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Interleukin-1 beta (IL-1β) induces endothelial dysfunction and reduces nitric oxide (NO) production. IL-1β also enhances adhesion molecule expression and induces arteriosclerosis. Conversely, high-density lipoprotein (HDL) induces endothelial NO synthase (eNOS), paraoxonase-1 (PON-1) activity, and maintains vascular health. Diet-derived β-carotene prevents arteriosclerosis, but its mode of action is not understood. The purpose of this study was to examine the HDL-like mechanisms of β-carotene in endothelial cells. We added IL-1β and/or β-carotene to cultured human endothelial cells and examined its effects on the regulation of HDL signal transduction pathways using RT-PCR, real-time PCR, Western blot (WB), and endothelial-U937 adhesion analysis. IL-1β decreased the expression of Ca2+/calmodulin-dependent kinase II (CaMKII), eNOS, PON-1, phosphatidylinositol 3-kinase (PI3K), PSD-95/Dlg/ZO-1 (PZK1), and liver kinase B1 (LKB1). Conversely, it increased the expression of intercellular adhesion molecule-1 (ICAM-1), and monocyte chemoattractant protein 1 (MCP-1). In contrast, β-carotene increased the expression of CaMKKII, PI3K, PZK1, LKB1, eNOS, PON-1, and reduced the expression of ICAM-1 and MCP-1. β-carotene also induced phospho-AMP-activated protein kinase (p-AMPK), phospho-eNOS and PON-1 proteins. Importantly, β-carotene upregulated the IL-1β-mediated decrease of CaMKKII, PZK1, LKB1, eNOS and PON-1. β-carotene inhibited IL-1β-mediated cell adhesion of U937 to endothelial cells. The effect of β-carotene was reversed by a CaMKK inhibitor, STO-609. These findings indicate that β-carotene regulates the expression of PON-1, eNOS and adhesion molecules via CaMKK pathway activation. β-carotene may contribute to the functional maintenance of vascular endothelial cells in a manner similar to HDL, protecting them against stimuli such as IL-1β.

The early stages of vascular endothelial dysfunction enhance angiogenic stimulation and strongly influence vascular rearrangement. The aim of this study was to determine whether a short period of high glucose (HG, 30 mM glucose) plus tumor necrosis factor alpha (TNFα) treatment or reoxygenation after hypoxia (H/R) alters the expression levels of apelin in human endothelial cells. In addition, we also examined the effects of the dietary flavonoid apigenin on apelin expression. Human endothelial cell lines were treated with HG plus TNFα or subjected to H/R. The expression levels of genes and proteins were then assessed by the reverse transcriptase polymerase chain reaction, Western blotting and immunofluorescence analyses. The expression level of apelin was significantly higher in the HG group following exposure to reoxygenation or TNFα. Reoxygenation after hypoxia decreased the expression levels of apelin and fatty acid transport protein (FATP) 1 compared with those observed during hypoxia alone and normoxia in a normal glucose concentration. Inversely, apigenin augmented H/R-reduced apelin and FATP1 expression in endothelial cells. Based on our findings, we propose that the early stages of endothelial disorder subtly influence angiogenesis and that HG and H/R stimulate vascular rearrangement and are involved in fatty acid uptake. Furthermore, dietary apigenin might improve the expression of angiogenic genes and fatty acid uptake.

The antibiotic heliquinomycin inhibited cellular DNA replication at IC(50) of 2.5 µM without affecting level of chromatin-bound MCM4 and without activating the DNA replication stress checkpoint system, suggesting that heliquinomycin perturbs DNA replication mainly by inhibiting the activity of replicative DNA helicase that unwinds DNA duplex at replication forks. Among the DNA helicases involved in DNA replication, DNA helicase B was inhibited by heliquinomycin at IC(50) of 4.3 µM and RECQL4 helicase at IC(50) of 14 µM; these values are higher than that of MCM4/6/7 helicase (2.5 µM). These results suggest that heliquinomycin mainly targets actions of the replicative DNA helicases. Gel-retardation experiment indicates that heliquinomycin binds to single-stranded DNA. The single-stranded DNA-binding ability of MCM4/6/7 was affected in the presence of heliquinomycin. The data suggest that heliquinomycin inhibits the DNA helicase activity of MCM4/6/7 complex by stabilizing its interaction with single-stranded DNA.

我が国におけるゴマ葉の食習慣はほとんどないが，アジアやアフリカではゴマ葉は食用として，あるいは伝統薬・民間薬として利用されている．このゴマ葉はポリフェノールを豊富に含むものの，その成分研究はほとんど行われておらず，不明な点が多い．そこで，ゴマ若葉中の豊富なポリフェノールに着目し，抗酸化性と活性成分について検討した結果，以下の知見を得た．（1） 3つのラジカル消去測定（DPPHラジカル消去活性，ABTSラジカル消去活性，スーパーオキシドアニオンラジカル消去活性）により評価したところ，どの測定法においてもゴマ若葉は大麦若葉やケールよりも高い抗酸化性を示し，その活性は桑葉に次ぐものであった．この活性は含有ポリフェノール量に依存した．（2） ポリフェノール含量当たりで抗酸化性を評価したところ，どの測定法においてもゴマ若葉と桑葉の活性差は小さくなり，中でもDPPHラジカル消去活性ではゴマ若葉は桑葉よりも強い活性を示したことから，ゴマ若葉には強い抗酸化性を示すポリフェノール成分が含まれると考えられた．（3） DPPHポストカラム法を用いて，含有成分並びに活性関与成分を調べたところ，ゴマ若葉中には少なくとも9つのポリフェノール成分が含まれ，うち4つが主要な活性成分であると考えられた．（4） 各種スペクトル分析の結果，主要活性成分の一つはフェニルプロパノイド配糖体であるアクテオシドであり，ゴマ若葉乾燥粉末中に1.2％含まれることが判明した．アクテオシドは様々な生理機能を有し，種々の薬用植物に存在することが明らかにされているが，豊富に含む植物は希である．本研究により，ゴマ若葉にアクテオシドが豊富に含まれることが判明したことから，今後アクテオシドを指標としたゴマ若葉の有効利用が期待される．

Three iridoids (lamalbid (I1), sesamoside (I2) and shanzhiside methyl ester (I3)) and seven polyphenols (cistanoside F (P1), chlorogenic acid (P2), pedalitin-6-O-laminaribioside (P3), pedaliin (P4), isoacteoside (P6), pedalitin (P7) and martynoside (P8)) were identified in young sesame leaves (Sesamum indicum L.) other than the acteoside (P5) reported previously. P3 was a new compound, and I1, I3, P2 and P8 were found in a species of Sesamum for the first time. HPLC analyses revealed that the compounds I1 (0.29–1.75% of dry leaves), I2 (0.38–0.87%), I3 (0.04–1.07%), P4 (0.01–2.05%) and P5 (0.13–4.86%) were present primarily in young sesame leaves and were found in plants cultivated on different farms (plant height, 30–70 cm). Of the identified compounds, P5 and P6 showed high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, oxygen radical absorbance capacity (ORAC), and in vitro antiglycation activities. Given its content, P5 makes a major contribution to the biological activities of young sesame leaves. The compounds were examined at six different growth stages of plants cultured in a greenhouse to determine the optimum harvest stage and for end-use assessment. P5 accumulated in the leaves during growth, and the content reached a maximum of 12.9% of dry leaves in the 4th stage (plant height, 74.5±9.7 cm), which is one of the highest percentages reported in plants from nature.

Diabetes mellitus is associated with increased endothelial dysfunction and development of atherosclerotic vascular diseases. In contrast, an increased intake of dietary flavonoids is associated with a decreased risk of cardiovascular diseases. Here we demonstrate that high glucose (HG) and tumor necrosis factor alpha (TNFalpha) result in the expression of adhesion molecules and junctional molecules on endothelial cells (EC) within a short time. Simultaneously, we examined the regulatory effects of several dietary flavonoids. We demonstrated the short-term expression of adhesion molecules in a human EC line cultured with normal glucose (5.5 mM), HG (30 mM) and TNFalpha (10 ng/ml) by reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry and adhesion assay. The expression of intercellular adhesion molecule-1 (ICAM1) and vascular cell adhesion molecule-1 (VCAM1) increased, but that of occludin decreased. Apigenin strongly inhibited the expression of VCAM1, IkappaB kinase (IKK) alpha and IKKepsilon/IKKi, and suppressed the adhesion of U937 cells. From the structure and inhibitory activity of several dietary flavonoids, it was recognized that a double bond between apigenin and the third hydroxyl group was required for inhibition of gene expression. HG and TNFalpha induced the expression of cell adhesion molecules and reduced that of occludin in EC. These flavonoids modified the expression of cloudin 5 and occludin. These results demonstrated that apigenin inhibits HG- and TNFalpha-induced adhesion molecule expression and that flavonoids regulate the expression of junctional molecules in human EC. It is suggested that apigenin inhibited the expression of several genes through inhibition of IKKs.

We examined the possibility that the expression of adhesion molecules is regulated differently in cultured astrocytes from stroke-prone spontaneously hypertensive rats (SHRSP/IZM) rats than in those from Wistar Kyoto rats (WKY/IZM) by tumor necrosis factor-alpha (TNF-) or hypoxia and reoxygenation (H/R) and the inhibitory effects of apigenin. It was found that the expression of vascular cell adhesion molecule-1 (VCAM-1) by TNF- in astrocytes isolated from SHRSP/IZM was increased compared with that in WKY/IZM. The expression of monocyte chemotactic protein-1 (MCP-1) mRNA induced by H/R in SHRSP/IZM astrocytes was increased compared with that in normal oxygen concentrations. Apigenin strongly attenuated TNF--induced VCAM-1 mRNA and protein expression and suppressed the adhesion of U937 cells and SHRSP/IZM astrocytes. These results suggest that the expression levels of adhesion molecules during H/R affect disease outcome and can drive SHRSP/IZM to stroke. It is suggested that apigenin regulates adhesion molecule expression in reactive astrocytes during ischemia.

DPPHラジカル消去活性測定法を用いて，2成分間の活性に及ぼす効果（相乗効果，相加効果，相殺効果）について検討した．11種の酸化防止剤55通りの組み合わせでは，36通りにおいて統計上相乗効果，1通りで相殺効果と判定される結果が得られた．一方，24種の化合物276通りの組み合わせ（うち15通りは重複）では，74通りにおいて相乗効果，61通りで相殺効果が得られた．しかし，これらの多くの組み合わせによる効果は弱く，相加効果をわずかに上回る，あるいは下回る程度であり，2割以上の活性増強が認められた組み合わせは14通り，2割以下の活性低下が認められた組み合わせは33通りであった．一方，α-トコフェロールとの組み合わせのうち6通りで，p-クマル酸との組み合わせのうち4通りで2割以上の活性増強が観察され，バニリン酸との組み合わせのうち17通りで，p-クマル酸との組み合わせのうち12通りで2割以下の活性低下が観察された．

Effects of several nonthermal disinfection processes, including high pressure carbon dioxide treatment (HCT), gaseous chlorine dioxide treatment (GCT) and pulsed xenon treatment (PXT), on the inactivation of microorganisms and antioxidants in ten cut vegetables (red paprika, green pepper, cucumber, cabbage, lettuce, mizuna, celery, onion, carrot, and bean sprout) were investigated. HCT (6 or 10 MPa, 10 min, 35°C) showed similar inactivation effects on microorganisms using conventional NaOCl treatment (100 ppm, 10 min) by controlling pressure. However, HCT caused a loss of more antioxidants such as vitamin C and phenolic compounds in cut vegetables under severe conditions. In addition, HCT softened the leaf vegetables, cabbage, lettuce, and mizuna. All samples after PXT (500 J, 10 times) had the same antioxidant contents and DPPH radical scavenging activities as the untreated intact samples, suggesting PXT could be a disinfection method that does not influence the antioxidants in cut vegetables. For inactivation of microorganism, PXT decreased the viable cell count by about 10-fold, but not significantly. On the other hand, GCT (10 ppm, 30 min) showed almost the same microorganism inactivation as NaOCl treatment. The loss of vitamin C and phenolic compounds due to GCT were negligible. These results suggest that GCT might be a promising nonthermal disinfection treatment for cut vegetables.

Adenosine, which accumulates rapidly during ischemia due to the breakdown of ATP, has beneficial effects in many tissues. We examined whether adenosine induces the production of glial cell line-derived neurotrophic factor (GDNF) in cultured astrocytes. We evaluated GDNF mRNA expression and GDNF production in astrocytes cultured with adenosine and the adenosine selective receptor agonists 5-(N-ethylcarboxamido) adenosine (NECA), N(6)-cyclopentyladenosine (CPA) and 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamindo-adenosine hydrochloride (CGS 21680). Moreover, we examined the possibility that the expression of GDNF is regulated differently in cultured astrocytes from the stroke-prone spontaneously hypertensive rat (SHRSP) than in those from Wistar Kyoto rats (WKY). In this study, we confirmed that adenosine and the selective A(2B) adenosine receptor agonist NECA induced the expression of GDNF in cultured astrocytes. The A(2B) receptor antagonist alloxazine was able to inhibit the increase in extracellular GDNF produced by adenosine. Furthermore, the amounts of GDNF produced were significantly reduced in astrocytes of the adenosine-treated SHRSP compared with those of WKY. These results indicate that adenosine induces the expression of GDNF, and adenosine A(2B) receptors participate in the regulation of GDNF levels in astrocytes. This expression was attenuated in astrocytes of SHRSP compared with those of WKY.

We examined the detection of recombinant DNA of genetically modified (GM) soybeans in heat-treated GM soybeans and commercial natto. Genomic DNA was extracted from heat-treated GM soybeans and natto using the cetyltrimethylammonium bromide (CTAB) or alkaline lysis methods. First, primer pairs amplifying the junction region between CTP and CP4EPSPS in recombinant soybean were designed; they gave PCR products of band sizes, 100, 110, 120, 130, 140, and 150 bp. When DNA solution extracted from heat-treated GM soybeans by the alkaline lysis method was applied to polymerase chain reaction (PCR), PCR products of the expected 100, 110, 120, 130, 140, and 150 bp were detected by agarose gel electrophoresis. However, PCR products were not detected in DNA extracted from heat-treated GM soybeans by the CTAB method, and no PCR products were detected in either extract from natto. Next, PCR using primer pairs amplifying the junction region between NOS and part of CP4EPSPS were designed; they gave PCR products of 100, 110, 120, 130, 140, and 150 bp. The expected PCR products from all DNA extracts were detected (heat-treated GM soybeans by CTAB, 100–140 bp; heat-treated GM soybeans by alkaline lysis, 100–150 bp; natto by CTAB, 100–130 bp; and natto by alkaline lysis, 100–150 bp). These results indicate that judicious selection of DNA extraction methods and target sequences is important to detect DNA from natto and recombinant DNA can be detected in natto.

Summary Five different coloured sweet peppers (Capsicum annuum cv. Signal), white, green, yellow, orange and red were analysed for total carotenoids, α-tocopherol, sugars (glucose, fructose and sucrose), organic acids (citric and ascorbic acids) and anti-oxidant properties. The mature fruits, ‘Signal Red’, ‘Signal Orange’ and ‘Signal Yellow’ contained higher carotenoids, α-tocopherol, sugars and organic acids than the immature fruits, ‘Signal Green’ and ‘Signal White’. Among the mature fruits, ‘Signal Red’ was the highest in total carotenoids [9.15 mg (100 g)−1 of fresh weight] while ‘Signal Orange’ was the highest in α-tocopherol [5.40 mg (100 g)−1 of fresh weight]. ‘Signal Red’ and ‘Signal Orange’ contained the most sugars and organic acids. The suppression of 2,2′-azobis (2,4-dimethylvaleronitrile) (AMVN)-induced oxidation of methyl linoleate by the acetone extracts from the coloured sweet peppers resulted as follows: ‘Signal Red’ > ‘Signal Orange’ ≈ ‘Signal Yellow’ > ‘Signal Green’ ≈ ‘Signal White’. The order of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities of the acetone extracts was similar to that of suppression of methyl linoleate oxidation.

The stability of red radish extract to light, heat, and hydrogen peroxide at different pH values (3, 5, and 7) was examined, in which major anthocyanins were pelargonidin glycosides acylated with a combination of p-coumaric, ferulic, or caffeic acids. The light irradiation (fluorescence light, 5000 lx; at 25 degrees C) indicated that the red radish extract was more stable at lower pH than at higher pH. The HPLC analyses revealed that diacylated anthocyanins in the extract were more stable to light at pH 3 than monoacylated anthocyanins. No significant difference in degradation rates of acylated anthocyanins at pH 5 was observed, whereas anthocyanins acylated with p-coumaric or ferulic acids were more stable at pH 7 than ones with caffeic acids. The stability to heat (at 90-95 degrees C) showed a tendency similar to that for light. The number of intramolecular acyl units contributes to stability to light and heat at lower pH, whereas the characteristics of intramolecular acyl units influence the stability at higher pH. The degradation behavior of red radish extract to H2O2 were almost the same to those of light and heat, depending on the pH. However, HPLC analyses revealed that the stability of individual acylated anthocyanins were independent of the pH. These data suggest that the characteristics, the number, and the binding site of intramolecular acyl units affect the stability of anthocyanin to H2O2. DPPH radical scavenging activity of all acylated anthocyanins was higher than those of pelargonidin and perlargonidin-3-glucoside. The activity of acylated anthocyanins mostly depended on the activity of intramolecular acyl units (caffeic acidferulic acidp-coumaric acid). However, the activity was highly affected by the binding site of intramolecular acyl units even if anthocyanins have common acyl units.

We examined rapid and simple genomic DNA extraction method to detect material soybeans from natto (fermented soybeans) and soy sauce. Genomic DNA was extracted from natto and soy sauce by CTAB or alkaline lysis methods. Genomic DNA solutions from natto by both extraction methods were of adequate purity and yield for applying PCR, resulting that amplifications of the expected 100 bp fragment using the soybean specific primer pair were detected. When genomic DNA was extracted from soy sauce, the DNA solutions by alkaline lysis method were of a sufficient purity ( A 260 / A 280 = 1.5–2.0), but those by CTAB method were insufficient ( A 260 / A 280 = 1.2–1.7). In addition, the amplification products from the genomic DNA solutions by CTAB method were not detected. Thus, these results suggested that the desirable genomic DNA was not extracted from soy sauce by CTAB method, although it was possible to extract it by alkaline lysis method. Furthermore, when genomic DNA was extracted from natto and soy sauce using FTA ® card, which can preserve nucleic acids dried on the card just by applying a sample to it, the amplification was detected. It is found that an endogenous gene ( lectin 1 gene) of material soybeans can be extracted by rapid and simple methods, and detected from fermented foods as natto and soy sauce.

Six products were formed by reaction of ethynylestradiol (EE2) with sodium hypochlorite in buffered solutions. 4-Chloroethynylestradiol (4-ClEE2) and 2,4-dichloroethynylestradiol (2,4-diClEE2) were identified as the two major reaction products, using preparative HPLC, MS, and NMR. When EE2 reacted with chlorine at different pHs (pH 5, 7, and 9) or chlorine concentrations (0.2, 1, 2, and 5 mmol/l, corresponding to molar ratios to EE2, 1, 5, 10, and 25, respectively), the formation of 4-ClEE2 and 2,4-diClEE2 was observed under the above conditions, and the highest yields were 20 and 52 mol%, respectively. EE2 was consumed almost completely within 5 min of chlorination by addition of chlorine of more than 1 mmol/l (molar ratio to EE2, 5). On the other hand, the two products existed in highly chlorinated solutions after 60 min (4ClEE2, 1-6 mol%; 2,4-diClEE2, 3-25 mol%). The estrogenic activities of 4-ClEE2 by estrogen receptor alpha or beta binding assay were similar to those of the parent EE2, and the activities of 2,4-diClEE2 were lower about 10 times.

We examined the antioxidant effects of paprika pigments on oxidation of linoleic acid and on decoloration of the sample when stored at 37 degrees C in the dark or exposed to fluorescent light for 8 h per day. (1)H nuclear magnetic resonance with dioxane as an external proton reference was used to estimate the oxidative deterioration of linoleic acid. Oxidation was estimated by observing the ratio of the divinylmethylene proton signal area in linoleic acid vs the proton signal area in dioxane. The addition of paprika pigments suppressed the oxidation of linoleic acid during storage in the dark, and the effect was markedly increased with increasing concentrations (0.02, 0.2, and 2%). When the linoleic acid with added paprika pigments was exposed to light, only a slight suppression of oxidation was observed, and the color of the sample disappeared more rapidly than that in the dark. At the time of decoloration of the sample with added pigments, considerable oxidation of linoleic acid occurred. As the color change is due to degradation of the pigment, an increase in oxidation at the time of discoloration is consistent with the pigments functioning as antioxidants. The addition of alpha-tocopherol to paprika pigments stabilized degradation of the pigments by light. Although the addition of alpha-tocopherol to linoleic acid with added paprika pigments prolonged the decoloration of the sample under light, the prevention of oxidation under the light condition was not as effective as for the samples stored in the dark.

pH及び遊離塩素量を変化させ, ビスフェノール (BPA) と塩素とを反応させた。pH9.0ではpH5.0及び7.0に比べ速やかにBPAを消費し, 多くの塩素化BPA (ClBPA) を生成した。特に3, 3', 5, 5'-tetraClBPAの生成が顕著であった。一方, 遊離塩素濃度を増大させたところ, クロロフェノール類の生成増加が見られた。4つのClBPA類はBPAと同様にエストロゲンレセプターαに対する結合活性を示し, 3-ClBPA及び3, 3'-diClBPAの結合活性はBPAより強いものの, 3, 3', 5-triClBPAはBPAと同レベル, 3, 3', 5, 5'-tetraClBPA.はBPAより弱かった。一方, 3, 3', 5, 5'-tetraClBPAはAmes試験により変異原性を有することが明らかとなった。

Three peptide toxins (gigantoxins I–III) with crab toxicity were isolated from the sea anemone Stichodactyla gigantea by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T and their complete amino acid sequences were determined. Gigantoxins II (44 residues) and III (48 residues) have LD 50 (against crabs) of 70 and 120 μg/kg, respectively, and are analogous to the known type 1 and 2 sea anemone sodium channel toxins, respectively. On the other hand, gigantoxin I (48 residues) is potently paralytic to crabs (ED 50 215 μg/kg), although its lethality is very weak (LD 50 >1000 μg/kg). Interestingly, gigantoxin I has 31–33% homologies with mammalian epidermal growth factors (EGFs), with the same location of six cysteine residues. In accordance with the sequence similarity, gigantoxin I exhibits EGF activity as evidenced by rounding of A431 cells and tyrosine phosphorylation of the EGF receptor in the cells, although much less potently than human EGF. Gigantoxin I is the first example of EGF-like toxins of natural origin.

The relationship between the cardiac glycoside contents in Corchorus olitorius seeds and the seed color was examined. The seed color was assigned a shade (color value) (L value in UCS system). The dark grayish green seeds, showing lower L value, contained more cardiac glycosides than dark grayish yellow seeds showing higher L value. When the total cardiac glycoside contents were plotted against the L values, a positive correlation (r = ‐0.913) was observed. Also, there was a higher content ratio of strophanthidin glycosides (erysimoside and olitoriside) in the seeds showing lower L value, while there was a lower content ratio of digitoxigenin glycosides (coroloside and glucoevatromonoside) in the seeds showing lower L value.

A magenta subsidiary colour was isolated from commercial Food Blue No. 1 (B-1; Brilliant Blue FCF). The absorption maximum for this subsidiary colour at 580 nm is outside of the range of 614-628 nm found for other subsidiary colours and m,m-B-1. On the basis of MS and NMR analyses, the structure of the subsidiary colour was elucidated as the disodium salt of 2-[[4-[N-ethyl-N-(3-sulphophenylmethyl)amino]phenyl][4-oxo- 2,5-cyclohexadienylidene]methyl]benzenesulphonic acid. HPLC analyses revealed that 24 batches of commercial Food Blue No. 1 (three manufacturers) contain 0.1-0.8% (average: 0.5%) of the magenta subsidiary colour.

The antioxidant ability of capsanthin and the fatty acid esters was examined by measuring the free radical-oxidation of methyl linoleate. To assess radical scavenging effect, the production of meth...

Heliquinomycin was isolated as a part of a program designed to find inhibitors of DNA helicase from microbial sources. It was purified from the culture broth of Streptomyces sp. MJ929-SF2 by solvent extraction and serial chromatographies of centrifugal partition chromatography, Sephadex LH-20 and Capcell Pak C18 (HPLC). The isolated red powder was analyzed to have the molecular formula of C33H30O17. It inhibited partially purified DNA helicase from HeLa cell in a non-competitive manner with the inhibition constant (Ki) of 6.8 mM. Heliquinomycin exhibited biological activity against microorganisms including MRSA, and cultured cell lines.

Background The research emphasis in anti-cancer drug discovery has always been to search for a drug with the greatest antitumor potential but fewest side effects. This can only be achieved if the drug used is against a specific target located in the tumor cells. In this study, we evaluated Minichromosome Maintenance Protein 7 (MCM7) as a novel therapeutic target in cancer. Results Immunohistochemical analysis showed that MCM7 was positively stained in 196 of 331 non-small cell lung cancer (NSCLC), 21 of 29 bladder tumor and 25 of 70 liver tumor cases whereas no significant staining was observed in various normal tissues. We also found an elevated expression of MCM7 to be associated with poor prognosis for patients with NSCLC (P = 0.0055). qRT-PCR revealed a higher expression of MCM7 in clinical bladder cancer tissues than in corresponding non-neoplastic tissues (P < 0.0001), and we confirmed that a wide range of cancers also overexpressed MCM7 by cDNA microarray analysis. Suppression of MCM7 using specific siRNAs inhibited incorporation of BrdU in lung and bladder cancer cells overexpressing MCM7, and suppressed the growth of those cells more efficiently than that of normal cell strains expressing lower levels of MCM7. Conclusions Since MCM7 expression was generally low in a number of normal tissues we examined, MCM7 has the characteristics of an ideal candidate for molecular targeted cancer therapy in various tumors and also as a good prognostic biomarker for NSCLC patients.

Although hyperglycemia can induce diabetic vascular disorders, the mechanisms responsible for the early stages of this process are unknown. To determine the factor(s) that initially stimulate hyperglycemia and the preventive effects of polyphenols, we examined the effects of high glucose (HG) conditions and several dietary polyphenols on human endothelial cells (EC). The purpose of the present study was to investigate the augmentation of the expression of angiotensin II type I receptor (AT1R), cyclooxygenase-2 (COX-2), lectin-like oxidized LDL receptor-1 (LOX-1), prostacyclin/prostaglandin I 2 synthase (PGIS), and thromboxane A2 synthase (TXA2S) by tumor necrosis factor-alpha (TNFα) in HG conditions (30 mM) in human EC over a short period, and we also investigated the regulatory effects of 10 dietary flavonoids. HG plus TNFα strongly induced LOX-1 and AT1R expression in the EC. Furthermore, apigenin, kaempferol, chrysin, and flavone significantly inhibited HG plus TNFα-induced LOX-1 expression. The inhibition of LOX-1 expression by apigenin was found to require a flavone skeleton, the double bond found in its C-ring, and the absence of a third hydroxyl group from its B- and C-rings. These findings suggest that TNFα and HG regulate diverse cellular processes and promote endothelial dysfunction via the expression of LOX-1 and AT1R. Conversely, the inhibitory action of apigenin may be beneficial for the treatment of diabetic endothelial dysfunction.

Heliquinomycin, a novel microbial product, was found to inhibit a human DNA helicase enzyme isolated from HeLa S3 cells at concentrations of 5 to 10 micrograms/ml. In contrast, adriamycin, etoposide and cisplatin did not inhibit this enzyme at the concentrations tested. Furthermore, the replication and repair of SV40 chromosome were not affected at heliquinomycin concentration of 50 micrograms/ml. The topoisomerase II and I enzymes were inhibited at 30 micrograms/ml and 100 micrograms/ml of heliquinomycin, respectively. Heliquinomycin inhibited the growth of HeLa S3, KB, LS180, K562 and HL60 human tumor cell lines at IC50 values of 0.96 to 2.8 micrograms/ml. In addition, the growth of adriamycin and cisplatin resistant P388 cell lines were inhibited at similar concentrations. Heliquinomycin inhibited both DNA and RNA synthesis in cell culture but did not inhibit protein synthesis. HeLa S3 cells were arrested at the G2/M phase by heliquinomycin. These studies suggest that heliquinomycin is a selective inhibitor of a cellular DNA helicase and in turn, inhibits growth of tumor cell lines.

The acute toxicity of arsenocholine was examined in mice by oral administration and intravenous injection. The LD50 values of arsenocholine were 6.5 g kg−1 for oral administration and 187 mg kg−1 for oral administration and 187 mg kg−1 for intravenous injection. Decreases of respiration and spontaneous motility were observed in the mice dosed orally at 12 g kg−1. The animals exhibited ataxia and finally showed paralysis of the hind legs within 20 min of administration. When arsenocholine was administered orally to mice at 5 or 50 mg As kg−1, the greater part of the arsenic administered was recovered in urine within 96 h. The metabolite of arsenocholine in urine was identified as arsenobetaine by high-performance liquid chromatography-inductively coupled plasma emission spectrometry (HPLC ICP) and fast atom bombardment mass spectrometry (FAB MS). These results suggested that the major part of orally administered arsenocholine was absorbed from the gastrointestinal tract in mice and then rapidly excreted in urine with biotransformation.

The antibiotic heliquinomycin, which inhibits cellular DNA replication at a half-maximal inhibitory concentration (IC(50)) of 1.4-4 microM, was found to inhibit the DNA helicase activity of the human minichromosome maintenance (MCM) 4/6/7 complex at an IC(50) value of 2.4 microM. In contrast, 14 microM heliquinomycin did not inhibit significantly either the DNA helicase activity of the SV40 T antigen and Werner protein or the oligonucleotide displacement activity of human replication protein A. At IC(50) values of 25 and 6.5 microM, heliquinomycin inhibited the RNA priming and DNA polymerization activities, respectively, of human DNA polymerase-alpha/primase. Thus, of the enzymes studied, the MCM4/6/7 complex was the most sensitive to heliquinomycin; this suggests that MCM helicase is one of the main targets of heliquinomycin in vivo. It was observed that heliquinomycin did not inhibit the ATPase activity of the MCM4/6/7 complex to a great extent in the absence of single-stranded DNA. In contrast, heliquinomycin at an IC(50) value of 5.2 microM inhibited the ATPase activity of the MCM4/6/7 complex in the presence of single-stranded DNA. This suggests that heliquinomycin interferes with the interaction of the MCM4/6/7 complex with single-stranded DNA.

Arsenic compounds in the red alga Porphyra yezoensis were purified by gel filtration on Bio-Gel P-2; chromatographic behavior suggested that they were arsenic-containing ribofuranosides (arsenosugars). When partially purified arsenosugars were orally administered to mice, 86% and 13% of the arsenic administered were excreted in feces and urine, respectively, within 48 h. Arsenic compounds excreted in feces were identified as arsenosugars by gel filtration and highperformance liquid chromatography, while those excreted in urine were identified as methylarsonic acid, dimethylarsinic acid and arsenobetaine. On the other hand, upon intravenous administration of arsenosugars, a large part of the arsenic administered was excreted rapidly in urine; after 72 h, 92% of the arsenic administered was recovered in urine and 6% in feces. Similarly to the case of oral administration, methylarsonic acid, dimethylarsinic acid and arsenobetaine were detected in urine.

Red radish anthocyanin extract, which consists of 12 known acylated anthocyanins, was reacted with 2,2‘-azobis(2-amidinopropane) dihydrochloride (AAPH) to generate peroxyl radicals under acidic pH conditions at 37 °C. The reaction products were isolated using preparative HPLC, and their chemical structures were determined to be p-hydroxybenzoic acid (1), 6-O-(E)-p-coumaroyl-2-O-β-d- glucopyranosyl-α-d-glucopyranoside (3), p-coumaric acid (4), 6-O-(E)-feruloyl-2-O-β-d-glucopyranosyl-α-d-glucopyranoside (5), and ferulic acid (6). Some products were not identified. HPLC analyses of the mixture of acylated pelargonidin isolated from red radish and AAPH revealed that the acylated pelargonidins possess the radical scavenging ability on some common sites even if the characteristics of the intramolecular acyl units are different. Degradation rates of acylated pelargonidins and the formation rates of the resulting reaction products were found to be quite different. Keywords: Acylated anthocyanin; pelargonidin; per...

Hokosshi extract is obtained by ethanol extraction from the seeds of hokosshi (Psoralea corylifolia), which is used as a Chinese medicine. The constituents of hokosshi extract were analyzed. The main constituent was isolated using column chromatography, and identified as bakuchiol by TLC, LC/MS and NMR. Bavachinin A was also detected. In order to prepare a marker substance for hokosshi extract, bakuchiol was isolated from seeds of hokosshi using Sep-Pak cartridges. An analytical method for hokosshi extract in foods based on detection of bakuchiol was developed. Bakuchiol was extracted from food with 60 vol% ethanol. The extract was cleaned up using a Sep-Pak plus C18 cartridge, and bakuchiol was determined by HPLC. Seasoning and juice were spiked with hokosshi extract at 500 micrograms/g and analyzed by the proposed method. The recoveries of bakuchiol were 72-99%. The detection limit for the assay was 25 micrograms/g.

HPLC analysis revealed that eight subsidiary colors existed in commercial Food Green No. 3 (fast green FCF, FD & C Green No. 3). Among them, four subsidiary colors C, F, G, and H were isolated by using preparative HPLC and their structures were determined by MS and NMR. They were the disodium salt of 2-[[4-[N-ethyl-N-(3- sulfophenylmethyl)amino]phenyl][4-[N-ethyl-N-(4- sulfophenylmethyl)amino]phenyl]methylio]-4-hydroxybenzenesulfonic acid (abbreviated as m,p-G-3), the sodium salt of 2-[[(4-N-ethylamino)phenyl][4-[N-ethyl-N-(3- sulfophenylmethyl)amino]-phenyl]methylio]-4-hydroxybenzenesulfonic acid [abbreviated as HSBA-(EA) (m-EBASA)], the sodium salt of 2-[[(4-N-diethylamino)phenyl][4-[N-ethyl-N-(3- sulfophenylmethyl)amino]phenyl]-methylio]-4-hydroxybenzenesulfonic acid [abbreviated as HSBA-(di-EA) (m-EBASA)], and the sodium salt of 2-[[4-[N-ethyl-N-(phenylmethyl)amino]phenyl][4-[N-ethyl-N-(3- sulfophenylmethyl)-amino]phenyl]methylio]-4-hydroxybenzenesulfonic acid [abbreviated as HSBA-(EBA)(m-EBASA)], respectively. HSBA-(di-EA) (m-EBASA) was a subsidiary color newly found in commercial Food Green No. 3.

Antibacterial antibiotics, diperamycin (1) was produced in the culture broth of Streptomyces griseoaurantiacus MK393-AF2. Various spectroscopic analyses of 1 suggested that 1 belonged to a member of cyclic hexadepsipeptide antibiotic. Antibiotic 1 had potent inhibitory activity against various Gram-positive bacteria including Enterococcus seriolicida and methicillin-resistant Staphylococcus aureus.

Heliquinomycin, a novel microbial product, was found to inhibit a human DNA helicase enzyme isolated from HeLa S3 cells at concentrations of 5 to 10 micrograms/ml. In contrast, adriamycin, etoposide and cisplatin did not inhibit this enzyme at the concentrations tested. Furthermore, the replication and repair of SV40 chromosome were not affected at heliquinomycin concentration of 50 micrograms/ml. The topoisomerase II and I enzymes were inhibited at 30 micrograms/ml and 100 micrograms/ml of heliquinomycin, respectively. Heliquinomycin inhibited the growth of HeLa S3, KB, LS180, K562 and HL60 human tumor cell lines at IC50 values of 0.96 to 2.8 micrograms/ml. In addition, the growth of adriamycin and cisplatin resistant P388 cell lines were inhibited at similar concentrations. Heliquinomycin inhibited both DNA and RNA synthesis in cell culture but did not inhibit protein synthesis. HeLa S3 cells were arrested at the G2/M phase by heliquinomycin. These studies suggest that heliquinomycin is a selective inhibitor of a cellular DNA helicase and in turn, inhibits growth of tumor cell lines.

Crude extracts from hypobranchial glands of two species of muricid gastropods, Thais clavigera and Thais bronni , were highly lethal to mice. Regardless of species, a new choline ester was isolated as the major toxic principle and elucidated to be tigloylcholine, a structural isomer of senecioylcholine widely found in gastropod hypobranchial glands, by 1 H- and 13 C-NMR as well as FAB-MS. The i.v. ld 50 (mouse) of tigloylcholine was estimated to be 0.92 mg/kg.

The structure of heliquinomycin which was isolated from the culture broth of Streptomyces sp. MJ929-SF2 was studied by NMR spectroscopies, X-ray crystallographic analysis and degradation experiments. Heliquinomycin is the first member of glycosylated rubromycins and griseorhodins group antibiotics.

HPLC analysis revealed that five subsidiary colors were present in a commercial Food Blue No. 1 (Brilliant Blue FCF) product. Among them, major subsidiary colors C, D, and E were isolated. On the bases of spectroscopic analyses, their structures were identified as the disodium salt of 2-[[4-[N-ethyl-N-(3-sulfophenylmethyl)amino]phenyl][4-[N-ethyl-N-(4-sulfophenylmethyl)amino]phenyl]methylio]benzenesulfonic acid, the disodium salt of 2-[[4-[N-ethyl-N-(2-sulfophenylmethyl)amino]phenyl][4-[N-ethyl-N-(3-sulfophenylmethyl)amino]-phenyl]methylio]benzenesulfonic acid, and the sodium salt of 2-[[4-(N-ethylamino)phenyl][4-[N-ethyl-N-(3-sulfophenylmethyl)amino]phenyl]methylio]benzenesulfonic acid, respectively.