玉兰肌动蛋白基因的克隆与序列分析

本研究选取玉兰（Magnolia denudata Desr.)为材料，提取总RNA，利用设计的肌动蛋白编码区两端的特异性引物和5'RACE试剂盒，扩增出肌动蛋白编码的cDNA基因。以豌豆卷须肌动蛋白基因作探针，通过Southern杂交进行检测，证明同源性很高。将所获得序列克隆到pGEM-T载体上进行测序，测序结果在GenBank中进行查询，并与其他高等植物肌动蛋白基因序列进行了分析。     

In vitro metabolism of plant lignans: new precursors of mammalian lignans enterolactone and enterodiol.

The metabolism of the plant lignans matairesinol, secoisolariciresinol, pinoresinol, syringaresinol, arctigenin, 7-hydroxymatairesinol, isolariciresinol, and lariciresinol by human fecal microflora was investigated to study their properties as mammalian lignan precursors. The quantitative analyses of lignan precursors and the mammalian lignans enterolactone and enterodiol were performed by HPLC with coulometric electrode array detector. The metabolic products, including mammalian lignans, were characterized as trimethylsilyl derivatives by gas chromatography-mass spectrometry. Matairesinol, secoisolariciresinol, lariciresinol, and pinoresinol were converted to mammalian lignans only. Several metabolites were isolated and tentatively identified as for syringaresinol and arctigenin in addition to the mammalian lignans. Metabolites of 7-hydroxymatairesinol were characterized as enterolactone and 7-hydroxyenterolactone by comparison with authentic reference compounds. A metabolic scheme describing the conversion of the most abundant new mammalian lignan precursors, pinoresinol and lariciresinol, is presented.     

Sesquiterpenoids isolated from the flower buds of Tussilago farfara L. inhibit diacylglycerol acyltransferase

Inhibition of acyl CoA:diacylglycerol acyltransferase (DGAT), which is a key enzyme in triglyceride synthesis in eukaryotic organisms, has been proposed as one of the drug targets for treating obesity, type II diabetes mellitus, and metabolic syndrome. Bioassay-guided fractionation of EtOH extract of the flower buds of Tussilago farfara , using an in vitro DGAT enzyme assay, resulted in the isolation of four known sesquiterpenoids, tussilagonone (1), tussilagone (2), 7beta-(3-ethyl-cis-crotonoyloxy)-1alpha-(2-methylbutyryloxy)-3,14-dehydro-Z-notonipetranone (3), and 8-angeloylxy-3,4-epoxy-bisabola-7(14),10-dien-2-one (4). DGAT1 inhibitory activity was studied by in vitro DGAT assay using rat liver microsomes and HepG2 cell microsomes. They showed DGAT1 inhibition with IC(50) values of 99.2 (1), 18.8 (2), 47.0 (3), and 211.1 (4) microM (for rat liver microsomes) and >1 mM (1), 49.1 (2), 160.7 (3), and 294.4 (4) microM (for HepG2 cell microsomes), respectively. Compound 2 showed the most potent inhibition against microsomal DGAT1 derived from rat liver and human hepatocellular carcinoma HepG2 cells and also significantly inhibited triglyceride synthesis by suppressing incorporation of [(14)C]acetate or [(14)C]glycerol into triglycerides in HepG2 cells. These findings suggest that tussilagone is a potential lead compound in the treatment of obesity and type 2 diabetes.     

Grain-size effect on the structure and antiobesity activity of konjac flour

The effect of high-frequency oscillatory type ball-mill treatment on the structure and antiobesity activity of konjac flour was investigated. The grain size of konjac flour changed from 657.3 microm (d(50)) to 23.7 microm (d(50)) after 4 h of treatment. The structural change of the konjac flour with different grain size was characterized by using X-ray diffraction (XRD) differential scanning calorimetry (DSC). The results indicated that the crystallinity decreased and the diffraction peak drifted not only by when the crystallization region was reduced but also when the crystalline structure was changed. With the decrease of the grain size and crystallinity, the konjac flour grain, especially the 4 h milled konjac flour, swelled more rapidly and led to the improvement of the antiobesity effect. Compared with the native konjac flour, the 4 h milled konjac flour could significantly decrease the body weight and total wet weight of fat of nutritional obese rats (P < 0.05) and also decreased the contents of triglyceride, glucose, and high-density lipoprotein in blood of nutritional obese rat significantly (P < 0.05), which meant the grain-size effect of konjac flour improved its antiobesity activity notably.     

New lignans from the perisperm of Sesamum indicum

A procedure, based on XAD-4 adsorption resin, which permits the obtainment of enriched polyphenolic extracts from Sesamum indicum perisperm (coat) has been developed. Chemical analysis of the obtained extracts led to the identification of 16 lignans. Among them, two new lignans, (+)-saminol and (+)-episesaminone-9-O-beta-D-sophoroside, have been isolated. Additionally, the relative stereochemistry of (-)-sesamolactol, previously reported as todolactol A epimer, has been unequivocally defined using X-ray crystallography. The structures of all new compounds were determined by spectroscopic methods, mainly by the concerted application of 1D and 2D NMR techniques (HMQC, HMBC, and NOESY) and mass spectroscopy.     

In vitro evaluation of flavonoids from Eriodictyon californicum for antagonist activity against the bitterness receptor hTAS2R31

The leaves of the native North American plant, Eriodictyon californicum, were once used to mask the bitter taste of pharmaceuticals, an application currently of importance. Ten flavonoids (1-10) were isolated from the leaves of E. californicum, of which the structure and absolute configuration of 6-methoxyhesperetin (8) were assigned for the first time. In addition, the absolute configurations at C-2 were established for 4'-isobutyrylhomoeriodictyol (3) and 6-methoxyhomoeriodictyol (7). Using a cell-based assay, it was determined that the 7-methoxylated flavanones, sakuranetin (2) and 6-methoxysakuranetin (9), and the flavone, jaceosidin (10), are antagonists of hTAS2R31.     

马立克氏病病毒MEQ蛋白对MDV增殖影响的研究

在马立克氏病病毒MDV致病机理的研究中，弄清致病基因meq与病毒增殖之间的关系及其分子机制是十分重要的基础，以重组反转录病毒（RCAS-meq)感染鸡胚成纤维细胞（CEF），使源于MDV强毒参考株（vMDV)GA株的meq基因表达于宿主细胞内，然后再用GA株感染这些细胞。通过利用MDV强毒株特异的抗pp38单克隆抗体所进行的“黑斑”试验以确定MDV的增殖水平，并与未接种RCAS-meq的CEF进行比较。研究结果发现，细胞内表达的meq基国产物可促进GA株于体外培养细胞中的感染与增殖（病毒斑数增多）。根据试验的结果，作者认为meq基因在感染细胞内的表达水平是MDV增殖以及进而能致病、致瘤的分子基础。     

In vitro inhibitory effect on pancreatic lipase activity of subfractions from ethanol extracts of fermented Oats (Avena sativa L.) and synergistic effect of three phenolic acids

The purpose of the present work is to study the pancreatic lipase inhibitory effects of different subfractions (n-hexane, ethyl acetate (EA), n-butanol, and water) from ethanol extracts of nonfermented and fungi-fermented oats and to delineate the interactions of three primary phenolic acids in the EA subfractions. The EA subfraction showed the highest inhibitory effect on pancreatic lipase activity at 1.5 mg/mL compared to the other subfractions, regardless of whether the oats were fermented. Meanwhile, both of the EA subfractions of two fungi-fermented oats demonstrated more effective inhibitory activity than that of nonfermented oats. A positive correlation between the total phenolics content and inhibitory activity was found. The inhibitory ability of the EA subfraction from nonfermented or fermented oats also displayed a dose-dependent effect. The standards of caffeic, ferulic, and p-coumaric acids, mainly included in EA subfractions of fermented oats, also displayed a dose-dependent inhibitory effect. A synergistic effect of each binary combination of p-coumaric, ferulic, and caffeic acids was observed, especially at 150.0 μg/mL. Those results indicate that fungi-fermented oats have a more effective inhibitory ability on pancreatic lipase and polyphenols may be the most effective component and could be potentially used for dietary therapy of obesity.     

Studies on the effect of Amadoriase from Aspergillus fumigatus on peptide and protein glycation in vitro

Amadoriase I is a fructosyl amine oxidase from Aspergillus fumigatus that catalyzes the oxidation of Amadori products (APs) producing glucosone, H2O2, and the corresponding free amine. All the enzymes of this family discovered so far only deglycate small molecular weight products and are inactive toward large molecular weight substrates, such as glycated BSA or ribonuclease A. Therefore, they cannot be used to reverse protein glycation occurring in diabetes or in foods. In this paper, the effect of Amadoriase I added during the in vitro reaction between glucose and peptides having different polarities or proteins with molecular weights ranging from to 5 to 66 kDa was tested. The formation of APs was monitored by ESI-MS of intact glycated protein or peptides and by measuring the Nepsilon-(1-deoxy-d-fructos-1-yl)-L-lysine and furosine concentrations. Results showed that the formation of APs is reduced up to 80% when peptides and glucose are incubated in the presence of Amadoriase. The effect is more evident for hydrophobic peptides. In protein-glucose systems, the effect was dependent on the molecular weight and steric hindrance being negligible for BSA and at a maximum for insulin, where the formation of APs was reduced up to 60%. These findings indicate new potential applications of Amadoriase I as an efficient tool for inhibiting protein glycation in real food systems.     

Studies on the metabolism of the plant lignans secoisolariciresinol and matairesinol

The plant lignans secoisolariciresinol and matairesinol occur in numerous foods such as oil seeds, whole grains, vegetables, and fruits. We have studied the hitherto unknown oxidative metabolism of secoisolariciresinol and matairesinol in hepatic microsomes from untreated and Aroclor 1254-induced Wistar rats and from humans. Five oxidative metabolites of secoisolariciresinol and 10 oxidative metabolites of matairesinol were detected in rat liver microsomes, and their chemical structures were elucidated. The pathways in the metabolism of both secoisolariciresinol and matairesinol included aliphatic and aromatic hydroxylation, whereas oxidative demethylation was only observed for matairesinol. Human hepatic microsomes were able to metabolize secoisolariciresinol whereas matairesinol was only poorly metabolized. This study clearly shows that secoisolariciresinol and matairesinol are substrates of cytochrome P450-mediated metabolism. However, from preliminary experiments with rats dosed orally with secoisolariciresinol and matairesinol, it appears that the intestinal absorption and subsequent oxidative metabolism of these plant lignans occur only to a very small extent due to the highly efficient conversion of secoisolariciresinol and matairesinol to the mammalian lignans enterodiol and enterolactone by the gut microflora.     

In vitro anti-inflammatory and anti-proliferative activity of sulfolipids from the red alga Porphyridium cruentum

A sulfoglycolipidic fraction (SF) isolated from the red microalga Porphyridium cruentum was analyzed for fatty acid composition and assayed for ability to inhibit, in vitro, the generation of superoxide anion in primed leucocytes and the proliferation of a panel of human cancer cell-lines. Results demonstrated that SF contained large amounts of palmitic acid (26.1%), arachidonic acid (C20: 4 omega-6, 36.8%), and eicopentaenoic (C20:5 omega-3, 16.6%) acids, and noticeable amounts of 16:1n-9 fatty acid (10.5%). It strongly inhibited both the production of superoxide anion generated by peritoneal leukocytes primed with phorbol myristate acetate (IC(50): 29.5 microg/mL), and the growth of human colon adenocarcinoma DLD-1 and to a lesser extent of human breast adenocarcinoma MCF-7, human prostate adenocarcinoma PC-3, and human malignant melanoma M4 Beu cell-lines, and therefore might have a chemopreventive or chemotherapeutic potential, or both. It was found markedly more cytotoxic than sulfoquinovosyldiacylglycerols from plant used as a standard (STD), due to a stronger ability to inhibit DNA alpha-polymerase (IC(50): 378 microg/mL, vs 1784 microg/mL for STD). After a 48-h continuous treatment, IC(50) values for growth inhibition were in the range of 20-46 microg/mL instead of 94 to >250 microg/mL for STD, and those for inhibition of metabolic activity were in the range of 34-87 microg/mL instead of >250 microg/mL for STD. The higher anti-proliferative effect was observed on colon adenocarcinoma DLD-1 cells, and the weaker effect was observed on breast adenocarcinoma MCF-7.     

影响绵羊体外胚胎生产的主要因素

体外胚胎生产能够扩大胚胎来源,降低胚胎生产成本,同时有利于种质资源的国际间交流和拯救濒危动物,因此受到广泛关注(桑润滋,2002)。但目前完全体外化生产胚胎还有很多尚未解决的问题,如囊胚率较低、质量差。本研究着重于系统地探讨体外受精中的一些影响因素,以期为改善绵羊体外胚胎生产技术提供有用的参考依据。     

樱桃离体叶片高效再生的影响因素

利用基因工程技术将外源的抗病基因导入受体植物,使受体获得抗病性,已成为抗病育种的一个新途径。叶片是基因工程操作中常用的受体材料,本实验以几个樱桃树种、品种为试材,系统研究了影响樱桃叶片再生的几个因素,旨在为基因转化提供一个高效、稳定的植株再生体系。1材料和方法1     

In vitro and in vivo release of aroma compounds from yellow-fleshed kiwifruit

Comparisons were made between the aroma volatiles of the yellow-fleshed kiwifruit, "Hort16A", at two different stages of eating ripeness: firm and soft. The firm fruit contained a small number of aroma compounds that the soft fruit did not contain. In general, however, the largest difference between the two firmness categories was in the levels of esters, with the soft fruit containing higher concentrations and a larger number of esters than the firm fruit. In vitro analysis directly after maceration using atmospheric pressure chemical ionization mass spectrometry (APCI-MS) showed the relative importance of the most intense aromas between fruit at the two different firmness stages and was used to compare the release rates of aromas. A comparison of the aroma concentrations from gas chromatography mass spectrometry (GC-MS) and APCI-MS headspace analyses showed that the APCI-MS headspace showed less bias toward enzymatically generated lipid degradation compounds. A GC-sniffing study showed that many of the most intense compounds, acetaldehyde, hexanal, ethyl butanoate, and (E)-2-hexenal but not ethanol, showed odor activity in macerated fruit. In addition, dimethyl sulfide (DMS), a volatile present at very low levels in the fruit, also appeared to be an important contributor to the odor. In vivo analyses also showed much higher levels of aroma compounds in the soft fruit compared to the firm fruit, with evidence of persistence of some compounds, including DMS. There were a number of similarities between the breath profiles of the two panelists, which confirmed the importance of DMS in "Hort16A" aroma.     

In vitro effect of malathion and endosulfan on the LH-induced oocyte maturation in the common CARP,Cyprinus carpio (L.)

Effect of malathion (organophosphorus insecticide) and endosulfan (organochlorine insecticide) on in vitro LH-induced oocyte maturation was investigated in the oocytes of common carp, Cyprinus carpio. In control incubation, LH at the concentration of 10 μg mL^?1 induced 41.2 ± 1.6% of germinal vesicle breakdown (GVBD). When the oocytes were incubated with malathion at the concentrations of 1000, 500, 100, and 50 ppb together with LH it could induce only 13.4 ± 0.4, 14.0 ± 1.0, 12.9 ± 3.5, and 18.1 ± 3.9% of GVBD, respectively. Similarly, when the oocytes were incubated with endosulfan at the concentrations of 0.5, 0.1, 0.05, and 0.01 ppb together with LH, it induced only 12.8 ± 1.6, 8.8 ± 1.2, 20.9 ± 2.1 and 26.0 ± 2.2% of GVBD, respectively. The significance of the result obtained were discussed on the basis of available literature.     

In vitro antioxidant and antiproliferative activities of selenium-containing phycocyanin from selenium-enriched Spirulina platensis

Both selenium and phycocyanin have been reported to show potent cancer chemopreventive activities. In this study, we investigated the in vitro antioxidant and antiproliferative activities of selenium-containing phycocyanin (Se-PC) purified from selenium-enriched Spirulina platensis. The antioxidant activity of Se-PC was evaluated by using four different free radical scavenging assays, namely, the 2,2'-azinobis-3-ethylbenzothiazolin-6-sulfonic acid (ABTS) assay, 1,1-diphenyl-2-picryhydrazyl (DPPH) assay, superoxide anion scavenging assay, and erythrocyte hemolysis assay. The results indicated that Se-PC exhibited stronger antioxidant activity than phycocyanin by scavenging ABTS, DPPH, superoxide anion, and 2,2'-azobis-(2-amidinopropane)dihydrochloride free radicals. Se-PC also showed dose-dependent protective effects on erythrocytes against H 2O 2-induced oxidative DNA damage as evaluated by the Comet assay. Moreover, Se-PC was identified as a potent antiproliferative agent against human melanoma A375 cells and human breast adenocarcinoma MCF-7 cells. Induction of apoptosis in both A375 and MCF-7 cells by Se-PC was evidenced by accumulation of sub-G1 cell populations, DNA fragmentation, and nuclear condensation. Further investigation on intracellular mechanisms indicated that depletion of mitochondrial membrane potential (DeltaPsi m) was involved in Se-PC-induced cell apoptosis. Our findings suggest that Se-PC is a promising organic Se species with potential applications in cancer chemoprevention.     

成年鲁西牛肌内前脂肪细胞的分离培养

建立牛肌内前脂肪细胞体外培养方法，旨在更深入地研究组成肌肉内脂肪组织的肌内前脂肪细胞的增殖分化特性及其影响因素。选取来自成年育肥鲁西黄牛第6与第7肋骨间的肌肉内脂肪组织，利用Ⅰ型胶原酶消化法分离获得牛肌内前脂肪细胞。培养出的细胞成分均一，增殖旺盛，分化率高，经形态学动态变化观察、生长曲线、油红O脂肪染色提取法及对胰岛素和地塞米松反应的测定，证明是功能活跃的前脂肪细胞，并在体外重现了其增殖分化过程，同时经染色体核型分析证明体外培养的细胞倍性正常，可以用于后续研究。本试验成功分离得到了成年育肥牛肌内前脂肪细胞，这为下一步研究肌内脂肪的沉积机制，改善牛肉品质奠定了基础。     

In vitro inhibition of human cGMP-specific phosphodiesterase-5 by polyphenols from red grapes

A moderate consumption of red wine may reduce the risk of cardiovascular diseases via wine-derived phenolic compounds. A variety of biological mechanisms have been proposed for wine-derived phenolic compounds including nitric oxide-mediated vasorelaxation. This study examined whether the vasodilating effect of wine-derived phenolic compounds was associated with the inhibition of phosphodiesterases (PDEs) and, in particular, PDE5. For this purpose, human recombinant PDE5A1 isoform was prepared by expression of the full-length cDNA of PDE5A1 into COS-7 cells. Red wine and the extracts from grape skin inhibited PDE5A1 activity, whereas the seed extracts had a negligible effect. The mixture of anthocyanins inhibited the enzyme activity (IC50 = 11.6 microM), with malvidin-3-O-beta-glucoside (IC50 = 35.4 microM) and malvidin (IC50 = 24.9 microM) the most potent among the monoglucosides and aglycons, respectively. trans-Resveratrol and trans-piceid exhibited negligible effects, whereas hydroxycinnamates were completely inactive. These results indicate that polyphenols-induced vasorelaxation may also be sustained by smooth muscle PDE inhibition by anthocyanins present in red wines and grapes.     

In vitro antioxidant activities of edible artichoke (Cynara scolymus L.) and effect on biomarkers of antioxidants in rats

Artichoke (Cynara scolymus L.), an edible vegetable from the Mediterranean area, is a good source of natural antioxidants such as vitamin C, hydroxycinnamic acids, and flavones. The antioxidant activity of aqueous-organic extracts of artichoke were determined using three methods: (a) free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH(*)) scavenging, (b) ferric-reducing antioxidant power (FRAP), and (c) inhibition of copper(II)-catalyzed in vitro human low-density lipoprotein (LDL) oxidation. In addition, the present study was performed to investigate the ability of the edible portion of artichoke to alter in vivo antioxidative defense in male rats using selected biomarkers of antioxidant status. One gram (dry matter) had a DPPH(*) activity and a FRAP value in vitro equivalent to those of 29.2 and 62.6 mg of vitamin C and to those of 77.9 and 159 mg of vitamin E, respectively. Artichoke extracts showed good efficiency in the inhibition in vitro of LDL oxidation. Neither ferric-reducing ability nor 2,2'-azinobis(3-ethylbenzothiazolin-6-sulfonate) radical scavenging activity was modified in the plasma of the artichoke group with respect to the control group. Among different antioxidant enzymes measured (superoxide dismutase, gluthatione peroxidase, gluthatione reductase, and catalase) in erythrocytes, only gluthatione peroxidase activity was elevated in the artichoke group compared to the control group. 2-Aminoadipic semialdehyde, a protein oxidation biomarker, was decreased in plasma proteins and hemoglobin in the artichoke-fed group versus the control group. In conclusion, the in vitro protective activity of artichoke was confirmed in a rat model.     

In vitro antioxidant properties and phenolic composition of Salvia virgata Jacq. from Turkey

Antioxidant activities and phenolic compositions of the active fractions of Salvia virgata Jacq. (Lamiaceae) from Turkey were examined. The aerial part of S. virgata was extracted with different solvents in an order of increasing polarity such as hexane, ethyl acetate, methanol, and 50% aqueous methanol using a Soxhlet apparatus. Water extract was also prepared from S. virgata by reflux. All solvent fractions were investigated for their total phenolic contents, total flavonoids, flavonols, qualitative-quantitative compositions (by HPLC-PDA analysis), iron(III) reductive activities, free radical scavenging activities (using DPPH*), and effect upon linoleic acid peroxidation activities; also, the peroxidation level was determined by the TBA method. The results of activity tests given as IC50 values were estimated from nonlinear algorithm and compared with standards, viz., butylated hydroxytoluene, ascorbic acid, and gallic acid. Polar fractions were found to be more active for free radical activity whereas nonpolar fractions protected the peroxidation of linoleic acid. Rosmarinic acid was the most abundant component in the extracts, followed by caffeic acid and lutelin-7- O-glycoside.