Evaluation of estrogenic/antiestrogenic activity of ellagic acid via the estrogen receptor subtypes ERalpha and ERbeta

Ellagic acid is a plant-derived polyphenol, possessing antioxidant, antiproliferative, and antiatherogenic properties. Whether this compound has estrogenic/antiestrogenic activity, however, remains largely unknown. To answer this question, we first investigated the ability of ellagic acid to influence the activity of the estrogen receptor subtypes ERalpha and ERbeta in HeLa cells. Cells co-transfected with an estrogen response element (ERE)-driven luciferase (Luc) reporter gene and an ERalpha- or ERbeta-expression vector were exposed to graded concentrations of ellagic acid. At low concentrations (10(-7) to 10(-9) M), this compound displayed a small but significant estrogenic activity via ERalpha, whereas it was a complete estrogen antagonist via ERbeta. Further evaluation revealed that ellagic acid was a potent antiestrogen in MCF-7 breast cancer-derived cells, increasing, like the pure estrogen antagonist ICI182780, IGFBP-3 levels. Moreover, ellagic acid induced nodule mineralization in an osteoblastic cell line (KS483), an effect that was abolished by the estrogen antagonist. Endometrium-derived epithelial cells (Ishikawa) showed no response to the natural compound by using a cell viability assay (MTT). These findings suggest that ellagic acid may be a natural selective estrogen receptor modulator (SERM).     

Procyanidins as antioxidants and tumor cell growth modulators

Five procyanidin fractions with different structural complexities were obtained after fractionation of a grape seed extract. The procyanidin fraction's abilities to inhibit lipid peroxidation induced by 2,2'-azobis-2-methyl-propanimidamide dihydrochloride in a liposomal membrane system were examined. The antioxidant capacities of all fractions were evaluated through monitoring oxygen consumption and by measuring the formation of conjugated dienes. All tested fractions provided protection of membranes against peroxyl radicals by increasing the induction time of oxidation. This effect increased up to fraction II but decreased with the increase of the structural complexity of further procyanidin fractions, possibly due to steric hindrance effects exhibited by the more complex fractions. In addition, the antiradical properties and the reducing power of these fractions were determined by using 2,2-diphenyl-1-picrylhydrazyl and ferric reducing/antioxidant power methods, respectively. Moreover, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide reduction and DNA synthesis were measured in Michigan Cancer Foundation 7 (MCF-7), a human breast cancer cell line, treated with catechin or procyanidin fractions in order to evaluate the effect of these compounds on cell viability and proliferation. The results obtained showed that at 30 microg/mL, fractions I and II decreased cell viability and proliferation, which was not observed with 60 microg/mL of the same fractions. Catechin was also able to decrease cell viability and proliferation at 30 and 60 microg/mL. It is interesting to notice that the procyanidin fractions that exhibited higher antioxidant activity were the same to affect cell viability and proliferation.     

猪Resistin基因真核表达载体的构建及在哺乳动物细胞中的表达

应用PCR技术从含有猪resistin基因cDNA的质粒RSTN-T中扩增目的基因片段，将其克隆至pcDNA3.1(+)表达载体中，经酶切及测序鉴定后，采用LipofectaminTM 2000 转染Hela细胞，用G418筛选稳定表达的Hela细胞株。以RT-PCR、Tricine-SDS-PAGE、Western blot分析其表达情况。测序结果表明成功构建了真核表达载体pcDNA3.1－RSTN，经G418筛选获得了Hela细胞克隆。用RT-PCR、Tricine-SDS-PAGE、western blot技术检测到了RSTN在Hela细胞中的转录与表达。重组质粒pcDNA3.1－RSTN的成功构建和表达为进一步研究猪resistin 的DNA疫苗奠定了基础。     

Use of caseinophosphopeptides as natural antioxidants in oil-in-water emulsions

Chelators are valuable ingredients used to improve the oxidative stability of food emulsions. Caseins and casein peptides have phosphoseryl residues capable of binding transition metals. Thus, the ability of enriched caseinophosphopeptides to inhibit lipid oxidation in corn oil-in-water emulsions was investigated. Enriched caseinophosphopeptides (25 microM) inhibited the formation of lipid oxidation at both pH 3.0 and 7.0 as determined by lipid hydroperoxides and hexanal. Calcium (0-100 mM) had no influence on the antioxidant activity of the enriched caseinophosphopeptides. Casein hydrolysates were more effective inhibitors of lipid oxidation than the enriched caseinophosphopeptides at equal phosphorus content. Thus, antioxidant properties might not be uniquely attributed to chelating metals by phosphoseryl residues but also by scavenging free radicals. Overall, the observed antioxidant activity of casein hydrolysates means they could be utilized to decrease oxidative rancidity in foods.     

红色荧光标记载体pBacA4DsRed1的构建及其表达

以首次克隆的家蚕Bombyx mori 肌动蛋白（actin4, A4）启动子、红色荧光蛋白基因DsRed1和多聚腺苷酸识别序列SV40为元件，经多次克隆将其插入到 piggyBac转座载体中，构建成pBacA4DsRed1转基因表达载体。将该载体显微注射到胚盘形成前期的蚕卵中，在荧光显微镜下观察其发光情况，结果发现：在胚胎早期发育的第三天，检测到蚕卵内发出较强的红色荧光。由于蚕卵没有红色自发荧光，用DsRed1标记背景干扰小，对注射蚕种无特别选择，在荧光显微镜下观察到明显的红色荧光，表明pBacA4DsRed1载体构建正确且能在蚕卵中表达。红色荧光蛋白(RFP)基因丰富了报告基因的种类，并且与绿色荧光蛋白(GFP)基因一样，可以作为理想的报告基因应用于家蚕的功能基因研究。     

Lipid oxidation in emulsions as affected by charge status of antioxidants and emulsion droplets

The influence of charge status of both lipid emulsion droplets and phenolic antioxidants on lipid oxidation rates was evaluated using anionic sodium dodecyl sulfate (SDS) and nonionic polyoxyethylene 10 lauryl ether (Brij)-stabilized emulsion droplets and the structurally similar phenolic antioxidants gallamide, methyl gallate, and gallic acid. In nonionic, Brij-stabilized salmon oil emulsions at pH 7.0, gallyol derivatives (5 and 500 microM) inhibited lipid oxidation with methyl gallate > gallamide > gallic acid. In the Brij-stabilized salmon oil emulsions at pH 3.0, low concentrations of the galloyl derivatives were prooxidative or ineffective while high concentrations were antioxidative. In SDS-stabilized salmon oil emulsions, oxidation rates were faster and the galloyl derivatives were less effective compared to the Brij-stabilized emulsions. Differences in antioxidant activity were related to differences in the ability of the galloyl derivatives to partition into emulsion droplets and to increase the prooxidant activity of iron at low pH.     

Cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside as primary phenolic antioxidants in black raspberry

Anthocyanin constituents in black raspberries (Rubus occidentalis L.) were investigated by HPLC-DAD, and their involvement as potent, significant antioxidants in black raspberries was demonstrated by three common antioxidant assays (FRAP, DPPH, ABTS) in this study. Five anthocyanins were present in black raspberries: cyanidin 3-sambubioside, cyanidin 3-glucoside, cyanidin 3-xylosylrutinoside, cyanidin 3-rutinoside, and pelargonidin 3-rutinoside. Their identities and structures, with particular emphasis on cyanidin 3-xylosylrutinoside, were confirmed by NMR spectroscopy. Two of these anthocyanins, cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside, predominated, comprising 24-40 and 49-58%, respectively, of the total anthocyanins in black raspberries. On the basis of both potency and concentration, cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside were found to be the significant contributors to the antioxidant systems of black raspberries. These findings indicate that these two anthocyanin compounds may function as the primary phenolic antioxidants in black raspberries. These two compounds exhibit potential biological activities that may be exploited in conjunction with other naturally occurring bioactive compounds in black raspberry fruit-based products used in clinical trials for the treatment of various types of cancer.     

Tetrahydro-beta-carboline alkaloids occur in fruits and fruit juices. Activity as antioxidants and radical scavengers

Tetrahydro-beta-carbolines are biologically active alkaloids that occur and accumulate in mammalian tissues, fluids, and brain, but their ultimate origin or biological role is still uncertain. Four tetrahydro-beta-carboline alkaloids: 1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid, 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid, 1-methyl-1,2,3,4-tetrahydro-beta-carboline, and 6-hydroxy-1-methyl-1,2,3,4-tetrahydro-beta-carboline, are found as naturally occurring substances in some fruit and fruit juices. These compounds occur in the microg/g level in those products, and a characteristic and distinct profile appears to exist depending on the type of fruit and juice involved. Thus, 1-methyl-1,2,3,4-tetrahydro-beta-carboline may appear in tomato, tomato juice, and kiwi; 6-hydroxy-1-methyl-1,2,3,4-tetrahydro-beta-carboline in bananas, pineapple, tomato, and their corresponding juices; and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid in oranges and grapefruits, although it also occurred in most juices. Fruit-occurring tetrahydro-beta-carboline alkaloids acted as antioxidants and free radical scavengers in the ABTS assay when compared with ascorbic acid and Trolox. This suggests that tetrahydro-beta-carboline alkaloids might act as antioxidants when absorbed and accumulated in the body, contributing to the antioxidant effect of fruit products containing these compounds.     

Effect of apple cell walls and their extracts on the activity of dietary antioxidants

The effect of dietary fiber in the form of apple cell walls and pectin extracts on natural antioxidants was examined. Cell walls (CW), isolated from apples ( Malus domestica Borkh. cv. "Pacific Rose"), were incubated with ascorbic acid (AA) or quercetin in N-2-hydroxyethylpiperazine- N'-2-ethanesulfonic acid (HEPES) buffer (pH 6.5) at 37 degrees C for 2 h. The resulting supernatants were characterized by a ferric reducing antioxidant power (FRAP) assay and cyclic voltammetry (CV). The experiments were repeated with pectin isolated from the apple cell walls and commercial pectins and showed that polysaccharide preparations stabilized AA effectively but offered little protection against quercetin oxidation. The water-soluble components from cell walls appeared to be responsible for the observed effects of cell-wall polysaccharide preparations on antioxidant activity.     

Comparison of quercetin and a non-orthohydroxy flavonol as antioxidants by competing in vitro oxidation reactions.

Two structurally related flavonols, quercetin and morin, along with protocatechuic acid (PA), beta-resorcylic acid (DHBA), and phloroglucinol carboxylic acid (PCA), which represent quercetin and morin degradation products, were assessed with respect to their antioxidant potency by chemical comparisons in competing oxidation reactions. The measurement of the antioxidant capacity was performed with the beta-carotene bleaching method, and the compounds were also tested with respect to their abilities to prevent lipid, protein, and DNA oxidation. The effect of concentration was also considered. The results obtained strongly suggested that quercetin is a powerful antioxidant in every system used, whereas morin is a much weaker antioxidant and in some cases may also have pro-oxidant action. PA and PCA were always inferior antioxidants compared to the parent molecule quercetin; DHBA and PCA exhibited activities comparable to that of morin in reaction comparisons.     

Comparative effects of phytosterol oxides and cholesterol oxides in cultured macrophage-derived cell lines

The cytotoxicity of cholesterol and a mixture of beta-sitosterol/campesterol (50%/40%) and their oxides was examined in a cultured-derived macrophage cell line, C57BL/6. Cell numbers, lactate dehydrogenase (LDH) leakage, protein content, lipid uptake, and mitochondria dehydrogenase activity were determined after exposure of cell mononlayers to sterols and sterol oxides at a concentration of 200 microg/mL for up to 120 h. Results indicate that the oxides of cholesterol, beta-sitosterol, and campesterol exhibited similar patterns of toxicity as indicated by LDH leakage, cell viability, and mitochondria dehydrogenase activity. Greatest cell damage was associated with treatments containing 5 alpha,6 alpha-epoxide or cholesterol oxides, followed by beta-sitosterol/campesterol oxides, cholesterol, and beta-sitosterol. The oxides of beta-sitosterol/campesterol caused less LDH leakage and less of an effect on protein content. Results of this study demonstrate that phytosterols contained in vegetable oils, when subjected to frying conditions, do oxidize and may cause cellular damage in an in vitro cell line similar to cholesterol oxides, although less severe.     

Furan formation from lipids in starch-based model systems, as influenced by interactions with antioxidants and proteins

The formation of furan upon sterilization of a lipid-containing starch gel was investigated in the presence of various antioxidants, namely, α-tocopherol, β-carotene, and ascorbic acid, with and without proteins. Results indicated that α-tocopherol did not significantly influence furan formation from oxidized lipids. β-Carotene, suggested previously to be a furan precursor itself, did influence the generation of furan in a concentration-dependent manner, although to a limited extent. Surprisingly, the presence of lipids seemed to limit the furan generation from β-carotene. Interestingly, the addition of ascorbic acid to the emulsions containing soybean or sunflower oils considerably enhanced the formation of furan from these oils. This was also the case when fresh oils were applied, shown previously to be nearly unable to generate furan. This observation can be explained by an intensified ascorbic acid degradation stimulated by the presence of lipids.     

Antiproliferative lactams and spiroenone from adlay bran in human breast cancer cell lines

Two new lactams, coixspirolactam D (1) and coixspirolactam E (2), and a new spiroenone, coixspiroenone (3), together with seven known compounds, coixspirolactam A (4), coixspirolactam B (5), coixspirolactam C (6), coixlactam (7), coixol (8), ethyl dioxindole-3-acetate (9), and isoindol-1-one (10), and two neolignans, zhepiresionol (11) and ficusal (12), were isolated from the bioactive subfraction of adlay bran ethanolic extract (ABE). Compounds 9 and 10 are the first isolates from natural resources. The structures of new compounds were identified by spectroscopic methods, including infrared (IR) spectrum, 1D and 2D nuclear magnetic resonance (NMR), and mass spectrum (MS). All of the isolated compounds were tested for antiproliferative effects on MCF-7, MDA-MB-231, and T-47D cells. Results showed that compounds 1, 3, 4, 6, and 7 at 50 μM significantly inhibited MCF-7 cell proliferation by 30.2, 19.2, 21.0, 13.5, and 32.4%, respectively; compounds 2, 4, and 7 significantly inhibited T-47D cells at 50 μM by 20.7, 24.8, and 28.9%; and compounds 1, 2, and 12 significantly inhibited MDA-MB-231 cells at 50 μM by 47.4, 25.3, and 69.3%, respectively. In conclusion, ABE has antiproliferative activities, and this effect is partially related to the presence of lactams and spiroenone.     

Competition between Rhizobium strain NGR234 and Bradyrhizobium strain CP283 for nodulation in Siratro investigated with the GUS reporter gene

The use of the GUS reporter gene was found to be very suitable for studying the competitiveness of the Rhizobium strain NGR234 and Bradyrhizobium strain CP283 for nodulation in siratro. However, the expression from the transposon mTn5SSgusA20 declined in the nodules of old plants, particularly the nodules at 35 d after inoculation and onward. Siratro inoculated with both gusA-marked NGR234 and CP283 strains showed a similar nodulation and acetylene reduction activity (ARA) to those of their parental strains. No major changes in nodulation and symbiotic properties in these marked strains were observed. When the Rhizobium strain NGR234 and Bradyrhizobium strain CP283 were inoculated separately to siratro, both of them were found equally effective for nodulation in the plant. But when the Rhizobium strain NGR234, and gusAmarked Bradyrhizobium strain CP283 were co-inoculated to the plants in a ratio of 1:1, the strain NGR234 occupied 75% of the nodules, while, the strain CP283 occupied only 25%, irrespective of the growth stages and inoculum concentrations. Similar results were also observed in the plants in which the Bradyrhizobium strain CP283 and gusA-marked Rhizobium strain NGR234 had been co-inoculated. Thus, the Rhizobium strain NGR234 was more competitive for nodulation in siratro.     

转Bt基因玉米自交系及其杂交种的抗螟性鉴定

采用基因枪法将Bt基因(GFM CrylA)导入东北春玉米自交系铁7922的幼胚中,获得转基因植株。经过连续3年的田间接卵、室内饲喂玉米螟鉴定,获得稳定抗虫转基因系。以此为亲本与常规自交系配制了四密25Bt、通单24Bt、吉单209Bt3个杂交种。转基因自交系和杂交种的抗螟性鉴定结果表明：转基因系间抗虫性差异极显著,同一转基因系单株间和配制的3个杂交种间抗虫性也存在差异;与对照相比,转基因系配制的杂交种抗虫性明显提高,差异显著。农艺性状鉴定结果显示,转基因系配制的杂交种与对照在株高、穗长、穗行数、行粒数、百粒重上无显著差异。本研究认为,GFM CrylA(Bt)基因可用于玉米的抗螟性改良,也为转Bt基因玉米自交系能够直接用于育种提供了依据。     

Genetic diversity in European accessions of the Barley Core Collection as detected by isozyme electrophoresis

Genetic diversity in 79 European accessions of the Barley Core Collections was surveyed using isozyme electrophoresis. A total of 26 alleles were observed at the ten isozyme loci. All loci were polymorphic except Pgd-1 which was monomorphic. The comparison of the results with those of previous studies indicates that most of the alleles occurring in the European Barley are also observed in this set of the European Barley Core Collections. Only five alleles (Est-1 Al; Est-5 Ag, Te; Pgi-1 C and Ndh-2 B) were absent. Nine of 26 alleles were rare alleles, which were detected only in one or two accessions. Moreover, most of rare alleles were detected in 6-rowed winter barley. It is very important to include rare alleles for maximising the genetic variations in core collections. In the set of European Barley Core Collection, 6-rowed barley contained larger diversity than 2-rowed barley; winter type contained larger diversity than spring type. The cluster analysis separated 79 accessions into three major groups. Group I is more complex and comprised 2-rowed spring, 2-rowed winter and 6-rowed winter barley. In this group, 18 accessions in the cluster A and 14 accessions in the cluster B possessed identical genotypes as judged from the ten isozyme data. Principal coordinate analysis could not clearly separate the spring cultivars from the winter barley lines, as well as not separate 2-rowed from 6-rowed barley.     

Quantification of carotenoid and tocopherol antioxidants in Zea mays.

Recent investigations into carotenoid and tocopherol biological activity in mammalian systems indicate that these antioxidants are associated with the prevention of degenerative diseases. Both carotenoids and tocopherols can be found in corn kernel tissue. A replicated survey of 44 sweet and dent corn lines was conducted to determine qualitative and quantitative variability of lutein, zeaxanthin, beta-cryptoxanthin, alpha-carotene, and beta-carotene, as well as the alpha-, delta-, and gamma- forms of tocopherol. The primary carotenoids in fresh market sweet corn were found to be lutein and zeaxanthin, with the gamma form dominating among the tocopherols. Mean values among the genotypes were observed to range from 0 to 20.0 and 2.4 to 63.3 microg/g dry weight for lutein and gamma-tocopherol, respectively, indicating variability among genotypes in genes regulating the metabolism of these compounds. The observed genetic variability suggests profound differences in potential health promotion among genotypes and supports the feasibility of developing germplasm with enhanced levels of these antioxidant compounds at dosages that could promote health among the consuming public.     

Lipid peroxidation by "free" iron ions and myoglobin as affected by dietary antioxidants in simulated gastric fluids

Grilled red turkey muscle (Doner Kabab) is a real "fast food" containing approximately 200 microM hydroperoxides, homogenized in simulated gastric fluid and oxidized more rapidly at pH 3.0 than at pH 5.0, after 180 min, producing 1200 and 600 microM hydroperoxides, respectively. The effects of "free" iron ions and metmyoglobin, two potential catalyzers of lipid peroxidation in muscle foods, were evaluated for linoleic acid peroxidation at pH 3.0 of simulated gastric fluid. The prooxidant effects of free iron ions on linoleic acid peroxidation in simulated gastric fluid was evaluated in the presence of ascorbic acid. At low concentrations of ascorbic acid, the effects were prooxidative, which was reversed at high concentrations. In the presence of metmyoglobin, ascorbic acid with or without free iron enhanced the antioxidative effect. Lipid peroxidation by an iron-ascorbic acid system was inhibited totally by 250-500 microM catechin at pH 3.0. The catechin antioxidant effect was determined also in the iron-ascorbic acid system containing metmyoglobin. In this system, catechin totally inhibited lipid peroxidation at a concentration 20-fold lower than without metmyoglobin. The ability of catechin to inhibit lipid peroxidation was also determined at a low pH with beta-carotene as a sensitive target molecule for oxidation. The results show that a significant protection was achieved only with almost 100-fold higher antioxidant concentration. Polyphenols from different groups were determined for the antioxidant activity at pH 3.0. The results show a high antioxidant activity of polyphenols with orthodihydroxylated groups at the B ring, unsaturation, and the presence of a 4-oxo group in the heterocyclic ring, as demonstrated by quercetin.     

Binding of phthalate plasticizers to human serum albumin in vitro: a multispectroscopic approach and molecular modeling

As endocrine-disrupting chemicals, a few frequently used phthalate plasticizers were banned or restricted for use as additives in food in some countries. The interaction mechanisms between three phthalate plasticizers with human serum albumin (HSA) were studied by fluorescence (quenching, synchronous, and three-dimensional), UV-vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy, in combination with molecular modeling under simulative physiological conditions, respectively. The results obtained from fluorescence quenching data revealed that the plasticizers-HSA interaction altered the conformational strcture of HSA. Meanwhile, the alterations of HSA secondary structure in the presence of phthalate plasticizers were investigated. The binding distances for the plasticizers-HSA system were provided by the efficiency of fluorescence resonance energy transfer. Furthermore, the thermodynamic analysis implied that hydrophobic forces were the main interaction for the plasticizers-HSA system, which agreed well with the results from the molecular modeling study.     

Root-shoot interaction as a limiting factor of biomass productivity in new tropical rice lines

The objective of the present study was to identify the limiting factors in biomass productivity of new tropical rice lines (panicle weight type) by analyzing the relationship between root and shoot activity. Five field experiments using three new lines, IR65598-112-2, IR65600-42-5-2, and IR68544-29-2-1-3-1-2, and one of the highest-yielding indica rice varieties (panicle number type), IR72, were conducted at the International Rice Research Institute from 1997 to 2002. Specific absorption rate of N (SARN , mg N g^-1 root dry weight d^-1), specific stem sap exudation rate (sap exudation rate, g exudate g^-1 root dry weight 12 h^-1) and specific root respiratory rate (root respiratory rate, μmol O₂ g^-1 root dry weight h^-1) were determined as indices of root activity. Relative growth rate (RGR, mg g^-1 d^-1) was used as an index of shoot activity. Compared with IR72, the new lines showed lower RGR and SAR_N values during the early growth stages. In contrast, during the late growth stages, these activities were higher in the new lines than in IR72. The SAR_B and sap exudation rate showed a linear correlation with RGR at successive growth stages, regardless of the genotypes and growth conditions. These findings indicated that active root-shoot interaction was maintained throughout the growth period in high-yielding tropical rice, including the new lines and IR72. Therefore, it was considered that SAR_N and the sap exudation rate are useful indices of root activity regulating RGR in the new lines. However, the root respiratory rate could not be used for selection, because the relationship between RGR and the root respiratory rate changed with the growth conditions. Our findings support the hypothesis that root and shoot activities during maturation are important factors affecting the productivity of the new lines, which have not yet been able to attain the targeted yield.