重组荞麦胰蛋白酶抑制剂理化性质的研究

荞麦胰蛋白酶抑制剂(BuckwheatTrypsinInhibitor,BTI)是存在于荞麦种子中的一种抗营养因子。本文经过原核表达及两步层析纯化得到高纯度的重组荞麦胰蛋白酶抑制剂rBTI。通过分析表明,重组荞麦胰蛋白酶抑制剂与天然荞麦胰蛋白酶抑制剂的性质类似。rBTI的相对分子质量约为11kDa,等电点约为6.2。rBTI对胰蛋白酶有较强的抑制作用,抑制摩尔比(以BApNA为底物)为1:1.5。rBTI的最适pH值为8.0,在pH3.0～8.0之间有较好的热稳定性,100℃加热120min仍保持70%的胰蛋白酶抑制活性。     

Regulating inhibitory activity of potato I-type proteinase inhibitor from buckwheat by rutin and quercetin

This study aims to investigate the effects of two flavonoids, rutin and quercetin, on inhibitory activity of recombinant buckwheat trypsin inhibitor (rBTI). We found that rutin and quercetin could quench the florescence of rBTI through the static quenching process. We also observed that upon binding to rutin or quercetin, rBTI underwent conformational changes. The results also suggested that rutin and quercetin bind to two different sites on rBTI through different interactions: rutin binds to rBTI through van der Waals forces and hydrogen bonds, whereas quercetin binds through hydrophobic interactions. Rutin and quercetin also markedly deactivated the trypsin inhibitory activity (TIA) of rBTI, while quercetin exhibited higher inactivation effect on rBTI than rutin due to its structure. Finally, the molecular docking revealed the molecular binding between the flavonoids and rBTI. These findings can be useful for the understanding of how flavonoid affects the inhibitory of rBTI.     

rBTI的制备及其对果蝇寿命的影响

构建含荞麦胰蛋白酶抑制剂（rBTI）表达质粒pExSecI-BTI的工程菌,在100 L发酵罐中放大发酵,发酵产物经热处理及离子交换层析纯化后,获得rBTI产品,其纯度达到95%以上。用含不同质量浓度rBTI（0、20、50 μg/mL）的培养基饲养果蝇,并分别检测果蝇的最高寿命,平均寿命和半数死亡时间等。初步研究rBTI对果蝇寿命的影响。结果显示,rBTI能够显著延长果蝇的寿命。在实验剂量范围内,果蝇的寿命与rBTI质量浓度呈现正相关。抗氧化酶活性检测发现,实验组果蝇体内的SOD和CAT活性均显著升高。rBTI延缓果蝇衰老的作用可能是通过刺激机体的氧化应激能力来实现的。     

一种胰蛋白酶亲和材料的制备及应用

基于抑制剂与酶的特异亲和作用,将具有良好稳定性的荞麦胰蛋白酶抑制剂(Buckwheat trypsin inhibitor,BTI)固定化,制备成一种胰蛋白酶的亲和吸附剂,实现胰蛋白酶的高效纯化。通过原核表达、Ni2+-NTA亲和层析和Superdex G-25凝胶过滤技术得到电泳纯的BTI,以溴化氰活化琼脂糖凝胶(CNBr-Sepharose CL-4B)作为亲和层析载体,制备亲和吸附剂BTI-Sepharose,检测其对胰蛋白酶的特异性吸附作用,并进一步研究BTI-Sepharose对胰蛋白酶吸附及解吸附的条件。制备的BTI-Sepharose对胰蛋白酶具有特异吸附性,可用于胰蛋白酶的亲和纯化。缓冲液p H对BTI-Sepharose与胰蛋白酶的吸附及解吸附均有重要影响,二者吸附作用的最适pH为7.2,在pH为3.5时两者能够完全分离,且不影响胰蛋白酶的生物活性。试验制备的BTI-Sepharose可实现一步层析制备高纯度胰蛋白酶,为胰蛋白酶的高效纯化及新型胰蛋白酶的研究开发提供理论依据。     

Synergistic cytotoxicity induced by α-solanine and α-chaconine

α-Solanine and α-chaconine are well-known potato toxins, but the mechanism of the synergistic cytotoxic effect of these alkaloids has been little clarified. This study confirmed their synergistic cytotoxic effects on C6 rat glioma cells by three different cell viability tests, namely WST-1 (water-soluble tetrazolium) assay sensitive to intracellular NADH concentration, menadione-catalysed chemiluminescent assay depending on both NAD(P)H concentration and NAD(P)H:quinone reductase activity, and LDH (lactate dehydrogenase) assay sensitive to the release of LDH from damaged cells. The maximum cytotoxic effect was observed at a ratio of 1:1 between α-solanine and α-chaconine at micromolar concentrations. The cytotoxic effects of these alkaloids were observed immediately after incubation and were constant after 30 min, suggesting that rapid damage of plasma membrane causes the lethal disorder of metabolism.     

蜂胶醇提物对人肝癌细胞Hep G2增殖及凋亡的影响

为探讨蜂胶醇提物对人肝癌细胞Hep G2 增殖和凋亡的影响,采用MTT 法检测蜂胶醇提物对Hep G2 细胞增殖的抑制作用,用荧光倒置显微镜和流式细胞仪分析蜂胶醇提物对Hep G2 细胞凋亡的影响。结果表明：12.5～200μg/mL 质量浓度的蜂胶醇提物作用24、48h 后,均能不同程度地抑制Hep G2 细胞的增殖,呈明显的时间、剂量依赖关系,半数抑制质量浓度(IC50)分别是115、78μg/mL。作用8h 后,Hep G2 细胞出现不同程度的凋亡,凋亡率由6.36% 上升到21.9%,呈现出剂量依赖关系。故蜂胶醇提物可显著抑制人肝癌细胞Hep G2 的生长,诱导其凋亡。     

Structure-dependent membrane interaction of flavonoids associated with their bioactivity

Plant foods contain various flavonoids with nutraceutical and health benefits. Structurally different flavonoids were compared by the potency to interact with liposomal membranes in the context of their mode of action. A series of fluorescence polarisation measurements showed that flavonoids (1–10 μM) structure-dependently acted on the deeper regions of lipid bilayers to decrease membrane fluidity. Their comparative effects on cell-mimetic membranes, consisting of unsaturated phospholipids and cholesterol, characterised the structure–membrane interactivity relationship: 3-hydroxylation of the C ring, non-modification of the B ring and 5,7-dihydroxylation of the A ring led to the greatest membrane interactivity, followed by 3′,4′-dihydroxylation of the B ring. Galangin and quercetin, meeting such a structural requirement, inhibited the proliferation of tumour cells at 10–100 μM, together with rigidifying cell membranes, but not membrane-inactive flavonoids. The structure-dependent membrane interaction, which modifies the fluidity, is mechanistically associated with flavonoid bioactivity in a membranous lipid phase.     

Anti-invasion and apoptosis induction of chlorella (Chlorella sorokiniana) in Hep G2 human hepatocellular carcinoma cells

The effects of 80% ethanolic extract derived from commercial granule chlorella (GPE) on cell viability, invasion capacity and apoptosis in human hepatoma cell line (Hep G2 cells) were investigated. The results demonstrated that GPE decreased cell viability, induced apoptosis and showed invasion inhibitory effects in the Hep G2 cells. GPE-triggered apoptosis was confirmed by 4′-6-diamidino-2-phenyindole (DAPI) staining and comet assay. GPE promoted an increase of reactive oxygen species (ROS) and Ca²⁺, and loss of mitochondrial membrane potential (ΔΨm) accompanied by cytochrome c release that was due to the decrease of Bcl-2 in the Hep G2 cells. GPE also induced the protein levels of apoptosis-inducing factor (AIF), increased the levels of caspase-3, -8 and -9, and stimulated the levels of fatty acid synthase (Fas) and Fas ligand (FasL) in the Hep G2 cells. Additionally GPE inhibited invasion of Hep G2 cells by down-regulation of the expression of matrix metalloproteinase (MMP)-2 and -9. Furthermore, cellular glutathione content and superoxide dismutases (SOD) activities were significantly reduced and thiobarbituric acid-reactive substances (TBARS) levels were significantly increased after GPE treatment. These results suggest that GPE can induce cytotoxicity on Hep G2 cells and inhibit the invasive capacity of malignant cells.     

高盐刺激嗜碱性粒细胞产生促炎因子及其分子机制

本研究以食物过敏重要的效应细胞嗜碱性粒细胞（KU812）为对象，探究高盐条件对嗜碱性粒细胞脱颗粒以及相关细胞因子表达的影响，并初步探讨其分子机制。首先利用酶联免疫吸附试验（enzyme linked immunosorbent assay,ELISA）和实时荧光定量聚合酶链式反应（real-time fluorescence quantitative polymerase chain reaction,qPCR）分析高盐条件下KU812细胞脱颗粒释放生物活性介质和促炎细胞因子的变化；并用q PCR分析血清和糖皮质激素调节蛋白激酶1(serum and glucocorticoid-regulated kinase 1,SGK1)抑制剂和P38抑制剂对高盐条件下KU812细胞产生白细胞介素（interleukin,IL）-4的影响。结果表明，高盐条件并不能促进KU812细胞脱颗粒释放生物活性介质组胺和β-氨基己糖苷酶，但能促进其产生促炎因子IL-6和肿瘤坏死因子（tumor necrosis factor,TNF）-α；高盐条件也可以促进KU812细胞产生IL-4，同时伴随着SGK1基因...     

苦荞麦蛋白酶抑制剂研究进展

蛋白酶抑制剂(PI)对癌症、肺气肿、胰腺炎、HIV多种疾病具有一定疗效,且在抗虫基因 工程中可作为理想元件,具有广阔应用前景;苦荞麦蛋白酶抑制剂(BWPI)属于丝氨酸PI,含有丰 富胰蛋白酶抑制剂,相关研究并不多,但目前已引起广大科研者研究兴趣。该文对已有研究成果进 行总结,并从BWPI提取方法、理化性质、初级结构、生理功能及研究存在问题等方面进行介绍,以 供相关研究人员参考。     

Improving the antioxidant activity of buckwheat (Fagopyrum tataricm Gaertn) sprout with trace element water

Trace element water (TEW) (100, 200, 300, 400 and 500 ppm) was used to grow buckwheat (Fagopyrum tataricm Gaertn) to evaluate whether the beneficial effects of trace elements on the antioxidant activity could be accomplished with the supplement of TEW. At 300 ppm, TEW significantly increased the Cu, Zn and Fe contents in buckwheat sprout, but not the Se and Mn contents. The levels of rutin, quercitrin and quercetin did not differ between buckwheat sprouts grown in TEW and de-ionized water (DIW). The ethanolic extract from buckwheat sprout grown in 300 ppm TEW showed higher DPPH radical scavenging activity, ferrous ion chelating activity, superoxide anion scavenging activity and inhibitory activity toward lipid peroxidation than that grown in DIW. The extract of the TEW group also enhanced intracellular superoxide dismutase activity and resulted in lower level of reactive oxygen species in human Hep G2 cells.     

Caco-2细胞模型用于乳源ACE抑制肽LL、LPEW的小肠吸收研究

通过建立并验证Caco-2细胞模型,分析血管紧张素转化酶(angiotensin-I converting enzyme,ACE)抑制肽LL、LPEW在小肠中的转运量,研究LL、LPEW的小肠吸收机制。从细胞形态、跨膜电阻和碱性磷酸酶活性3个方面验证Caco-2细胞模型可用性。分析ACE抑制肽LL、LPEW的Caco-2细胞转运量,LL的表观渗透系数(P_(app))为(275.17±8.28)×10~(-7 )cm/s,肠道吸收良好;LPEW的P_(app)为(5.13±1.49)×10~(-7) cm/s,相比于LL,肠道吸收量较低。加入旁路转运促进剂去氧胆酸钠、内吞抑制剂渥曼青霉素(Wortmannin)、肽转运载体竞争性抑制剂Gly-Pro,对比无抑制剂时LL的转运量,分析得到LL的跨膜转运机制可能为内吞途径。加入ATP能量生成抑制剂叠氮化钠、多药耐药蛋白抑制剂MK-571、P-糖蛋白抑制剂维拉帕米,对比无抑制剂时肽LL的转运量,得出LL没有外排作用,所以LL肠道吸收较好。     

The interaction of sesamol with DNA and cytotoxicity,apoptosis, and localization in HepG2 cells

Sesamol, a nutritional antioxidant phenolic compound present in sesame seed, has a potential therapeutic molecule effect against cancers. In this study, the interaction between sesamol and DNA was investigated by employing ultraviolet/visible (UV/Vis), fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR), and molecular modeling. The fluorescence analysis indicated that the fluorescence quenching mechanism of sesamol by calf thymus DNA (ctDNA) occurred through static quenching. The UV/Vis, CD, FT-IR spectra and molecular docking results implied that the primary binding mode was minor groove binding. Furthermore, the intracellular interaction of sesamol with DNA and its bioactivity effect were explored. The cell activity results demonstrated that sesamol induced hepatic cell line (HepG2) death. The acridine orange (AO)/ethidium bromide (EB) staining assay and DNA fragmentation confirmed that sesamol could efficiently induce the apoptosis of HepG2 cells. Moreover, addition of sesamol to HepG2 cells resulted in nuclear localization, as visualized by confocal microscopy.     

Changes in lipoxygenase isozymes and trypsin inhibitor activity in soybean during germination at different temperatures

Influence of germination temperature on lipoxygenase isozymes and trypsin inhibitor activity, the two undesirable components in soybean for human consumption, is not yet reported in soybean sprouts. Two Indian soybean genotypes were incubated for 144 h in a seed germinator at two different temperatures (25 and 35 °C) and the activities of lipoxygenase isozymes and trypsin inhibitor were monitored in the germinating seedlings every 24 h. Lipoxygenase-I as well as lipoxygenase-II + III were degraded continuously over the 144 h and the rate of degradation, of both the classes of lipoxygenase, was faster at the higher germinating temperature (35 °C) in both the genotypes. Trypsin inhibitor was also degraded continuously during germination upto 144 h and the degradation was faster at higher germination temperature. Protein extracts of seedlings of different periods, developed at different temperatures, and analyzed using polyacrylamide gel electrophoresis, indicated that the original Kunitz inhibitor band (R_f = 0.75) declined continuously in intensity during germination at both temperatures in both genotypes, and a new band (R_f = 0.72) possessing trypsin inhibitor activity appeared at 48 h at 35 °C, while it appeared at 72 h at 25 °C. Early appearance of a modified form of Kunitz inhibitor, a degraded product of native form, at 35 °C as compared to 25 °C, confirms that the faster quantitative reduction at higher temperature is due to faster degradation of the original Kunitz inhibitor form at higher temperature.     

荞麦芽不同提取部位细胞毒性的实验研究

试验对荞麦芽不同极性提取部位的细胞毒性进行研究,为开发荞麦芽的保健作用提供依据。试验过程中采用体外培养的肺癌细胞(A549)、胃癌细胞(AGS)、乳腺癌细胞(MCF-7)、肝癌细胞(Hep3B)、结肠癌细胞(Colo205)为试验对象,利用SRB试验方法对荞麦芽不同极性提取部位抑制肿瘤细胞生长效果进行研究。结果:荞麦芽乙酸乙酯和丁醇提取物具有较高的抑制肿瘤细胞生长效果。在浓度为1.0mg/mL时,荞麦芽乙酸乙酯提取物显示出的抗肿瘤细胞(A549、AGS、MCF-7、Hep3B、Colo205)活性分别为70.3%、94.8%、79.6%、82.3%、73.2%。因此表明荞麦芽不同极性提取部位均具有一定的抑制肿瘤细胞生长的作用。     

Characterisation of trypsin and α-chymotrypsin inhibitors in Australian wattle seed (Acacia victoriae Bentham)

Crude water extracts from Australian wattle seed (Acacia victoriae Bentham) and their salt (ammonium sulphate)-precipitated fractions were analysed for trypsin and α-chymotrypsin (chymotrypsin) inhibitor activity, using gel electrophoresis and spectrophotometric methods. Three different bands with molecular weight 30.20, 38.03 and 39.81 kDa were active, with the 50% salt-precipitated fraction exhibiting highest activity and number of active bands. The same proteins also appeared to be responsible for both trypsin and chymotrypsin inhibitor activity. To establish conditions for the inactivation of these inhibitors, whole seed and uncoated (dehulled) cotyledon were subjected to different heat treatments. Moist heat treatment at 100 °C for 30 s was sufficient to inactivate both protease inhibitors although the trypsin inhibitor was found to be more heat-resistant than was the chymotrypsin inhibitor. Soaking overnight, before thermal treatment, improved the trypsin inhibitor activity but enhanced the efficiency of thermal inactivation in both inhibitors.     

原花青素B2诱导乳腺癌MCF-7细胞凋亡及其作用机制

本研究旨在探讨原花青素B2（procyanidin B2,PCB2）对人乳腺癌MCF-7细胞的抗肿瘤作用及其机制。采用噻唑蓝法分析PCB2在不同浓度和不同处理时间下对MCF-7细胞存活率的影响;通过激光共聚焦显微镜检测细胞内Ca2＋浓度变化并观察Hoechst 33342染色后的细胞凋亡形态;采用流式细胞分析仪检测PCB2对MCF-7细胞凋亡率、周期、胞内活性氧（reactive oxygen species,ROS）水平及线粒体膜电位变化的影响;通过Western blot检测PCB2对MCF-7细胞凋亡相关蛋白表达量的影响。结果表明,当浓度在10～80 μmol/L范围内时,PCB2能显著降低MCF-7细胞的存活率（P＜0.05）,且呈浓度依赖性,处理24、48、72 h时对MCF-7细胞存活率的半抑制浓度分别为114.82、57.15 μmol/L和55.64 μmol/L。PCB2能显著增加MCF-7细胞凋亡率、胞内ROS水平（P＜0.05）、细胞核荧光强度,并破坏Ca2＋平衡,同时显著降低线粒体膜电位（P＜0.05）,并将细胞周期阻滞在G0/G1期进而诱导细胞凋亡。PCB2可上调Bax、细胞色素c、Caspase-12和Caspase-3蛋白的相对表达水平,下调Bcl-2蛋白相对表达水平。综上可知,PCB2具有抗肿瘤作用,其机制与激活线粒体和内质网介导的凋亡通路、周期阻滞和细胞内ROS水平增加有关。     

Effect of antioxidants on the apoptosis of CHO cells and production of tissue plasminogen activator in suspension culture

The effects of antioxidants on the apoptosis of Chinese hamster ovary (CHO) 1-15,500 cells and production of tissue plasminogen activator (tPA) in suspension culture were investigated. After cell growth to 2 x 10(5) cells/ml in Ham's F12 medium containing 10% newborn bovine serum (NBS) in a spinner bottle, CHO cells were maintained for 6 d in Ham's F12 medium containing 0 or 0.4% NBS and 10 mM antioxidants, namely, l-ascorbic acid 2-phosphate (VCP) or the reduced form of glutathione (GSH). The viable cell concentrations at day 6 in the serum-free culture with GSH and in the low-serum culture wiht VCP or GSH were 0.57, 1.04 and 1.69 x 10(5) cells/ml, respectively, while those in the serum-free and low-serum cultures without the antioxidants were only 2.33 and 1.17 x 10(3) cells/ml, respectively. The percentages of apoptotic cells in the serum-free and low-serum cultures with VCP (73.2, 44.6%) and GSH (76.9, 38.6%) measured using a flowcytometer after annexin V-FITC/propidium iodide staining were markedly lower than those in the cultures without antioxidants (96.3, 92.5%). The percentage of cells having a high mitochondrial membrane potential among the viable cells in the cultures with antioxidants determined using a flowcytometer after 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide staining was clearly higher than those in the cultures without the antioxidants. The production of tPA in the serum-free and low-serum media with VCP (0.282, 2.92 mg/l) or GSH (1.01, 1.61 mg/l) was markedly higher than that in the cultures without the antioxidants (0.275, 0.689 mg/l). Consequently, the suppression of apoptosis through the maintenance of the membrane potential of mitochondria by VCP or GSH resulted in a marked increase in tPA production by CHO cells in the serum-free and low-serum cultures.