related to apoptosis, transcription regulation and immune response. These results clearly indicate that pDNA in the lipoplex increases the cytotoxicity of CL as a result of inducing apoptosis. The fundamental principle for gene therapy is to deliver gene-based therapeutics to target cells for specific gene targeting with minimal cytotoxicity. Our results suggest the possibility that cytotoxicity induced by lipofection, accompanied by gene changes, could intrinsically exacerbate, attenuate or even mask the desired effects of gene-based therapy.
The potential for protein therapy, such as the use of antibodies, and vaccines is now well accepted. However, it is difficult to enhance efficiency in protein therapy without a suitable delivery system for delivering proteins to target sites. Here we describe the development of protein delivery system, which is capable of cytoplasmic delivery as well as efficient packaging. The multifunctional envelope-type nano device (MEND), which was originally developed for the delivery of nucleic acids such as plasmid DNA and oligodeoxynucleotides, can also be applied to protein delivery. In this study, the green fluorescent protein (GFP), a model protein, was condensed with stearyl octaarginine (stearyl R8) to form a nano particle, which was then coated with a lipid membrane, thus permitting R8 to be introduced for efficient cellular uptake and controlled intracellular trafficking. The packaging efficiency of the MEND was significantly higher than that of conventional liposomes, because the GFP can be encapsulated a condensed form. According to confocal laser scanning microscopy, the MEND is internalized efficiently and escapes from the acidic compartment to efficiently release GFP into the cytosol. These results indicate that the MEND can serve as a useful cytoplasmic delivery system for protein therapy.
In a previous study, we hypothesized that some type 2 diabetes mellitus susceptible genes may be up/down-regulated in white blood cells (WBC) of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, reflecting their up/down-regulation in major insulin-target tissues such as the liver before the onset of diabetes. We identified 57 potential candidate genes for predicting diabetes. In this study, we examined this hypothesis further by extending the experimental conditions from before the onset (6 weeks) to after the onset (24 weeks) of diabetes that type 2 diabetes mellitus susceptible genes are co-regulated in WBC, reflecting their expression in the liver. Using rat oligo DNA microarrays, we found that 48 genes are up/down-regulated in OLETF rats compared to control Long-Evans Tokushima Otsuka (LETO) rats in WBC and liver under fasting or insulin administration conditions. Twenty nine and 33 genes were up/down-regulated in both WBC and livers, respectively, under fasting and insulin administration conditions, respectively. Eight out of 29 genes in fasting condition and 12 out of 33 genes in insulin administration conditions have been reported to be type 2 diabetes mellitus susceptible genes and the remainder have not been reported to be related to type 2 diabetes mellitus. These results support our hypothesis that the expression levels of type 2 diabetes mellitus related genes in WBC are reflective of those in the liver after the onset of diabetes.
Clinical studies had suggested that there were sex differences in pharmacological and side effects of pioglitazone. However, there are few studies on the sex differences of peroxisome proliferator-activated receptor (PPAR) γ expressions in rat adipose tissues. We investigated the sex differences in peroxisome proliferator-activated receptor (PPAR) γ expressions in rat adipose tissues. Subcutaneous abdominal adipose tissues and perigonadal adipose tissues were obtained from male and female Wistar rats (12 weeks of age). Expressions of PPARγ protein were determined by Western blot analysis with anti-PPARγ antibody. Vaginal smear check was performed in female rats. We obtained adipose tissues from females, according to the different phases of the estrous cycle. Both PPARγ1 and γ2 subtypes were expressed in subcutaneous adipose tissues. Expressions of PPARγ2 in subcutaneous adipose tissues were significantly higher in males than in females. On the other hand, expressions of PPARγ2 in perigonadal adipose tissues were significantly higher in females than in males. Expressions of PPARγ2 in perigonadal adipose tissues in females significantly decreased during diestrus. It can be suggested that the sex hormones might affect the expressions of PPARγ2 in adipose tissues.
Pairs of forward and reverse primers and TaqMan probes specific to each of 15 human sulfotransferases were prepared. The mRNA expression level of each target enzyme was analyzed in total RNA from single and pooled specimens of various human tissues (adrenal gland, bone marrow, brain, colon, heart, kidney, liver, lung, pancreas, peripheral leukocytes, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid gland, trachea, and uterus) by real-time reverse transcription PCR using an ABI PRISM 7700 Sequence Detection System. The mRNA expression profiles of the sulfotransferases in these 23 different human tissues were used to identify the tissues exhibiting high transcriptional activity for these enzymes. These results provide valuable information for studies concerning the human carbohydrate sulfotransferase and tyrosylprotein sulfotransferase genes in various tissues.
Ethanol is widely used as a pharmaceutical excipient for the solubilization of many hydrophobic drugs for injections. However, there are only few studies about drug interaction with pharmaceutical excipients in the body after injection. In this study, the effect of ethanol (500 mM) or several alcohols (500 mM) on the stereoselective binding of warfarin enantiomers to fatty acid-free human serum albumin (HSA) or proteins of commercial albumin preparations was investigated. An ultrafiltration method was used for the separation of unbound warfarin enantiomers. By the addition of ethanol or 1-propanol, the unbound fraction of the S-enantiomer was decreased. On the other hand, the unbound fraction of the R-enantiomer was increased by the addition of ethanol or 1-propanol. Unbound fractions of both the S- and R-enantiomer were decreased by 2-propanol. In various commercial albumin preparations, unbound fractions of both the S- and R-enantiomer were increased by ethanol. The different effects of ethanol among fatty acid-free HSA and commercial albumin preparations were observed.
The aim of this study was to characterize and compare the percutaneous penetration kinetics of lidocaine (L) and prilocaine (P) in two local anesthetic formulations by in vivo microdialysis coupled with HPLC. The microdialysis system for studying lidocaine and prilocaine was calibrated by a no-net-flux method in vitro and retrodialysis method in vivo, respectively. A dosage of 0.2 g/cm2 of an in-house P–L formulation (2.5% lidocaine and 2.5% prilocaine, methylcellulose-based) and commercially available Eutectic Mixture of Local Anesthesia (EMLA, 2.5% lidocaine and 2.5% prilocaine, carbopol-based) was separately but symmetrically applied in the dorsal region of pigs. Saline (0.9%, w/v) was perfused into the linear microdialysis probe at a flow rate of 1.5 μl/min. Dialysate was collected upon topical application up to 6 h at 20-min intervals and assessed by HPLC. The results demonstrated the area under the concentration–time curve (AUC0—6 h) of lidocaine and prilocaine in EMLA was 71.95±23.36 μg h/ml and 38.01±14.8 μg h/ml, respectively, in comparison to 167.11±56.12 μg h/ml and 87.02±30.38 μg h/ml in the P–L formulation. The maximal concentrations (Cmax) of lidocaine and prilocaine in the dermis were 29.2±9.08 μg/ml and 16.54±5.31 μg/ml in EMLA and 80.93±17.98 μg/ml and 43.69±12.87 μg/ml in the P–L formulation, respectively. This study indicates a well-calibrated microdialysis system can provide vital real-time information on percutaneous drug delivery and specifically a methylcellulose-based P–L formulation can increase percutaneous absorption of both lidocaine and prilocaine in pigs compared to carbopol-based EMLA.
In prion diseases, the normal cellular form of prion protein (PrPC) is converted into the disease-associated isoforms (PrPSc) which accumulate in the infected tissues. Although the precise mechanism of this conversion remains unsolved, drugs of various categories have been reported to reduce the accumulation of PrPSc in prion-infected cultured cells. We here show that AY-9944 (a 7-dehydrocholesterol reductase inhibitor) and U18666A (a 24-dehydrocholesterol reductase inhibitor) prevent PrPSc from accumulating in prion-infected mouse neuroblastoma cells (ScN2a), with an ED50 of about 0.5 μM and 10 nM, respectively. In order to evaluate the efficacy of these two inhibitors in vivo, C57BL/6J mice inoculated with mouse-adapted scrapie-prion received repetitive intraperitoneal injections of U18666A (10 mg/kg) or a mixture of U18666A (10 mg/kg) and AY-9944 (12 mg/kg). By contrast to the potent anti-prion effects observed in ScN2a cells, the in vivo trial was abortive with neither drug halting the progression of the disease.
We investigated the genotype distribution and allele frequency of C452T polymorphism of γ-glutamyl hydrolase (GGH) gene, which causes the decreased enzymatic activity affecting the efficacy of methotrexate (MTX), in a Japanese population. The polymerase chain reaction–restriction fragment length polymorphism assay was applied to determine the genotype of C452T polymorphism in 269 Japanese healthy individuals. The genotype distribution was as follows: C/C, 89.2% (n=240); C/T, 10.4% (n=28); T/T, 0.4% (n=1). The frequency of C and T allele was 0.944 and 0.056, respectively. The obtained genotype distribution was well agreed with those expected by Hardy–Weinberg equilibrium. The genotype distribution and allele frequency in a Japanese population were found to be similar to those of African–Americans but significantly different from Caucasians. Although the frequency of variant T allele in a Japanese population is not so high as compared to Caucasians, determination of C452T polymorphism of GGH may be useful for monitoring of efficacy and side-effects of MTX for treatment of diseases such as rheumatoid arthritis or childhood acute leukemia. To our knowledge, this is the first report about the examination of C452T polymorphism of GGH in a Japanese population.
We investigated the correlation between the flavonoid content and NO production inhibitory activity of fruit peel extracts using 20 citrus plants. The contents of seven flavonoids (naringin, naringenin, hesperidin, hesperetin, rutin, nobiletin, and tangeretin) were determined by HPLC analysis. Each citrus peel extract varied in flavonoid content, but the contents of nobiletin and tangeretin, which were contained in all 20 fruit peels, showed a positive and significant correlation with each other (r=0.879, p<0.0005 for immature fruit peels; r=0.858, p<0.0005 for mature fruit peels). All citrus peel extracts dose-dependently inhibited LPS-induced NO production in RAW 264.7 cells. This inhibitory effect was significantly and positively correlated with the content of nobiletin and tangeretin. Nobiletin showed a more potent NO production inhibitory activity (IC50=26.5 μM) compared to tangeretin (IC50=136.6 μM). This result supports the premise that nobiletin-rich citrus may provide protection against disease resulting from excessive NO production.
To induce various mutations randomly, PCRs with Mn2+ and with a mutagenic deoxyribonucleotide, 8-hydroxy-dGTP (8-OH-dGTP), were performed. Mutations were induced by deoxyribonucleotide imbalance plus 500 μM Mn2+ in the Mn2+-PCR, and the amplified DNA was inserted into a plasmid. The plasmid library obtained from the transformed bacterial cells was then used as the template in the next PCR, which was done with 50 or 100 μM 8-OH-dGTP. Four kinds of mutations, A:T→G:C and G:C→A:T transitions and A:T→T:A and A:T→C:G transversions, occurred with similar frequencies. These results suggest that this strategy will be useful in random PCR mutagenesis for the in vitro evolution of nucleic acids and proteins, and for analyses of residues in these biomolecules.
We fed mice food granules containing fermented soybean (natto in Japanese) powder (hereafter “natto granules”) for 14 d to investigate whether natto granules had any effects on mouse behavior. We noted an enhancement of locomotor activity in natto-granule-fed mice compared to control and soybean-pellet-fed mice. This enhanced locomotor activity was blocked by a low dose of haloperidol (1 μg/kg i.p.), a dopamine receptor antagonist, but not by methysergide, a serotonin 5-HT1/2 receptor antagonist. The results suggest that the enhanced locomotor activity induced by continuous intake of natto granules in mice is sensitive to haloperidol.