Our earlier investigations demonstrated the remarkable activation of cytochrome P-450 reductase and nitric oxide synthase by 7,8-diacetoxy-4-methylcoumarin, a model polyphenolic acetate by way of acetylation, catalyzed by the Calreticulin. Protein acetyltransferase action of Calreticulin was hence termed Calreticulin transacetylase (CRTAase). Nitric oxide synthase and nitrite reductase are now considered as parts of nitric oxide cycle. The activation of platelets nitric oxide synthase by 7,8-diacetoxy-4-methylcoumarin has already been demonstrated by us. Also, there are reports that certain proteins such as cytochrome P-450 reductase and cytochrome P-450 are endowed with the nitrite reductase activity in mammalian cells. Keeping these facts in view, we turned our attention to probe whether 7,8-diacetoxy-4-methylcoumarin could alter the levels of nitric oxide independent of the action of nitric oxide synthase in the human platelets model. The incubation of 7,8-diacetoxy-4-methylcoumarin and nitrite with platelets caused significant elevation of nitric oxide and cyclic guanosine monophosphate levels possibly due to the activation of nitrite reductase. Several polyphenolic acetates were similarly found to activate the nitrite reductase in tune with their affinities as substrate to CRTAase. N-ω-Nitro-L-arginine methyl ester, the inhibitor of nitric oxide synthase, failed to reverse such an effect of 7,8-diacetoxy-4-methylcoumarin. Clotrimazole which is known to be an inhibitor of nitrite reductase, effectively abolished the 7,8-diacetoxy-4-methylcoumarin mediated enhancement of nitric oxide levels in platelets as well as the nitric oxide mediated effects; such as cyclic guanosine monophosphate levels as well as adenosine diphospate induced platelets aggregation due to nitrite.

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We elucidated the protective effect of 7,8-dihydroxyflavone against hydrogen peroxide (H₂O₂)-induced DNA damage. We found that 7,8-dihydroxyflavone scavenges 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and intracellular reactive oxygen species (ROS). 7,8-Dihydroxyflavone with antioxidant effect prevented the H₂O₂-induced cellular DNA damage, as evidenced by comet tail, 8-hydroxy-2′-deoxyguanosine (8-OHdG) content, and phospho-histone H2A.X protein expression. Hence, 7,8-dihydroxyflavone was shown to protect cell via the inhibition of apoptosis induced by H₂O₂. This was substantiated by decreased apoptotic nuclear fragmentation, decreased sub-G₁ cell population, and decreased DNA fragmentation. Furthermore, 7,8-dihydroxyflavone activated the protein kinase B (PKB, Akt) signal pathway, which is a major survival signal pathway. In addition, LY294002, which is phosphatidylinositol 3 kinase (PI3K, upstream of Akt) inhibitor, attenuated the protective effect of 7,8-dihydroxyflavone against H₂O₂-induced cell damage. In conclusion, 7,8-dihydroxyflavone was shown to possess cytoprotective properties against oxidative stress by scavenging intracellular ROS and enhancing Akt activity.

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The major function of farnesoid X receptor (FXR) is to maintain bile acid and lipid homeostasis. Fxr-null mice, in which the levels of hepatic bile acid and lipid have been elevated, develop spontaneous liver tumors. We evaluated differences in hepatic bile acid and triglyceride concentrations, and in generation of oxidative stress between wild-type mice and Fxr-null mice. The hepatic levels of 8-hydroxy-2′-deoxyguanosine (8OHdG), thiobarbituric acid-reactive substance (TBARS) and hydroperoxides, oxidative stress-related genes, and nuclear factor (erythroid-2 like) factor 2 (Nrf2) protein in Fxr-null mice were significantly higher than those in wild-type mice. An increase in the hepatic bile acid concentration in Fxr-null mice fed a cholic acid (CA) diet resulted in an increase in the hepatic levels of hydroperoxides, TBARS and 8OHdG, whereas a decrease in the hepatic concentration in mice fed a diet containing ME3738 (22β-methoxyolean-12-ene-3β,24(4β)-diol) resulted in a decrease in these oxidative stress marker levels. A good correlation was observed between the hepatic bile acid concentrations and the hepatic oxidative stress marker levels, although there was no significant correlation between the hepatic triglyceride concentrations and oxidative stress. The results show that oxidative stress is spontaneously enhanced in Fxr-null mice, which may be attributable to a continuously high level of hepatic bile acids.

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Cell malignancy is negatively correlated with the expression of thrombomodulin (TM), a thrombin receptor expressed on the surface of various cells, including tumor cells. TM accelerates thrombin-activatable fibrinolysis inhibitor (TAFI) activation catalyzed by thrombin. The active form of TAFI (TAFIa) contributes to inhibition of plasmin formation through its carboxypeptidase B (CPB)-like activity. Here, we examined whether TM- and tumor cell-dependent TAFI activation participates in controlling pericellular fibrinolysis and cell invasion. Human fibrosarcoma HT1080 cells retained the ability to activate both prothrombin and plasminogen, but did not express TM. HT1080 cells mediated activation of TAFI in plasma in the presence of soluble-type TM (sTM) through cell-dependent prothrombin activation. HT1080 cells stably expressing TM (TM-HT1080) mediated plasma TAFI activation without added sTM, but HT1080 (wild-type) and Mock-transfected HT1080 cells (Mock) did not. Production of TAFIa suppressed cell-mediated plasminogen activation. Matrigel invasion by wild-type and Mock cells was enhanced two-fold by diluted plasma (10%), whereas the plasma-induced invasion of TM-HT1080 cells (65% of wild-type invasion) was lower than those of wild-type and Mock cells. Cell invasion by TM-HT1080 was partially enhanced by addition of a TAFIa/CPB inhibitor. These results suggest that TM suppresses pericellular fibrinolysis and plasma-induced tumor cell invasion, and that it is mediated, at least in part, by plasma TAFI activation.

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The Maillard reaction contributes to the complications of diabetes and normal aging. Dihydropyrazines (DHPs), which are produced during the Maillard reaction, generate radicals and possess DNA strand-cleaving activities in vitro. In the present study, we evaluated the genotoxic and cytotoxic potentials of a DHP derivative, cyclohexyl-DHP, which is obtained as a mixture of two isomers, 2,3,5,6,7,8-hexahydroquinoxaline (endo-type) and 1,2,3,5,6,7-hexahydroquinoxaline (exo-type), fused with a cyclohexyl ring. Cyclohexyl-DHP caused DNA strand breaks in plasmid pUC18, especially in the presence of Cu²⁺. By using Escherichia coli mutant strains, we observed that cyclohexyl-DHP exposure strongly reduced the survival rate of a cytosolic sodium dodecyl sulfate (SOD)-deficient strain (sodA sodB), significantly reduced the survival rates of DNA repair-deficient strains (recA and uvrB) and mildly reduced the survival rate of a catalase-deficient strain (katE katG) compared with the survival rate of the wild-type strain. Addition of Cu²⁺ enhanced the cell killing ability of cyclohexyl-DHP. The frequency of mutations induced by cyclohexyl-DHP increased dose-dependently in the sodA sodB strain. Assays with the highly water-soluble tetrazolium salt WST-1 revealed that cyclohexyl-DHP strongly generated superoxide anions. Moreover, cyclohexyl-DHP elevated the protein carbonyl levels in E. coli. These findings indicate that cyclohexyl-DHP could potentially generate superoxide anions, and cause not only breakage of chromosomal DNA leading to mutagenic lesions but also induce damage to cellular proteins.

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Selenium deficiency has been reported to result in an extraordinary decrease of glutathione peroxidase (GSH-Px) and, reversely, an increase of detoxifying enzymes such as glutathione-S-transferase (GST), uridine-5′-diphosphate glucuronosyltransferase (UGT), nicotinamide-dependent quinine oxidoreductase (NQO1; DT-diaphorase), and epoxide hydrolase without significantly affecting cytochrome P450 activity. However, little is known about the effects on aldehyde oxidase 1 (AOX1) activity towards various kinds of aldehydes and N-heterocyclic aromatic compounds. The aim of this study is to clarify the effects of selenium deficiency on AOX1 in rats. As expected, selenium deficiency was confirmed by the extremely low activity of GSH-Px along with the increased activities of GST and DT-diaphorase. AOX1 activity towards vanillin and (S)-RS-8359 was increased by selenium deficiency, and that corresponded to an increase of AOX1 protein level but not to a decreased AOX1 mRNA level. It has been documented that the assembly of the catalytically active holoenzyme forms of the molybdo-flavoenzyme family is very complex and is controlled through transcriptional and translational events by many gene products. In addition, selenium deficiency has been known to cause oxidative stress that leads to an increase of AOX1 activity. Furthermore, aldehyde oxidase homolog 1 (AOH1) with properties similar to AOX1 is present in rodent liver. All the reports suggest that the mechanisms by which selenium deficiency increases AOX1 activity are highly complicated and investigated from different points of view.

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Phytochemicals are naturally present in a wide variety of plants, and have been suggested to exert a number of effects beneficial to human health. Several phytochemicals possess estrogenic activity through estrogen receptor α (ERα) and ERβ, and are, therefore, termed phytoestrogens. In this study, we examined whether various phytochemicals have agonistic and/or antagonistic activity against six human nuclear receptors (ERα, ERβ, androgen receptor (AR), glucocorticoid receptor (GR), thyroid hormone receptor α₁ (TRα₁) and TRβ₁) by in vitro reporter gene assays using Chinese hamster ovary cells. Of the 31 phytochemicals tested, including flavonoids, isoflavonoids, coumestan, lignans, catechins and their metabolites, 20 compounds showed estrogenic activity via ERα and/or ERβ, and we ranked these phytochemicals according to their estrogenic potency via ERα and ERβ. As a result, coumestrol and genistein strongly activated ERα and ERβ at very low concentrations of <1×10⁻¹⁰ M. Most phytochemicals showing estrogenic activity also exhibited agonistic activity against ERβ at lower concentrations than those for ERα, and two typical isoflavones, genistein and daidzein, in particular, showed a potent preference for ERβ. Further, we found that baicalein has ERβ antagonistic activity, and two compounds, enterolacton and O-desmethylangolensin, have AR antagonistic activity. Nevertheless, none of tested compounds showed AR agonistic activity together with GR, TRα₁ and TRβ₁ agonistic/antagonistic activity. These results suggest that various phytochemicals or their metabolites preferentially interact with ERα/β among the six nuclear hormone receptors tested, and that the ERβ agonistic activity, in particular, of these compounds may be associated with various beneficial effects on human health.

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To avoid the need to use animals to test the skin irritancy potential of chemicals and cosmetics, it is important to establish an in vitro method based on the reconstructed human epidermal model. To evaluate skin irritancy efficiently and sensitively, we determined the gene expression induced by a topically-applied mild irritant sodium dodecyl sulfate (SDS) in a reconstructed human epidermal model LabCyte^TM EPI-MODEL (LabCyte) using a DNA microarray carrying genes that were related to inflammation, immunity, stress and housekeeping. The expression and secretion of IL-1α in reconstructed human epidermal culture is known to be induced by irritation. We detected the induction of IL-1α expression and its secretion into the cell culture medium by treatment with 0.075% SDS for 18 h in LabCyte culture using DNA microarray, quantitative reverse-transcription polymerase chain reaction (RT-PCR) and ELISA. DNA microarray analysis indicated that the expression of 10 of the 205 genes carried on the DNA microarray was significantly induced in a LabCyte culture by 0.05% or 0.075% SDS irritation for 18 h. RT-PCR analysis confirmed that SDS treatment significantly induced the expressions of interleukin-1 receptor antagonist (IL-1RN), FOS-like antigen 1 (FOSL1), heat shock 70 kDa protein 1A (HSPA1) and myeloid differentiation primary response gene (88) (MYD88), as well as the known marker genes for irritation IL-1β and IL-8 in a LabCyte culture. Our results showed that a DNA microarray is a useful tool for efficiently evaluating mild skin irritation using a reconstructed human epidermal model.

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Combined treatment with dexamethasone and oncostatin M (DEX/OSM) or interleukin-6 (DEX/IL-6) resulted in the appearance of numerous large vacuoles in human fetal liver (HFL) cells and showed synergistic effects on the formation of vacuoles. The number of vacuoles formed by DEX, DEX/OSM, or DEX/IL-6 was significantly suppressed by RU-486, a glucocorticoid receptor antagonist. On the other hand, the size of vacuoles formed by OSM, IL-6, DEX/OSM, or DEX/IL-6 was significantly decreased to about 65% by madindoline A (MDL-A), which is a non-peptide antagonist of gp130 and an inhibitor of cytokines, such as IL-6, mediated by gp130 homodimerization, while RU-486 did not affect the size of vacuoles. Expression of IL-6 mRNA in HFL cells was markedly induced by OSM. Expression of IL-6R mRNA was induced by DEX. These results indicate that DEX contributes to the formation of vacuoles through glucocorticoid receptors and that OSM and IL-6 contribute to enlargement of these vacuoles.

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Hwanggunchungyitang (HGCYT) is a newly designed herbal drug formula for the purpose of treating auditory diseases. A number of heavy metals have been associated with toxic effects to the peripheral or central auditory system. Cadmium (Cd²⁺) is a heavy metal and a potent carcinogen implicated in tumor development through occupational and environmental exposure. However, the auditory effect of Cd²⁺ is not poorly understood. The purpose of the present study was to investigate whether HGCYT prevent the ototoxic effects induced by Cd²⁺ in auditory cell line, HEI-OC1. HGCYT inhibited the cell death, reactive oxygen species generation (ROS), activation of caspase-9, and extracellular signal-related kinase (ERK) induced by Cd²⁺. In addition, we observed that cochlear hair cells in middle turn were damaged by Cd²⁺. However, HGCYT prevented the destruction of hair cell arrays of the rat primary organ of Corti explants in the presence of Cd²⁺. These results support the notion that ROS are involved in Cd²⁺ ototoxicity and suggest HGCYT therapeutic usefulness, against Cd²⁺-induced activation of caspase-9 and ERK.

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Diabetic nephropathy (DN) has become the leading cause of end stage renal failure, and prevention or retardation of DN has become a major goal in biomedical research. In this study, Tanshinone IIA, a component extracted from Salvia miltiorrhiza, was studied in experimental rats in which DN was induced by streptozotocin (STZ) treatment. The DN rats were treated with 10 mg/kg of Tanshinone IIA for 12 weeks to analyze its reno-protective effect with different parameters. Renal hypertrophy and 24-h urinary protein excretion were ameliorated by Tanshinone IIA. Moreover, advanced glycation end-products (AGEs), angiotensin II (Ang II), transforming growth factor β₁ (TGF-β₁), collagen IV, and monocyte/macrophage (ED-1) either in the serum or kidney were significantly reduced. These results suggest that Tanshinone IIA might have protective effects on several pharmacological targets during the progression of DN, and could be a potential drug for the prevention of DN.

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The present study investigated the effect of 4[(5,6,7,8-tetrahydro-5,5,8,8,-tetramethyl-2-naphthalenyl)carbamoyl] benzoic acid (Am-80), a synthetic retinoid, on spinal cord injury (SCI) in rats. Treatment with Am-80 (orally and subcutaneously) significantly promoted recovery from SCI-induced motor dysfunction. On day 28 after injury, the lesion cavity was markedly reduced, while the expression of myelin basic protein (MBP; myelin), βIIItubulin (neuron), and glial fibrillary acidic protein (GFAP; astrocyte) was increased, in comparison with SCI controls. Interestingly, expression of neurotrophin receptor, tyrosine kinase B (TrkB) was over 3-fold higher after Am-80 treatment than in SCI controls. A lot of TrkB-positive cells as well as brain-derived neurotrophic factor (BDNF)-positive ones were observed around the injured site. Am-80 (10 μM) combined with BDNF (100 ng/ml) promoted extensive neurite outgrowth and TrkB gene expression by cultured SH-SY5Y cells, as did all-trans retinoic acid (ATRA). Thymidine incorporation was dramatically suppressed, but there was little effect on cell viability. These findings suggest that Am-80 has the potential to be used for treating neurodegenerative disorders, including SCI. Its efficacy may be partly ascribed to promotion of cell viability and differentiation of neural stem cells through increased TrkB expression.

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The role of glutamine dipeptide (GDP) to prevent against prolonged insulin induced hypoglycemia (IIH) in overnight fasted rats was investigated. The glycemia was measured 0, 2, 4, 8, and 10 h after an intraperitoneal (i.p.) injection (1 U/kg) of Detemir insulin. Because the lowest glycemia was obtained 4 h after insulin administration, the experiments were done at this time. The hypoglycemia obtained 4 h after insulin injection was partially prevented with increasing oral doses of GDP (1.56, 3.12, 6.25, 12.5 mg/kg). The best result was obtained with 12.5 mg/kg. However, from this dose (25.0, 50.0, 100.0 mg/kg), the values of glycemia progressively decreased (p<0.05). The effect of GDP to prevent prolonged IIH was mediated, partly at least, by an intensification of liver gluconeogenesis. Moreover, the increased portal availability of GDP, L-alanine and L-glutamine after GDP administration also contributed to the IIH prevention, since the rate of gluconeogenesis progressively augmented with the infusion of increasing concentrations of these substances. However, after getting the maximal value, the rate of liver gluconeogenesis decreased (p<0.05) if a more elevated concentration of L-alanine or L-glutamine was infused. On the other hand, the liver gluconeogenesis during the infusion of increasing concentrations of GDP was unchanged. Because, GDP did not directly inhibit liver gluconeogenesis, but an inhibition of this metabolic pathway was observed with low ammonia concentrations (from 0.062 mM) it is possible that the ammonia from the catabolism of GDP by extra hepatic tissues could inhibit liver gluconeogenesis. This mechanism could help to explain the lower glycemia obtained with more elevated doses of oral GDP.

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Effects of goshuyuto, a traditional Japanese medicine, on vascular constriction were examined using isolated strips of rat aorta. Goshuyuto (1×10⁻⁵ to 1×10⁻³ g/ml) caused constriction of aorta strips in a dose-dependent manner. The vasoconstrictive effects of goshuyuto were significantly inhibited by pretreatment with prazosin, an adrenergic α₁ receptor antagonist. The constrictive effects were partially inhibited by pretreatment with BRL15572, a 5-HT_1D antagonist, and ketanserin, a 5-HT_2A antagonist. However, the constrictive effects were not inhibited by pretreatment with SB216641, a 5-HT_1B antagonist, or propranolol, an adrenergic β receptor antagonist. In addition, aqueous extracts of Evodiae Fructus, one of the constituent medicinal herbs of goshuyuto, caused constriction of aorta strips strongly, but aqueous extracts of Zizyphi Fructus, Ginseng Radix, and Zingiberis Rhizoma, the other constituents of goshuyuto, did not have much effect on the vascular response of rat aorta strips. Also, synephrine, which is one of the ingredients of Evodiae Fructus, constricted the rat aorta. These results suggest that goshuyuto constricts rat aorta strips and that the mechanisms involve the adrenergic and/or serotonergic receptors. Also, it may be suggested that Evodiae Fructus and synephrine play the important role in the vasoconstrictive effects of goshuyuto on rat aorta strips.

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The aim of this study was to evaluate the effects of Anemarrhena asphodeloides BUNGE (AA) on cholinergic memory deficits in mice. This agent has previously been used as an antipyretic, anti-inflammatory, anti-diabetic, and antidepressant in traditional Chinese medicine. Mangiferin was isolated from AA and showed a dose-dependent inhibition of acetylcholinesterase (AChE) activity (IC₅₀ value, 62.8 μM). Cholinergic dysfunction was induced in mice by administering scopolamine, and the animals were then tested using the passive avoidance test as well as the Morris water maze test. Mangiferin (20 mg/kg, p.o.) significantly reversed scopolamine-induced deficits in the passive avoidance test, and also improved escape latencies in training trials and increased swimming times in the Morris water maze test (p<0.05). Mangiferin also reduced acetylcholine and tumor necrosis factor (TNF)-α levels induced by scopolamine in mice brain (p<0.05) and inhibited nuclear factor (NF)-κB activation in scopolamine or TNF-α-stimulated BV-2 microglial cells. These results suggest that mangiferin can improve long-term cholinergic memory deficits by AChE inhibition or cholinergic receptor stimulation and inhibition of NF-κB activation.

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Sarcandra glabra was a renowned herb traditionally used as herbal tea or food supplement to enhance mental efficiency and to recover from stress or fatigue in China. We investigated the effects of Sarcandra glabra extract (SGE), with chemical composition clearly showed by HPLC fingerprint as quality control, on immunologic response including natural killer (NK) cell activity and its antioxidative capacity in splenocytes obtained from restraint mice. Our results found that daily oral administration of SGE (125, 500 mg/kg/d) for 5 consecutive days to restrained mice alleviated the stress-induced reduction of the number of lymphocytes, the balance of CD4⁺ T/CD8⁺ T and NK cell activity per spleen. SGE also significantly decreased the level of lipid peroxidation and increased oxygen radical absorbance capacity (ORAC) in splenocytes. These results indicated that SGE modulate stress-attenuated immunologic response, at least, partially explained by improving antioxidative capacity in immunocytes.

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We have cloned an earthworm-derived Factor Xa (FXa) inhibitor, with an excellent inhibitory specificity from the midgut of the Eisenia andrei. We designate this inhibitor eisenstasin. An eisenstasin-derived small peptide (ESP) was synthesized and we examined whether ESP played an essential role in FXa inhibition. Compared to antistasin-derived small peptides (ASP) originating from leech, ESP primarily exhibited a high level of FXa inhibition in chromogenic peptide substrate assays and revealed an approximately 2-fold greater inhibition of FXa cleavage of a target protein than ASP. This suggests that ESP could be an effective anti-coagulant that targets FXa during the propagation step of coagulation. ESP also inhibited proteinase-activated receptor 2-mediated FXa activation, which may trigger endothelial inflammation. Endothelial nitric oxide (NO) was significantly reduced by ESP (p<0.0001), indicating that protease-activated receptor-2 (PAR-2) was effectively inactivated. We also found that ESP reduced the expressions of pro-inflammatory cytokines (IL-1α, IL-1β, IL-8, IL-16, MCP-1, MIP-1α and MIP-1β) by cultured cells treated with both ESP and FXa. Our results provide the first evidence that ESP might interrupt coagulation cascades by inhibiting FXa, and thereby may effectively control the bidirectional alternation between coagulation and inflammation.

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Serotonin 2C receptor (5-HT2CR) mRNA has been reported to receive editing at 5 nucleotide positions (named sites A—E) which are located inconsecutively on the nucleotide sequence encoding the 2nd intracellular loop of the receptor protein. To clarify the physiological role of 5-HT2CR mRNA editing, we investigated developmental changes in editing frequencies at sites A—E in the rat cerebral cortex and primary cultured cortical neurons. The editing at sites A and B increased in parallel with the rat brain development and reached a plateau of 80—100% frequency at postnatal days 1—3. Although editing frequency at site C was low compared to those detected at other sites except site E during a developmental period, it reached the maximal value of 30% during a first 7-d period after birth and then decreased gradually to the negligible level at PN49. Site D exhibited almost constant susceptibility (about 60%) to editing, while no editing at site E was occurred during rat brain development. Similar changes during development in editing frequencies at these sites were observed in primary cultured cortical cells during the cultivation period. These findings indicated that editing sites A—D on 5-HT2CR mRNA have different susceptibility and that the frequencies at these sites are not always constant during development.

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The glottic movement is important for generating efficient airflow in respiration and related reflexes such as coughing. The superior laryngeal nerve (SLN) innervates the cricothyroid muscle and regulates the glottic aperture. A total of 620 laryngeal motoneurons (LMNs) providing efferent fibers to the SLN were retrogradely identified with fluorescent wheat germ agglutinin (WGA) in 8 guinea pigs. The WGA-labeled cells were distributed in the ventrolateral part of nucleus ambiguus, extending 1.8—2.2 mm lateral to the midline, 0.8—1.2 mm from the ventral surface and 1.7—2.6 mm rostral to the obex. Double staining revealed that 28.4% of LMNs (44 out of 155 cells examined) were immunopositive to N-methyl-D-aspartate (NMDA) receptors and 29.2% of LMNs (55 out of 185 cells) to μ-opioid receptors. The present study demonstrates phenotypic variation in the synaptic transmission to the LMNs in guinea pigs. These results suggest modulation of the glottic movement by μ-opioid and NMDA mechanisms.

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Phosphatidylinositol 3-kinase (PI3K) has been implicated in a variety of diseases including cancer. A number of PI3K inhibitors have recently been developed for use in cancer therapy. ZSTK474 is a highly promising antitumor agent targeting PI3K. We previously reported that ZSTK474 showed potent inhibition against four class I PI3K isoforms but not against 140 protein kinases. However, whether ZSTK474 inhibits DNA-dependent protein kinase (DNA-PK), which is structurally similar to PI3K, remains unknown. To investigate the inhibition of DNA-PK, we developed a new DNA-PK assay method using Kinase-Glo. The inhibition activity of ZSTK474 against DNA-PK was determined, and shown to be far weaker compared with that observed against PI3K. The inhibition selectivity of ZSTK474 for PI3K over DNA-PK was significantly higher than other PI3K inhibitors, namely NVP-BEZ235, PI-103 and LY294002. These results indicated that ZSTK474 was the most specific agent among these PI3K inhibitors.

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Increased production and accumulation of melanin lead to hyperpigmentation disorders. Several inhibitors of tyrosinase, the key enzyme in melanin synthesis have been developed but exhibited lack of efficiency or some adverse side effects. Therefore, it appears very important to find new agents that will be able to promote inhibition of tyrosinase and pigmentation. In this study, some phenylalkylcinnamide molecules were synthesized and evaluated for their ability to act as tyrosinase inhibitors. Compounds 2 (IC₅₀=0.03 mM) and 12 (IC₅₀=0.028 mM) showed strong tyrosinase inhibitory potential comparable to standard hydroquinone (IC₅₀=0.037 mM). Taken together, compounds 2 and 12 can be considered as good candidates for further investigations to evaluate their effect on the inhibition of melanin synthesis and skin pigmentation.

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Sparassis crispa (SC), Hanabiratake in Japanese, is an edible mushroom with medicinal properties, that contains more than 40% β-D-glucan. It was concluded from results of the methylation study that β-D-glucan from SC (SBG) was composed of a backbone of β-(1→3)-linked D-glucopyranosyl residues, and had β-D-glucopyranosyl groups joined through O-6 and O-2 of D-glucose of the backbone. We purified SBG and investigated its anti-angiogenic functions and anti-metastatic effects on neoplasm using different animal models. The oral administration of the purified SBG suppressed B16-F10 cell-induced angiogenesis in the dorsal air sac assay using female ICR mice as well as vascular endothelial growth factor induced neovascularization in the Matrigel plug assay using female C57BL/6J mice. Furthermore, it suppressed the growth and numbers of the metastatic tumor foci in lung, along with the primary tumor growth in the spontaneous metastatic model using female C57BL/6J mice. From these results, it is apparent that the oral administration of SBG results in suppressive effect on tumor growth and metastasis in lung through the inhibition of tumor induced-angiogenesis. These effects are not a result of direct action on the endothelial cells because cell growth, migration and capillary-like tube formation were not affected in the human umbilical vein endothelial cells by SBG application. This is the first report showing that the oral administration of SBG is capable of suppressing angiogenesis and metastasis.

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Tyrosinase is a key enzyme for melanin biosynthesis, and hyperpigmentation disorders are associated with abnormal accumulation of melanin pigments, which can be reduced by treatment with depigmenting agents. A methanol extract of Astragalus membranaceus showed inhibitory activity against mushroom tyrosinase. The active compound was purified from the methanol extract of A. membranaceus and, following several chromatographic methods, was identified as calycosin via spectroscopic analysis. The results showed that calycosin exhibited tyrosinase inhibitory activity with an IC₅₀ value of 38.4 μM. Moreover, calycosin showed a melanin biosynthesis inhibition zone in a culture plate of Streptomyces bikiniensis, which is commonly used as an indicator organism. Furthermore, calycosin dramatically reduced melanin synthesis of Melan-a cells without any apparent cytotoxicity and reduced expression of melanogenic enzyme, tyrosinase. These results suggest that calycosin may be an effective skin-lightening agent that regulates the expression of melanogenic enzymes.

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The effect of hyperlipidemia (HL) on the pharmacokinetics of nelfinavir (NFV), a human immunodeficiency virus protease inhibitor, was investigated, focusing on the change of NFV distribution in plasma using poloxamer 407-induced HL model rats (HL rats). The plasma unbound fraction (f_u) in HL rats (0.20—0.39%) was significantly lower than the control (0.92—1.47%). Lipoprotein level in HL rats was about 5 times higher and low- and very low-density liproteins ratio were 1.7—4.5 times higher than the control. NFV recovery in the triglyceride-rich lipoprotein such as chylomicron, low- and very low-density liproteins fractions of HL rats were significantly higher. The area under the plasma concentration–time curve (AUC) of NFV after intravenous (i.v.: 5 mg/kg) and intraduodenal (i.d.: 30 mg/kg) administration to HL rats (i.v.: 6.12±0.48, i.d.: 24.4±2.2 μg·h/ml) were higher than the control (i.v.: 1.62±0.21, i.d.: 5.00±0.36 μg·h/ml). The steady state volume of distribution (Vd_ss) in HL rats (0.60±0.07 l/kg) was lower than the control (6.25±0.55 l/kg). Systemic availability (F) in HL rats (66.6%) was higher than the control (51.4 %). Directly absorbed NFV from the gastrointestinal tract to the lymphatic system in HL rats was about 2 times higher than the control. From the results of this study, it was concluded that the increase of AUC was caused by decreasing f_u and Vd_ss due to the increase of the triglyceride-rich lipoprotein level. In addition, it was suggested that the increase of absorbed NFV through the lymphatic system, which did not receive the first-pass effect, was one reason for the increase of F in HL rats.

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Progesterone (P) is an important hormone for the establishment of pregnancy, and its administration is useful for luteal insufficiency. Considering the problems of commercially available oral and injection drugs, hospital-formulated vaginal suppositories are clinically used. However, since the half-life of P suppositories is short, it is difficult to maintain its constant blood concentration. To sustain drug efficacy and prevent side-effects, we are attempting to develop sustained-release suppositories by examining the degree of sustained-release of active ingredients. In this study, we examined the combinations of granulation methods and release systems for the preparation of sustained-release granules of P, and produced 13 types of sustained-release granules. We also examined the diameter, content, and dissolution of each type of granules, and confirmed that the sustained-release of all types of granules was satisfactory. Among the sustained-release granules, we selected granules with a content and a degree of sustained-release suitable for sustained-release suppositories.

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RNA interference (RNAi) is a technology that specifically inhibits gene expression and is carried out by small 21–27-nucleotide double-stranded small interfering RNA (siRNA) or short hairpin RNA (shRNA). RNAi has become a very promising technology for genetic research, however problems remain with the delivery of siRNA into cells. SiRNA and shRNA are easily degraded by RNases in body fluids and are hardly able to permeate cell membranes because of hydrophilic, polyanionic macromolecules which make their bioavailability very low. We have focused on the third double-stranded RNA-binding domain (dsRBD3) from the Mus musculus Staufen protein in order to develop a non-viral, multifunctional, artificial virus-like delivery system. We constructed a dsRBD3 expression vector and the recombinant dsRBD3 was expressed as a fusion protein with affinity tags. Purified dsRBD3 was mixed with siRNA or shRNA at various ratios and then added to fetal bovine serum (FBS) to evaluate the inhibition of the degradation of double-stranded RNA (dsRNA) by RNase. Unexpectedly, dsRBD3 was not able to protect the siRNA against degradation by FBS, but shRNA was stabilized to some degree by dsRBD3.

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The effect of gender on theophylline clearance was investigated retrospectively in 96 Japanese pediatric patients (63 males and 33 females) ranging in age from 0.5 to 8 years and in weight from 6.3 to 36.8 kg. All patients received intravenous constant-rate infusion of aminophylline in the asthmatic acute phase. The theophylline clearances in males and females were 56.2±15.4 and 50.1±14.2 ml/h/kg for ages 0.5—<2 years, 58.7±18.8 and 48.3±6.5 ml/h/kg for ages 2—<4 years, and 65.7±12.0 and 52.1±16.8 ml/h/kg for ages 4—<9 years, respectively. At ages from 2 to 8 years, the theophylline clearance was 20% higher in males than in females (p<0.05). Our findings suggested that the initial dosage of theophylline should be adjusted according to the gender of pediatric patients and particularly in the case of infants.

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In this study, we examined the effect of sesquiterpene lactones isolated from Calea urticifolia and Tanacetum parthenium (feverfew) on melanogenesis in mouse B16 melanoma cells. In response to 3-isobutyl-1-methylxanthin (IBMX), B16 melanoma cells underwent differentiation characterized by increased melanin biosynthesis. Treatment of sesquiterpene lactones at lower concentration (1 μM) significantly blocked IBMX-induced melanogenesis, but did not induce the inhibitory activity of cell growth. Among them, 2,3-epoxyjuanislamin exhibited a potent inhibitory effect on melanogenesis. Treatment of B16 cells with 2,3-epoxyjuanislamin elicited significant decreases in tyrosinase protein and mRNA levels. These results demonstrated that the inhibitory effects of sesquiterpene lactones on melanin biosynthesis may be due to the suppression of tyrosinase expression.

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As reported previously, cationic liposomes formulated with dioleoylphosphatidylethanolamine (DOPE) and N,N-methyl hydroxyethyl aminopropane carbamoyl cholesterol (MHAPC-liposomes) achieved efficient gene transfection in the mouse lung following intratracheal injection. We have studied here the role of surfactants, mannosylerythritol lipid-A (MEL-A) and polysorbate 80 (Tween 80), in affecting gene transfection of MHAPC-lipoplexes (complex with pCMV-luc DNA) in A549 cells and in the mouse lung. MEL-A increased gene transfection of MHAPC-lipoplexes significantly in vitro and slightly in the mouse lung, while Tween 80 decreased it both in vitro and in vivo. As assessed by confocal laser scanning microscopy and fluorescence imaging, MEL-A might faciliate gene dissociation from MHAPC-lipoplexes with fluorescein-labeled oligodeoxynucleotide (FITC-ODN) after internalization into the cells and retained the lipoplexes in the mouse lung for prolonged time, while Tween 80 was inefficient to deliver foreign gene into target cells and in the lung. These results demonstrated that MEL-A is advantageous to Tween 80 in the modification of cationic liposomes as gene delivery vectors in the lung.

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