Phosphoinositides are a family of phosphorylated derivatives of the membrane lipid phosphatidylinositol. These lipids are highly concentrated in distinct pools located in a cell's plasma membrane, endosomes or nucleus, where they function as ligands for phosphoinositide-binding proteins. Protein domains that bind phosphoinositides include the pleckstrin homology (PH) domain, the phox homology (PX) domain and the Fab1p-YOPB-Vps27p-EEA1 (FYVE) domain. These domains are found in many proteins involved in intracellular signaling, membrane trafficking and cytoskeletal rearrangement. Recent studies have identified potential links between alterations to various signaling pathways involving phosphoinositides and the etiology of many human diseases.

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The phosphoinositide kinase, phosphatidylinositol 4-phosphate 5-kinase (PIP5K), produces the versatile phospholipid phosphatidylinositol 4,5-bisphosphate (PI4,5P₂), through which PIP5K plays crucial roles in a wide variety of cell functions. So far, three PIP5K isozymes and splicing variants have been identified. We speculate that each PIP5K isozyme or splicing variant is activated in a tempo-spatially different manner, due to the existence of activators or recruiters specific to each isozyme: this tempo-spatially different activation of PIP5K produces PI4,5P₂ at different compartments of the cell at different times, which phenomenon may be responsible for the apparent multifunction of PI4,5P₂/PIP5K. Accumulating evidence supports this notion that each PIP5K isozyme is activated by its specific activator and plays a crucial role in a unique cell function. In this article, we describe recent advances regarding the PIP5K isozyme-specific activation mechanisms and physiological functions.

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Inositol phospholipids phosphorylated on D3-position of their inositol rings (3-phosphoinositides) are known to play important roles in various cellular events. Activation of PI (phosphatidylinositol) 3-kinase is essential for aspects of insulin-induced glucose metabolism, including translocation of GLUT4 to the cell surface and glycogen synthesis. The enzyme exists as a heterodimer containing a regulatory subunit and one of two widely-distributed isoforms of the p110 catalytic subunit: p110α or p110β. Activation of PI 3-kinase and its downstream AKT has been demonstrated to be essential for almost all of the insulin-induced glucose and lipid metabolism such as glucose uptake, glycogen synthesis, suppression of glucose output and triglyceride synthesis as well as insulin-induced mitogenesis. Accumulated PI(3,4,5)P₃ activates several serine/threonine kinases containing a PH (pleckstrin homology) domain, including Akt, atypical PKCs, p70S6 kinase and GSK.
In the obesity-induced insulin resistant condition, JNK and p70S6K are activated and phosphorylate IRS-proteins, which diminishes the insulin-induced tyrosine phosphorylation of IRS-proteins and thereby impairs the PI 3-kinase/AKT activations. Thus, the drugs which restore the impaired insulin-induced PI 3-kinase/AKT activation, for example, by suppressing JNK or p70S6K, PTEN or SHIP2, could be novel agents to treat diabetes mellitus.

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Recent advances in our understanding of the molecular basis of mammalian host immune responses to microbial invasion suggest that the first line of defense against microbes is the recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs). Phosphoinositide 3-kinase (PI3K) is thought to participate in the TLR signaling pathway. The activation of PI3K is commonly observed after stimulation with various TLR ligands. The resultant activation of a serine-threonine protein kinase Akt leads to the phosphorylation of glycogen synthase kinase (GSK)-3β, which is active in resting cells but is inactivated by phosphorylation. GSK-3β has been linked to the regulation of a multitude of transcription factors, including NF-κB, AP-1, NF-AT, and CREB either negatively or positively. Thus, the altered activity of GSK-3β causes diverse effects on cytokine expression. Generally, activation of PI3K results in the inhibition of proinflammatory events such as expression of IL-12 and TNF-α. Thus, PI3K is a negative regulator of TLR signaling. Among the members of the Class I PI3K family, p85/p110β appears to be the subtype activated on TLR ligation, but the molecular basis for this specificity has yet to be elucidated.

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The tumor suppressor phosphatase and tensin homolog (PTEN) functions as a phosphoinositide 3-phosphatase, that antagonizes phosphatidylinositol 3-kinase action, and negatively regulates cell proliferation and survival signals. Inactivation of PTEN by loss-of-function mutations gives rise to deregulated hyperproliferation of cells, leading to oncogenic transformation. Recent studies have identified a number of upstream regulatory factors for PTEN and unveiled that the impairment in the PTEN regulatory system potentially becomes a causal factor for oncogenic transformation of cells. This article will review the PTEN inactivation mechanism which is linked to human tumorigenesis, particularly focusing on recent research progress in PTEN regulators.

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Emodin has numerous biochemical and pharmacological activities, though information about its intestinal absorption and first-pass metabolism is scarce. The purpose of this study was to evaluate intestinal absorption and metabolism of luminally administered emodin in an isolated rat small intestine using the method of LC/MS/MS. About 22.55% of the administered emodin appeared at the vascular side, chiefly as free emodin (12.01%), but some emodin glucuronide (8.69%) and sulfate (1.84%) were also detected. Free glucuronide (5.23%) and sulfate (1.08%) moieties were found in the luminal perfusate. This model serves as a valuable tool for understanding intestinal handling of emodin, and our results confirm absorption and first-pass metabolism of emodin in the rat small intestine. Phase II metabolic enzymes such as glucuronyl transferase or sulfate transferase may also play an important role in the first-pass metabolism of emodin in the small intestine, which may ultimately reduce the bioavailability (and thus the efficacy) of orally administered emodin.

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Background: Despite increasing evidence that membrane type 1 matrix metalloproteinase (MT1-MMP), matrix metalloproteinase-2 (MMP-2), and cyclooxygenase-2 (COX-2) are involved in the pathogenesis of atherosclerosis, the possible links among these enzymes remain unclear. Accordingly, we investigated the distribution of MT1-MMP, MMP-2, and COX-2 immunohistologically in the atherosclerotic lesions of hypercholesterolemic (WHHLMI) rabbits. Methods and Results: Distribution of MT1-MMP, MMP-2, and COX-2 was examined by immunohistochemical staining using sixty cross sections of the ascending-arch and thoracic aortas prepared from 4 WHHLMI rabbits. MT1-MMP and MMP-2 staining was prominently observed in the macrophage-rich regions of the atheromatous lesions, and was positively correlated with morphological vulnerability (r=0.63 for MT1-MMP; r=0.60 for MMP-2; p<0.0001). MT1-MMP staining was positively correlated with MMP-2 staining (r=0.61, p<0.0001). COX-2 staining was also the highest in the macrophage-rich regions of the atheromatous lesions, with relatively high staining levels in other more stable lesions. Conclusions: Co-distribution of MT1-MMP, MMP-2, and COX-2 was demonstrated in grade IV atheroma, indicating a possible link among these enzymes in the destabilization of atherosclerotic plaques. The relatively high COX-2 distribution in other more stable lesions may indicate its additional roles in the stabilization of atherosclerotic lesions. The present findings in hypercholesterolemic rabbits should help advance our understanding of the pathophysiology of atherosclerosis and provide useful information for the development of new therapeutic and diagnostic (imaging) agents that target MMPs and COX-2 in atherosclerosis.

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Estrogen receptor (ER) is a member of the nuclear receptor superfamily, and functions as a ligand-dependent transcription factor with roles in cell growth and differentiation. In addition to endogenous estrogen, 17β-estradiol (E₂) and artificial antagonists, many suspected environmental estrogenic chemicals are reported to bind to ER, with various affinities and transcriptional responses. ER is also an allosteric protein and shows a positive cooperative interaction with E₂. Cooperativity affects inter-subunit interaction, and while ligand-bound ER interacts with coactivators, antagonist-bound ER does not. We therefore hypothesized that ligand-binding characteristics influence coactivator recruitment to the ER dimer, and thereby affect transcriptional activity. We investigated the interaction between ER and human Steroid Receptor Coactivator-1 (SRC-1), in the presence of compounds exhibiting various Hill coefficients. In the case of both ER subtypes (ERα and ERβ), the Hill coefficients of the compounds tested correlated with the affinity of the ER-ligand complex to SRC-1, with the exception of ERβ-4-n-nonylphenol and ER-antagonist complexes. This is the first report to investigate the relationship between Hill coefficients of ligand binding and coactivator interaction with the ER-ligand complex. We also examined the proteolytic digestion of ER using trypsin, in the presence and absence of compounds with various Hill coefficients, to investigate ligand-dependent conformational changes in ER. We used not only agonists and antagonists but also compounds of weak biological activity (partial agonists). Our results shed light on the subtle modulation of transcriptional activation by chemical agents.

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Three isoforms of anionic chum salmon trypsin (ST-1, ST-2, and ST-3) were purified from the pyloric caeca of chum salmon (Oncorhynchus keta). The molecular weights of the three isoforms were about 24 kDa as determined by SDS-PAGE. The isoelectric points of ST-1, ST-2, and ST-3 were 5.8, 5.4, and 5.6, respectively. The apparent K_m values of two isoforms (ST-1 and ST-2) for BAPA (benzoyl-L-arginine-p-nitroanilide) hydrolysis at 5, 15, 25 and 35 °C were slightly higher than that of the main isoform ST-3, depending on temperature. The turnover numbers, k_cat, of ST-1 and ST-2 were about twice as high as that of ST-3. Consequently, the catalytic efficiencies (k_cat/K_m) of ST-1 and ST-2 were more efficient than ST-3. There were marked differences in both apparent K_m and k_cat values of three anionic chum salmon trypsins as compared to bovine cationic trypsin. K_m values of all chum salmon trypsins were approximately 10 times lower than those of bovine trypsin, depending on the temperature. The k_cat values of all chum salmon trypsins were about 2- to 5-fold higher than those of bovine trypsin; therefore, the catalytic efficiencies (k_cat/K_m) of chum salmon trypsin were 20- to 40-fold more efficient than those of bovine trypsin. On the other hand, k_cat/K_m values of ST-1 for TAME (tosyl-L-arginine methyl ester) hydrolysis were lower than those of bovine trypsin, whereas k_cat/K_m values of ST-2 and ST-3 were comparable to those of bovine trypsin, depending on the temperature.

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A method for the determination of digoxin in human serum using a liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) technique is reported. Digoxin and the internal standard, [21,21,22-²H₃]digoxin, were extracted from 250 μl of human serum using a solid phase extraction cartridge (Oasis HLB) and analyzed by LC/ESI-MS/MS in the selected reaction monitoring mode. The intra- and inter-assay reproducibility and accuracy were satisfactory within the quantification range of 0.20—3.20 ng/ml. The concentrations of digoxin in the serum samples obtained from digitalized patients (n=19) were in the range of 0.25—2.84 ng/ml, which were compared to those obtained by radioimmunoassay.

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Develop a simple and reliable assay method to detect ginseng stem and leaf saponins (GSLS) in methanol and rat plasma by liquid chromatography-electrospray ionization mass spectrometry in scan mode, and construct the fingerprints of GSLS reference substances and plasma samples. In order to screen the active constituents of GSLS, analysis and comparison were carried out between the LC/ESI-MS profiles of blank rat plasma and rat plasma samples obtained after oral administration of GSLS. Thirty-one compounds were detected and 10 of them were identified in the fingerprints of reference substances and spiked plasma sample. Furthermore, 12 compounds (C7, C8, C14, C15, C18, Re, C24, Rb₁, Rc, Rb₂, Rb₃, Rd) were absorbed easily and some new compounds were generated after oral administration of GSLS, which might be the metabolites of GSLS. These absorbed components and new compounds may be the main bioactive components of GSLS.

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The quantification of tenofovir, a nucleoside reverse transcriptase inhibitor prescribed once daily, in human plasma is important due to a recent increase in its use. HPLC, however, can not easily detect and quantify tenofovir because of interfering peaks. Therefore, we developed a rapid and conventional LC-MS method, validated by estimating the precision and accuracy for inter- and intraday analysis in the concentration range of 0.019—1.567 μg/ml. The calibration curve was linear in the described concentration range. Average accuracy ranged from 95.9 to 100.7%. Relative standard deviations of both inter- and intraday assays were less than 11.6%. Recovery of tenofovir was more than 80.2%. This novel method provides a conventional, accurate and precise way to determine tenofovir in human plasma samples.

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Phenyl-substituted dihydropyrazines (Ph-DHPs) are derivatives of 2,3-dihydro-5,6-dimethylpyrazine (Me-DHP). Upon the addition of Cu²⁺, Me-DHP inhibits the growth of Escherichia coli by generating hydroxyl and carbon-centered radicals that cause DNA strand breakage. Here, we investigated the toxic effect of Ph-DHPs in several DNA repair-deficient or detoxifying enzyme-deficient mutant strains. Ph-DHPs caused cytotoxic and genotoxic damage, but, in a sodA sodB strain, the effects in the presence or absence of Cu²⁺ were different than those of Me-DHP. Our results suggest that the action of the generated superoxide anion in the interior side of the cell is remarkable.

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Platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) acetylhydrolase (PAF-AH) activity has been identified in bovine colostrum and high levels of this activity are found in early colostrum (within 24 h after parturition). In this study, PAF-AH in early colostrum was purified by ammonium sulfate precipitation, and sequential use of butyl-Toyopearl 650M, DEAE-Sepharose, heparin-Sepharose, hydroxyapatite, chelating-Sepharose and Mono Q HPLC column chromatography. This enzyme is a monomeric polypeptide with a molecular weight of approximately 45 kDa on 12.5% SDS-PAGE. The V_max and K_m for PAF-AH were 87.6 μM and 7.96 nmol/min/mg respectively. This enzyme was inhibited by phenylmethylsulfonyl fluoride, iodoacetamide and p-bromophenacylbromide, suggesting that both serine and histidine residues are required for enzyme activity. It was not inactivated by NaF or dithiothreitol. The purified enzyme did not degrade phospholipids with a long chain fatty acyl group at the sn-2 position. Accordingly, this enzyme is distinct from phospholipase A₂. In addition, PAF-AH selectively hydrolyzed oxidatively modified phosphatidylcholine. Furthermore, this enzyme was shown by Western blot analysis using antibody to human plasma PAF-AH to be plasma type PAF-AH. These results clearly demonstrate that 45 kDa plasma type PAF-AH activity exists in bovine colostrum.

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Dog liver contains an oligomeric NADPH-dependent carbonyl reductase (CR) with substrate specificity for alkyl phenyl ketones, but its endogenous substrate and primary structure remain unknown. In this study, we examined the molecular weight and substrate specificity of the enzyme purified from dog liver. The enzyme is a ca. 100-kDa tetramer composing of 27-kDa subunit, and reduces all-trans-retinal and α-dicarbonyl compounds including isatin, which are substrates for pig peroxisomal tetrameric carbonyl reductase (PTCR). In addition, the dog enzyme resembles pig PTCR in inhibitor sensitivity to flavonoids, myristic acid, lithocholic acid, bromosulfophthalein and flufenamic acid. Furthermore, the amino acid sequence of dog CR determined by protein sequencing and cDNA cloning was 84% identical to that of pig PTCR and had a C-terminal peroxisomal targeting signal type 1, Ser-His-Leu. The immunoprecipitation using the anti-pig PTCR antibody shows that the dog enzyme is a major form of soluble NADPH-dependent all-trans-retinal reductase in dog liver. Thus, dog oligomeric CR is PTCR, and may play a role in retinoid metabolism as a retinal reductase.

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Angiogenesis is important for promoting cardiovascular disease, wound healing, and tissue regeneration. We investigated the effects of Korean red ginseng water extract (KRGE) on angiogenesis and its underlying signal mechanism. KRGE increased in vitro proliferation, migration, and tube formation of human umbilical vein endothelial cells, as well as stimulated in vivo angiogenesis without increasing VEGF expression. KRGE-induced angiogenesis was accompanied by phosphorylation of ERK1/2, phosphatidylinositol 3-kinase (Akt), and endothelial nitric oxide synthase (eNOS) as well as an increase in NO production. Inhibition of PI3K activity by wortmannin completely inhibited KRGE-induced angiogenesis and phosphorylation of Akt, ERK1/2, and eNOS, indicating that PI3K/Akt activation is an upstream event of the KRGE-mediated angiogenic pathway. The MEK inhibitor PD98059 blocked KRGE-induced ERK1/2 phosphorylation without affecting Akt and eNOS activation. However, the eNOS inhibitor N^G-monomethyl-L-arginine effectively inhibited tube formation, but partially blocked proliferation and migration as well as ERK phosphorylation, without altering Akt and eNOS activation, revealing that the eNOS/NO pathway is partially involved in ERK1/2 activation. This study demonstrated that KRGE stimulates in vitro and in vivo angiogenesis through the activation of the PI3K/Akt-dependent ERK1/2 and eNOS signal pathways and their cross talk.

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We previously reported that, in Jurkat human T cells, the topoisomerase II inhibitor etoposide enhances sialidase activity and reduces cell surface sialic acid levels at an early stage of apoptosis and that the decreases in sialic acid are suppressed by the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid [Azuma Y., et al., Glycoconj. J., 17, 301—306 (2000)]. In the current studies, we treated Jurkat cells with etoposide and examined the changes in the cell surface levels of gangliosides GM1, GM2, GM3, GD1a, and GD3 at physiological pH using anti-ganglioside antibodies. We also examined the sialidase activity on the cell surface using 4-methylumbelliferyl N-acetylneuraminic acid and measured the mRNA expression of the plasma membrane-associated sialidase Neu3 and the lysozomal Neu1 using real-time PCR. We found an increase in GM3 and a decrease in GD3 during the early stage (4 h) of etoposide-induced apoptosis that preceded the increase in cell surface exposure of phosphatidylserine (4 to 6 h). The caspase 3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde significantly suppressed changes in GM3 and GD3 and blocked the enhanced cell surface sialidase activity. Furthermore, etoposide caused a gradual up-regulation of Neu3 mRNA expression but not Neu1 mRNA expression. Enhanced Neu3 mRNA expression was suppressed in the presence of caspase 3 inhibitor. These results indicate that Neu3 is up-regulated in Jurkat cells undergoing etoposide-induced apoptosis through intracellular signaling events downstream of caspase 3 activation and that enhanced Neu3 activity is closely related to the changes of cell surface ganglioside composition.

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Yeast Pif1 DNA helicase is the prototype member of a helicase subfamily participating in the maintenance of telomere, ribosome, and mitochondria DNAs. The Pif1 DNA helicase family is highly conserved from yeast to human, but the biochemical nature of human homologues remains to be clarified. To this end, we investigated the transcriptional unit of human Pif1 gene and its encoded protein hPif1. The results showed that the hPif1 gene product has at least two isoforms consisting of the conserved helicase motif and differential C-terminal regions derived from alternative splicing of the gene transcript. Deletion mutant analysis showed that Pif1 helicase has nuclear localization signal and mitochondria targeting signal at the N-terminal and C-terminal regions, respectively. In HeLa cells, hPif1 helicase expression was induced by the release of cells from serum starvation, suggesting that hPif1 has roles in the S phase. Consistently, the down regulation of the hPif1 helicase by RNA interference with siRNA caused a cell cycle delay at the S phase. These findings suggest that hPif1 in the nucleus may be involved in chromosome maintenance in association with DNA replication, while the function of hPif1 remains to be clarified.

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Cycloprodigiosin hydrochloride (cPrG·HCl), a compound isolated from a marine bacterium, acts as an immunosuppressant and an anti-cancer drug. We have previously reported that cPrG·HCl suppressed the transcriptional activation of nuclear factor (NF)-κB. Here we studied the effect of cPrG·HCl on activation of another transcription factor, activator protein 1 (AP-1). cPrG·HCl potently suppressed AP-1 activity induced by tumor necrosis factor (TNF) α and phorbol myristate acetate (PMA). cPrG·HCl did not inhibit any of the mitogen-activated protein kinase (MAPK) families, whereas it did suppress transcriptional activation of AP-1 induced by constitutively activated mutants of MEKK1 or Ras. cPrG·HCl inhibited neither TNFα- or PMA-induced DNA-binding of AP-1 nor co-activator p300-induced activation of AP-1. Taken together, cPrG·HCl suppresses AP-1-dependent gene expression downstream of MAPK group through the inhibition of the transcription activation step of the AP-1 promoter complex.

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In the present study, we investigated synergic anticandidal effect of epigallocatechin-O-gallate (EGCG) in a murine model of disseminated candidiasis caused by Candida albicans. In addition, its mechanism was examined. In the animal system, EGCG-given BALB/c mice group intraperitoneally (i.p.) before intravenous (i.v.) inoculation with viable C. albicans yeast cells survived longer than diluent-received (control) mice group (p<0.05). EGCG treatment inhibited the hyphal formation from the yeast form of C. albicans, causing growth-inhibition of the candidal cells. In experiments determining synergic effect, mice given diluent (control), Amp B (amphotericin B; 0.5 mg/kg of body weight), or EGCG (2 mg/kg) had mean survival times (MST) of approximately 10.9, 11.7, and 13.9 d, respectively. However, mice administered combination of Amp B (0.5 mg/kg) plus EGCG (2 mg/kg) had a MST value of 42.1 d, surviving an average of app. 30 d longer than the Amp B alone-received mice groups. The MST value from the combination-treated mice groups was much greater than MST value from mice groups that received four times the Amp B dose. These results indicate that EGCG, which has anticandidal activity causing blockage of the hyphal formation, has the synergism combined with Amp B against disseminated candidiasis.

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Shiga toxin (Stx) exerts toxic activity by binding to glycosphingolipids, mainly globotriaosyl (Gb₃) ceramide, on the surface of target cells. The inhibition of toxin-receptor binding is a promising therapeutic approach to prevent Stx-mediated diseases. In this study, we synthesized monovalent Stx-ligands of phosphatidylethanolamine dipalmitoyl-Gb₃ (Gb₃-PEDP) and galabiosyl (Gb₂)-PEDP and we examined their neutralizing activity against Stx-1 and Stx-2 in vitro. Both Gb₃-PEDP and Gb₂-PEDP strongly neutralized the cytotoxicity of Stx-1 and Stx-2. It is likely that the mechanism of neutralization involved formation of liposomes and consequently clustering of sugar units. We propose monovalent Gb₃-/Gb₂-derivatives conjugated with phosphatidyl residue as a novel class of Stx-neutralizing agent.

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The purpose of the present study was to investigate the effect of Ganoderma lucidum polysaccharide (GLPS), a major active component in Chinese medicinal fungus, on cytochrome P450 metabolic activity in Bacillus Calmette Guérin (BCG)-induced immune hepatic injury in rats. The enzyme kinetics of the probes including chlorzoxazone (CYP2E1), phenacetin (CYP1A2) and nifedipine (CYP3A) were evaluated by HPLC. The results showed that BCG-pretreatment (125 mg/kg) significantly increased serum levels of alanine transaminase (ALT), nitrite and malondialdehyde (MDA), inhibited activities of superoxide dismutase (SOD) and decreased P450 total content in microsomes (p<0.05). Administration of GLPS (50 and 200 mg/kg) reversed above hepatic injury stimulated by BCG in vivo. Moreover, GLPS dose-dependently inhibited activities of CYP2E1, CYP1A2 and CYP3A in hepatic microsomes in vitro, suggesting that inhibition of GLPS on P450 oxidative metabolism might participate in the hepatoprotective mechanism, and also suggested that pharmacokinetics might be changed by drug–herb interaction.

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The anti-allergic effect of an ethanol extract from Moutan Cortex was evaluated in some animal models. The Moutan Cortex extract (30, 100 mg/kg, i.p.) dose-dependently inhibited systemic anaphylactic shock induced by compound 48/80 in mice. It also inhibited dose-dependently the scratching behavior induced by compound 48/80 or histamine at a dose of 100 mg/kg. An increase in the vascular permeability induced by compound 48/80 or histamine was also inhibited by the Moutan Cortex. In addition, in vitro studies, the Moutan Cortex inhibited histamine release from rat peritoneal mast cells induced by compound 48/80. To investigate the active component of Moutan Cortex extract, it was suspended in water and extracted with EtOAc to yield EtOAc insoluble (A) and soluble (B) fractions. The effect of extract (B) was more potent than that of extract (A) in inhibiting histamine release. From these findings, it seems likely that the Moutan Cortex extract is effective in antagonizing certain pharmacological effects induced by compound 48/80, which is probably mediated by inhibiting the release of histamine from mast cells and antagonizing the effect on histamine. The main active component of Moutan Cortex is considered to be contained in extract (B). In conclusion, Moutan Cortex may be useful for the relief of symptoms of atopic dermatitis and other allergy-related diseases.

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Tyrosinase is a key enzyme for melanin biosynthesis and known to be sensitive to ultraviolet light in the presence of oxygen. Therefore, finding effective tyrosinase inhibitors, either from synthetic or natural sources, can be beneficial in the treatment of melanin-related disorders. We synthesized 4-(6-hydroxy-2-naphthyl)-1,3-bezendiol (HNB), a new family of hydroxyl substituted phenyl naphthalenes, as the isosteres of oxyresveratrol. This study investigated inhibitory effects of HNB on tyrosinase activity. HNB inhibited mushroom tyrosinase with an IC₅₀ value of 0.07 μM, which is more potent than the anti-tyrosinase activity of kojic acid (IC₅₀=38.24), a well-known tyrosinase inhibitor. The kinetic analysis of tyrosinase inhibition revealed that HNB is a competitive inhibitor (K_i 4.78×10⁻⁹ M at 0.125 μM and K_i 6.21×10⁻⁹ M at 0.25 μM). We further found that HNB also inhibited melanin production in B16F10 melanoma cells (B16 cells). In addition to tyrosinase inhibiting activity, melanin biosynthesis was inhibited by HNB in the B16F10 cells. These data strongly suggest that HNB can suppress the production of melanin via the modulation of tyrosinase activity.

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KNI-472 is the first anesthetic system for mice and rats to incorporate a ventilator. It consists of a newly developed syringe pump-type vaporizer and gas monitor that can deliver accurate concentrations of anesthetic gas at an extremely low airflow. In this study, we compared the hemodynamic effects of isoflurane anesthesia using KNI-472 and intraperitoneal pentobarbital anesthesia. In the isoflurane anesthetic group, Institute of Cancer Research (ICR) mice were anesthetized with 5% isoflurane, followed by endotracheal intubation. Subsequently, they were ventilated mechanically, and anesthesia was maintained with 2% isoflurane for a 60-min period using KNI-472. In the pentobarbital anesthetic group, the ICR mice were anesthetized by an intraperitoneal injection of sodium pentobarbital (70 mg/kg). In isoflurane anesthesia, the heart rate (HR) and mean blood pressure (MBP) were stable. In contrast, in pentobarbital anesthesia, MBP decreased in the first stage after the initiation of anesthesia, after which it gradually increased. The intra-group variability in the estimated skin blood flow (SBF) was higher in the pentobarbital anesthesia than that in the isoflurane anesthesia. The PaO₂ and PaCO₂ values at 15 min after the initiation of pentobarbital anesthesia revealed hypoxia and hypercapnia compared with isoflurane anesthesia. In this study, isoflurane anesthesia using KNI-472, unlike pentobarbital anesthesia, did not induce changes in MBP, SBF, or blood gases. The changes induced by pentobarbital anesthesia were attributed to a change in the depth of anesthesia with time. These results indicate that inhalation anesthesia using KNI-472 is suitable in research on the hemodynamic state in mice.

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Ginsenosides, active ingredients of Panax ginseng, exist as stereoisomers depending on the position of the hydroxyl group on carbon-20; i.e. 20(R)-ginsenoside and 20(S)-ginsenoside are epimers. We previously investigated the structure–activity relationship of the ginsenoside Rg₃ stereoisomers, 20-R-protopanaxatriol-3-[O-β-D-glucopyranosyl (1→2)-β-glucopyranoside], (20(R)-Rg₃) and 20-S-protopanaxatriol-3-[O-β-D-glucopyranosyl (1→2)-β-glucopyranoside], (20(S)-Rg₃) in regulating 5-HT_3A receptor-mediated ion currents (I_5-HT) expressed in Xenopus oocytes and found that 20(S)-Rg₃ rather than 20(R)-Rg₃ was more stronger inhibitor of I_5-HT. In the present study, we further investigated the effects of 20(R)-Rg₃ and 20(S)-Rg₃ on mouse 5-HT_3A receptor channel activity after site-directed mutations of 5-HT_3A receptor facilitation site, which is located at pre-transmembrane domain I (pre-TM1). 5-HT_3A receptor was expressed in Xenopus oocytes, and I_5-HT was measured using two-electrode voltage clamp technique. In wild-type, both 20(R)-Rg₃ and 20(S)-Rg₃ inhibited I_5-HT with concentration-dependent and reversible manner. Induction of 5-HT_3A receptor facilitation by point mutations of pre-TM1 amino acid residue R222 to R222A, R222D, R222E or R222T not only decreased EC₅₀ values for I_5-HT compared to wild-type but also abolished 20(R)-Rg₃-induced inhibition of I_5-HT. Those mutations also shifted the IC₅₀ values by 20(S)-Rg₃ into right direction by 2- to 4-folds compared with wild-type. These results indicate that 5-HT_3A receptor facilitation differentially affects 20(R)-Rg₃- and 20(S)-Rg₃-mediated 5-HT_3A receptor channel regulation.

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The effects of Polygala tenuifolia root fractions and the acyl groups of its constituents on the retrieval process of spatial cognition in rats were studied using an eight-arm radial maze task. Oral administration of a precipitate fraction (PTB) obtained by concentration of the n-BuOH-soluble portion from the extract of the roots significantly decreased the number of total errors (TEs) and that of working memory errors (WMEs) at doses of 100 mg/kg and 200 mg/kg. However, it caused no significant decrease in the number of reference memory errors (RMEs). In addition, the saponin-rich fraction (PTBM) obtained by purification of PTB also showed significant decreases in TEs and WMEs at a dose of 100 mg/kg. Among the cinnamic acid derivatives present as the acyl groups in the P. tenuifolia constituents, sinapic acid (SNPA) significantly decreased TEs and WMEs at doses of 10 to 100 mg/kg. These results indicated that P. tenuifolia extracts, PTB and PTBM, and SNPA had a beneficial effect on the memory impairment induced by dysfunction of the cholinergic system in the brain. The memory improvement in the scopolamine-induced memory impairment seen in the radial maze performance was due to improvement in the short-term memory. A contribution of some constituents other than SNPA to the memory improvement was also suggested.

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This study aimed to investigate the mechanism underlying the protective effects of manganese complexes of curcumin (Cp–Mn) and diacetylcurcumin (DiAc–Cp–Mn) on kainic acid (KA)-induced excitotoxicity in the rat hippocampus. Systemic injection of KA (10 mg/kg, i.p.) caused seizures and increased the expression of neurotoxic markers, immediate early genes [c-jun, cyclooxygenase 2 (COX-2), brain-derived neurotrophic factor (BDNF), and heat shock protein 70 (hsp70)] and a delayed response gene [inducible nitric oxide synthase (iNOS)], which were measured at 6 and 72 h after KA injection, respectively, in the hippocampus. Pretreatment with Cp–Mn (50 mg/kg, i.p.) and DiAc–Cp–Mn (50 mg/kg, i.p.) but not with curcumin (50 mg/kg, i.p.) delayed the onset of KA-induced seizure without affecting the seizure score. KA injection induced c-Fos immunoreactivity in DG, CA1, and CA3 hippocampal regions, the expression of which peaked at 6 h after injection. Cp–Mn and DiAc–Cp–Mn treatment significantly decreased c-Fos expression elicited by KA. Moreover, Cp–Mn and DiAc–Cp–Mn administration suppressed the KA-induced expression of c-jun, COX-2, BDNF, and iNOS mRNA, whereas curcumin attenuated only iNOS mRNA expression. No compounds tested had an effect on KA-induced hsp70 expression. It is therefore likely that in addition to radical scavenging and SOD-like activities, the suppression of potential neuronal injury marker expression by Cp–Mn and DiAc–Cp–Mn, contributes to the neuroprotective activities of these compounds, which are superior to those of curcumin, on KA-induced excitotoxicity in the hippocampus. These results suggest the beneficial effects of Cp–Mn, and DiAc–Cp–Mn on the treatment of excitotoxicity-induced neurodegenerative diseases.

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The apicomplexan parasites pathogens such as Plasmodium spp. possess an apicoplast, a plastid organelle similar to those of plants. The apicoplast has some essential plant-like metabolic pathways and processes, making these parasites susceptible to inhibitors of these functions. The main objective of this paper is to determine if phytotoxins with plastid target sites are more likely to be good antiplasmodial compounds than are those with other modes of action. The antiplasmodial activities of some compounds with established phytotoxic action were determined in vitro on a chloroquine (CQ) sensitive (D6, Sierra Leone) strain of Plasmodium falciparum. In this study, we provide in vitro activities of almost 50 such compounds, as well as a few phytoalexins against P. falciparum. Endothall, anisomycin, and cerulenin had sufficient antiplasmodial action to be considered as new lead antimalarial structures. Some derivatives of fusicoccin possessed markedly improved antiplasmodial action than the parent compound. Our results suggest that phytotoxins with plastid targets may not necessarily be better antiplasmodials than those that act at other molecular sites. The herbicides, phytotoxins and the phytoalexins reported here with significant antiplasmodial activity may be useful probes for identification of new antimalarial drug targets and may also be used as new lead structures for new antiplasmodial drug discovery.

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Japanese cedar pollen (Cryptomeria japonica, Cry j) is the most common allergen causing pollinosis in Japan. However, short ragweed pollen is used commonly as the antigen for experimentally-induced allergic conjunctivitis (EC) and Cry j-induced EC in mice has not been published. We actively immunized BALB/c mice with Cry j, and then performed a challenge with eye drops containing Cry j. We evaluated the early phase and late phase reactions in the conjunctiva, using Evans blue dye leakage and eosinophil infiltration, respectively. Significant inhibition of the early phase reaction was observed following pre-challenge with eye drops that block histamine H1 receptor in the conjunctiva. Thus, Cry j-induced EC appears to represent a suitable model for the study of pollinosis in Japan.

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Zizyphi Spinosi Semen (ZSS) has been widely used for the treatment of insomnia in oriental countries. This experiment was performed to investigate whether sanjoinine A, one of major alkaloid compounds of ZSS, has hypnotic effects and/or enhances pentobarbital-induced sleeping behaviors through the γ-aminobutyric acid (GABA)-ergic systems. Sanjoinine A itself did not induce sleeping at the higher dose used in this experiment. However, sanjoinine A prolonged sleeping time and reduced the sleeping latency induced by pentobarbital in a dose-dependent manner similar to muscimol, a GABA_A receptor agonist. Sanjoinine A also increased sleeping rate and sleeping time when administered combined with pentobarbital at a sub-hypnotic dosage and showed synergistic effects with muscimol in potentiating sleeping onset and enhancing sleeping time induced by pentobarbital. In addition, both sanjoinine A and pentobarbital increased chloride influx in primary cultured cerebellar granule cells. Sanjoinine A also showed similar effects with muscimol in potentiating chloride influx inducing effects of low dose pentobarbital. Sanjoinine A decreased GABA_A receptor α-subunit expression and increased γ-subunit expression, and had no effects on the abundance of β-subunits in primary cultured cerebellar granule cells, showing different subunit expression from pentobarbital. In addition, we found that sanjoinine A also enhanced expression of glutamic acid decarboxylase (GAD), but pentobarbital did not. In conclusion, sanjoinine A itself does not induce sleeping, but it augments pentobarbital-induced sleeping behaviors through the modification of GABA-ergic systems.

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Abnormal vascular smooth muscle cell (VSMC) proliferation and migration are involved in restenosis following percutaneous transluminal angioplasty (PTCA) as well as in the development and progression of atherosclerosis. We investigated the mechanisms underlying the inhibitory effect of the sesquiterpene 3-oxo-5αH,8βH-eudesma-1,4(15),7(11)-trien-8,12-olide (1) on rat VSMC proliferation and migration. VSMCs were isolated from rat aorta, and then the effect of 1 on cell proliferation and migration was examined using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and chemotaxis assays, respectively. Compound 1 had a potent inhibitory effect on fetal calf serum-induced VSMC proliferation. This effect correlated with reduced expression of cyclin D₁. In addition, 1 also inhibited platelet derived growth factor (PDGF)-induced migration of VSMCs. These results indicate that 1 is a promising candidate for additional biological evaluation to further define its potential as an inhibitory modulator of VSMC responses that contribute to restenosis following PTCA and to the development and progression of atherosclerosis.

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Cordyceps sinensis (CS) has been known as a component of traditional medicines that elicit various biological effects such as anti-fatigue, immunomodulatory, and hypoglycemic actions. Since it has been well-established that fatigue is closely related to depression, we used the tail suspension test (TST) in mice to examine the antidepressant-like effects of hot water extract (HWCS) and supercritical fluid extract (SCCS) of CS. Immobility time in the TST was reduced by administration of SCCS (2.5—10 ml/kg, p.o.) dose-dependently though it was not reduced by treatment with HWCS (500—2000 mg/kg, p.o.). Neither HWCS nor SCCS altered locomotor activity in the open field test, excluding the possibility that the effect of SCCS is due to activation of locomotion. Pretreatment with prazosin (an adrenoreceptor antagonist) or sulpiride (a dopamine D2 receptor antagonist) reduced the effect of SCCS on the immobility time. In contrast, pretreatment with p-chlorophenylalanine (p-CPA, a serotonin synthesis inhibitor) did not alter the anti-immobility effect of SCCS. The last finding is consistent with an additional observation that SCCS had no effect on head twitch response induced by 5-hydroxy-L-tryptophan in mice. Taken altogether, these results suggest that SCCS may elicit an antidepressant-like effect by affecting the adrenergic and dopaminergic systems, but not by affecting the serotonergic system.

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Cornuside is a bisiridoid glucoside compound isolated from the fruit of Cornus officinalis SIEB. et ZUCC. The present study was designed to examine the effects of cornuside on expression levels of cytokine-induced proinflammatory and adhesion molecules in the human umbilical vein endothelial cells (HUVECs). Cornuside treatment attenuated tumor necrosis factor-α (TNF-α)-induced nuclear factor-kappa B (NF-κB) p65 translocation in HUVECs. In addition, cornuside suppressed the expression levels of endothelial cell adhesion molecules including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) induced by TNF-α. TNF-α-induced monocyte chemoattractant protein 1 (MCP-1) expression was also attenuated by treatment of cornuside. These inhibitory effects of cornuside on proinflammatory and adhesion molecules were not due to decreased HUVEC viability as assessed by MTT test. Taken together, the present study suggests that cornuside suppresses expression levels of cytokine-induced proinflammatory and adhesion molecules in the human endothelial cells.

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A homology-based cloning strategy yielded a cDNA clone designated Dcga, presumably encoding α subunit of GTP-binding protein, from carrot (Daucus carota) seedlings. Molecular phylogenetic tree analysis of G protein α subunits from various biological sources suggested that, unlike in animal cells, classification of Gα proteins into specific subfamilies could not be applicable to the proteins from higher plants. The restriction digests prepared from genomic DNA of carrot showed one or two hybridized signals in Southern blot analyses, and the expression level of Dcga was appreciably decreased upon the exposure of carrot to high temperature or the prolonged treatment with salt. These results suggest that Dcga occurs as single or double copy genes in carrot genome, and its transcript might play specific roles in heat- and salt-induced responses of the plant.

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In this study the enhancement effect of Ferula szowitsiana roots' acetone extract on the antibacterial activity of penicillin G and cephalexin was evaluated against Staphylococcus aureus. Disk diffusion and broth dilution methods were used to determine the antibacterial activity of these antibiotics in the absence and presence of plant extract and its various fractions separated by TLC plate. The active component of plant extract involved in enhancement of penicillin G's and cephalexin's activities had Rf=0.336 on a TLC plate. The spectral data (¹H-, ¹³C-NMR) of this compound revealed that this compound was 7-[6-(β-carboxyethyl)-5-isopropylidene-1,2-dimethylcyclo-hexylmethoxy]coumarin (galbanic acid), previously isolated from Ferula assa-foetida. In the presence of sub-inhibitory concentration of galbanic acid (100 μg/ml) the MIC of penicillin G for S. aureus decreased from 64 to 1 (a sixteen four-fold decrease) and for cephalexin from 128 to 1 μg/ml (a one hundred twenty eight-fold decrease). The highest fold decrease in MIC was observed for cephalexin in combination of galbanic acid against test strain. These results signify that the low concentration of galbanic acid (100 μg/ml) potentiates the antimicrobial action of penicillin G and cephalexin suggesting a possible utilization of these compounds in combination therapy against S. aureus.

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We compared the pharmacokinetics of model compounds with different elimination processes between hypothermic and normothermic rats, to obtain basic information concerning drug therapy during hypothermia. Male Wistar rats were anesthetized with sodium pentobarbital and kept at temperatures of 37 °C (normothermic group) by heat lamp, and 32 °C or 28 °C (hypothermic group) by external cooling. We chose phenolsulfonphthalein (PSP), indocyanine green (ICG) and fluorescein isothiocyanate (FITC)-dextran (FD-4, Mw 4400) as model compounds to determine changes in clearance pathways during hypothermia therapy. The plasma concentrations of PSP as biliary, urinary and metabolic elimination type were increased significantly in the hypothermic group (32 °C, 28 °C) after i.v. administration at a dose of 1 mg, compared to the normothermic group (37 °C). Each PSP clearance (bile, urine and metabolites) in the hypothermic group was decreased, suggesting an influence of hypothermia on the active elimination process. The decreasing tendency was marked at a temperature of 28 °C. Moreover, the plasma concentrations of ICG as the biliary excretion type after i.v. administration to the hypothermic rats at a dose of 1 mg were higher with more than 50% decrease in the total body clearance compared to normothermic rats. On the other hand, there was almost no difference in the i.v. pharmacokinetics of FD-4 as the urinary excretion type between 37 °C and 32 °C. However, renal clearance of FD-4 was significantly decreased at a temperature of 28 °C. Accordingly, the change in pharmacokinetics of a drug in the hypothermic group could differ with the elimination processes.

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The effect of poly-L-arginine with a molecular weight of 35.5 kDa (PLA) on the ocular absorption of hydrophilic molecules after instillation was examined in rabbits in vivo. FITC-labeled dextran (3.8 kDa, FD-4) and pyridoxamine were used as model hyprophilic molecules for absorption. The potential toxicity of PLA was evaluated by microscopic observation of the cornea, production of TNF-α, and the thickness of the corneal epithelia and stroma. The concentration of pyridoxamine and FD-4 in aqueous humor 30 min after a single instillation of a solution of PLA was 29- and 16-fold higher than that without PLA, respectively, but the drug concentrations were not determined in the vitreous body. Repetitive instillation of PLA every 30 min for 150 min achieved 31.1- and 13.3-fold increases in pyridoxamine and FD-4 in aqueous humor, respectively. Furthermore, significant amounts of pyridoxamine and FD-4 were detected in the vitreous body after the repetitive instillation of PLA, even although very little of these drugs was detected in the vitreous body in the control eye without PLA. On the other hand, repetitive instillation of PLA did not induce any alteration of corneal epithelial and stromal thickness, production of TNF-α, and disruption of the epithelial and stromal morphologies and neutrophil infiltration. Our findings suggest that PLA may be useful in promoting drug delivery of hydrophilic drugs to the ocular tissues without producing any significant corneal damage and inflammation.

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Both polyethylenimine (PEI) polymers and cationic nanoparticles have been widely used for non-viral DNA transfection. Previously, we reported that cationic nanoparticles composed of cholesteryl-3β-carboxyamidoethylene-N-hydroxyethylamine and Tween 80 (NP-OH) could deliver plasmid DNA (pDNA) with high transfection efficiency. To increase the transfection activity of NP-OH, we investigated the potential synergism of PEI and NP-OH for the transfection of DNA into human prostate tumor PC-3, human cervices tumor Hela, and human lung adenocarcinoma A549 cells. The transfection efficiency with low-molecular PEI (MW 600) was low, but that with a combination of NP-OH and PEI was higher than with NP-OH alone, being comparable to commercially available lipofectamine 2000 and lipofectamine LTX, with very low cytotoxicity. Low-molecular weight PEI could not compact pDNA in size, but rather might help to dissociate pDNA from the complex and release pDNA from the endosome to cytoplasm by the proton sponge effect. Therefore, the combination of cationic cholesterol-based nanoparticles and a low-molecular PEI has potential as a non-viral DNA vector for gene delivery.

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In order to develop a model of liver metastasis of human gastrointestinal cancer cells, we examined the potential of 10 human colon and stomach cancer cell lines (HT-29, WiDr, HCT-116, HCT-15, HCC-2998, MKN7, MKN28, MKN45, MKN74 and St-4) to form liver metastases in nude mice. Among the cell lines, HCT-116 cells consistently formed gross liver metastases when injected into the spleens of nude mice. In contrast, other human colon and stomach cancer cells produced little or no liver metastasis. In order to analyze the high metastatic potential of HCT-116 cells, the adhesion potential was compared between HCT-116 cells and the other colon cancer cell lines. HCT-116 cells showed more efficient adhesion to fibronectin (FN) than other cells. Furthermore, FN enhanced haptotaxis of HCT-116 cells, but not of other colon cancer cells. The high adhesion potential to FN and enhanced haptotaxis may contribute, at least in part, to the high metastatic potential of HCT-116. To assess the value of this newly developed model of liver metastasis, we compared the ability of four anticancer drugs (fluorouracil, doxifluridine, paclitaxel and irinotecan) to inhibit the formation of liver metastases. Paclitaxel and irinotecan showed strong inhibition of liver metastasis but fluorouracil and doxifluridine showed only slight inhibition. Therefore, this model of metastasis may be useful for screening anti-liver metastatic reagents. These results indicate that the HCT-116 liver-metastasis model should be useful for analyzing the molecular mechanism of liver metastasis and for evaluating new anti-liver metastatic drugs.

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Food mycotoxin deoxynivalenol (vomitoxin, DON) produced by Fusarium graminearum and F. culmorum can induce rapid diminution of lymphoid tissues and lymphopenia in the growing chickens and mammals. We first investigated the direct acute effects of DON on the chick immune-related embryo tissues such as embryonic liver and spleen. Direct DON administration into the embryonic eggs caused toxin accumulation in liver in a time-dependent manner. Electron microscopic observation showed a notable accumulation of fat droplet in the liver tissue and the re-exposed hatched chicken showed more distinguishing enlarged fat globules, so-called fatty cysts like human steatosis. Regarding effects of deoxynivalenol on the chick embryonic spleen, fatty change was also observed in splenocytes. Functionally, mitogen-stimulated cellular and humoral lympho-proliferations were suppressed in the DON-treated embryo. Conclusively, acute direct exposure to deoxynivalenol in the chick embryo caused toxic histological alterations in the liver and spleen and suppressed in vitro lymphoblastogenesis.

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By using a simple, low-cost system of polystyrene centrifuge tubes we compared the secreted aspartyl proteases (Saps) secretion during the biphasic growth modes of Candida albicans using twenty-one clinical isolates. Our results indicate that biofilms of C. albicans consistently secrete more Saps than their planktonic counterparts.

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As abnormal behaviors such as jumping and falling from balcony were reported in patients aged 10 to 19 years who administrated oseltamivir phosphate (Tamiflu) for treatment influenza infection, the Ministry of Health, Labor and Welfare in Japan notified that, as a rule, Tamiflu should not be prescribed to patients aged 10 to 19 years. To examine the relationship between Tamiflu and abnormal behaviors, we investigated the effects of Tamiflu and its carboxylic acid metabolite, GS4071, on the central nervous system, that is, on 3 neurotransmitters (dopamine, serotonin, and norepinephrine) in presynapses (inhibition of re-uptake, promotion of release) and postsynapses (guanosine 5′-triphosphate (GTP) γS binding), using rat brain synaptosomes. Neither Tamiflu nor GS4071 influenced the re-uptake/release of the 3 monoamines or GTP binding in postsynapses.

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