红头豆芫菁成虫芫菁素含量的研究

芫菁素在红头豆芫菁体内主要贮存在雄虫的生殖腺和卵内。与野生群体两性芫菁素的平均含量相比较,刚交配过的雄虫失去体内７０％的芫菁素,其雌配偶体内芫菁素含量相应升高４３％。红头豆芫菁可用作中药材。经１１０℃烘干后的雄虫,用酸水解后提取的芫菁素含量比直接提取的含量增高４倍。     

乙醛脱氢酶2可减少氧化损伤诱导的心肌细胞凋亡

乙醛脱氢酶2 (aldehyde dehydrogenase 2, ALDH2)是线粒体特异性酶,已被证明参与氧化应激诱导的细胞凋亡,而在心肌细胞中的作用知之甚少。本研究旨在通过用特异性ALDH2抑制剂大豆苷抑制ALDH2活性来研究ALDH2在抗霉素A诱导的心肌细胞凋亡中的作用。应用抗霉素A和大豆苷诱导小鼠心肌细胞,然后测定ALDH2酶活性、细胞内活性氧(reactive oxy gen species, ROS)含量和细胞凋亡,应用RT-PCR和蛋白质印迹法(Western blotting)检测ALDH2 m RNA和蛋白表达。结果表明,抗霉素A (40μg/mL)可诱导新生心肌细胞凋亡,而大豆苷(50μmol/L)能有效地抑制ALDH2活性而对细胞凋亡没有影响,并且可显著增强抗霉素A诱导的心肌细胞凋亡(53.72%～71.33%, p<0.05)。与单独用抗霉素A处理的细胞相比,抗霉素A和大豆苷共处理的心肌细胞中活化的丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)信号传导途径(p38-MAPK)的磷酸化也显著增加。本研究初步表明,改变线粒体ALDH2活性可能是减少氧化损伤诱导的心肌细胞凋亡的潜在选择。     

短翅豆芫菁生物学特性研究

短翅豆芫菁 Epiauta aptera Kaszab系芫菁科豆芫菁属昆虫 ,为民间常用中草药。具有破血逐瘀 ,消症散结 ,壮阳利尿 ,攻毒等功效。主治症瘕、恶疮、闭经、疥癣等。近年来国内外医药专家多用芫菁素和以芫菁素为原料合成的一系列药物治疗肝癌、肺癌、乳腺癌以及直肠癌等均取得了较好的疗效 ,总有效率在 45 %～ 65 %。由于芫菁资源日趋枯竭 ,用药量不断增大 ,市场供求矛盾突出 ,为了开发利用新的药源 ,1 987～1 997年作者先后对四川、云南、广西等地的芫菁资源做了实地考察 ,并对其中分布广、贮量大的种类的生物学做了较为详细的研究 ,现将有关…     

局部力学刺激对黄瓜系统抗病性的诱导

病原真菌在侵入植物细胞过程中,除了分泌化学物质外还通过物理挤压细胞产生力学作用.用压应力作为力学信号,研究了局部力学刺激对黄瓜系统抗病性的诱导.结果表明,力学刺激可以诱导黄瓜系统抗病性的产生.当细胞壁与质膜间的黏附被Arg-Gly-Asp(RGD)阻断后,力学刺激对黄瓜系统抗病性的诱导几乎完全被减除.通过薄层色谱和液相色谱分析,发现力学刺激可以使植保素含量明显增加.这表明黄瓜植保素的积累可能是力学刺激诱导其产生抗性的原因之一.而细胞壁与质膜间的黏附被RGD阻断后,力学刺激只能诱导植保素的部分积累.即力学刺激对植保素积累的诱导依赖于细胞膜与细胞壁间的黏附.     

胞外钙离子以及细胞膜组分参与蓝光诱导拟南芥叶片花色素苷的积累

以野生型和hy4突变体拟南芥为材料,运用药物学方法研究可能参与蓝光诱导叶片花色素苷积累和CHS基因表达的信号组分。培养基中外施Ca2 、钙离子通道剂A23187、螯合剂EGTA、钙通道阻断剂尼群地平(nifedipine,Nif)以及异博定(verapermil)的实验证实,蓝光诱导13d龄叶片花色素苷积累和CHS基因表达需要胞外Ca2 的参与,而蓝光作用是由cry1(cryptochrome1)介导的。此外,质膜黄素蛋白抑制剂DPI(diphenylene iodonium)抑制蓝光诱导的花色素苷积累,质膜H -ATPase激活剂壳梭胞素(fusicoccin,FC)抑制蓝光反应,而抑制剂钒酸钠则起促进作用。CaM拮抗剂W7、Ca2 -ATPase抑制剂EB(erythrosine B)、G蛋白激活剂霍乱霉素(cholera toxin,CTX)以及抑制剂百日咳毒素(pertussis toxin,PTX)对蓝光下野生型与hy4的花色素苷积累都有影响。对药物实验的分析表明,质膜氧化还原系统、H -ATPase可能参与依赖于外源Ca2 的蓝光反应。     

新狼毒素A诱导人黑色素瘤A375细胞凋亡的机制研究

为探讨新狼毒素A抑制人黑色素瘤A375细胞增殖及诱导凋亡的作用。本实验采用噻唑蓝(MTT)法检测新狼毒素A对人黑色素瘤A375细胞增殖的影响,Hoechst33258法观察细胞形态,流式细胞仪检测细胞凋亡率和线粒体膜电位,Westernblot检测凋亡相关蛋白表达。结果显示新狼毒素A以时间剂量依赖性的方式显著抑制A375细胞的增殖;不同浓度新狼毒素A(0、15、30、45μmol/L)作用于A375细胞48h后细胞出现显著凋亡特征,新狼毒素A增加A375细胞的凋亡率且降低了线粒体膜电位,上调Bax、caspase-3和细胞色素C(CytochromeC)蛋白的表达,下调Bcl-2蛋白的表达。以上结果说明新狼毒素A抑制A375细胞增殖,诱导细胞凋亡,其机制可能与诱导线粒体途径的凋亡有关。     

细胞周期蛋白依赖激酶Roscovitine 诱导非小细胞肺癌A549 细胞凋亡及 其机制的研究

目的:探讨细胞周期蛋白依赖激酶(CDK)抑制剂Roscovitine(Ros)诱导非小细胞肺癌(NSCLC)A549细胞凋亡及其作用机制。方法:以不同浓度Ros(10μM、20μM、40μM)处理细胞24h,采用Annexin V-PI染色以流式细胞仪检测细胞凋亡,Westernblot法检测胞浆中和线粒体促凋亡蛋白Bax和Bad的表达,流式细胞仪检测线粒体膜电位(MMP)变化。结果:Ros以剂量依赖的方式诱导A549细胞凋亡,同时Bad和Bax在胞浆的含量随着Ros剂量的增加而减少,而在线粒体中却出现相反的结果,线粒体膜电位随Ros剂量的增大而降低。结论:Ros可通过促进Bax和Bad由胞浆向线粒体易位,诱导NSCLC A549细胞由线粒体途径发生凋亡。     

芫菁体内斑蟊素的合成、 转移和生物学功能

斑蟊素是芫菁科昆虫合成的一种防御物质, 已经被证实对多种癌症和其他疾病有着特殊的疗效。芫菁体内存在不同结合态的斑蟊素或斑蟊素衍生物, 包括斑蟊素酸镁、斑蟊素酸钙、羟基斑蟊素、甲基斑蟊胺和脱甲斑蟊素等。不同芫菁种类、不同发育阶段其斑蝥素合成量有显著的差异, 并且有着典型的性二型现象, 性成熟的雄成虫斑蝥素含量最高可达10%。关于斑蝥素的生物合成途径以及斑蝥素在虫体内的分布, 尽管有一些研究, 但仍然没有定论。本文从斑蟊素在芫菁体内的含量、分布、生物合成、代谢及生物学功能等方面对国内外的研究进行概括, 以期为充分发掘芫菁科昆虫资源、指导芫菁的人工养殖、合理的利用资源以及人工合成斑蟊素提供参考。     

植物苯丙氨酸解氨酶的研究——Ⅴ.PAL活性与尾穗苋黄化苗中苋红素积累的相关性

用不同因素诱导与抑制的方法较系统地探讨了尾穗苋黄化苗PAL活性与苋红素积累之间的关系。根据激动素诱导的时间曲线表明。PAL活性高峰较早于苋红素积累高峰;PAL活性及苋红素积累都随激动素浓度上升而上升。 光对激动素诱导的PAL活性有增效作用,对苋红素积累也有同样的增效作用。不同光质(蓝、红)对PAL活性的诱导结果表明:蓝光大于红光,同时蓝光下苋红素积累也大于红光。 用环己亚胺、放线菌素D及反式肉桂酸等处理激动素诱导的材料时,发现在各种抑制条件下PAL活性受抑的同时,苋红素合成也受抑制,且随着抑制剂浓度增加受抑程度也增加,可见PAL活性与苋红素积累之间有密切关系。本文提出了PAL可能参与苋红素双氢吲哚部分合成的设想,这与目前一般认为多巴是双氢吲哚直接前体的观点不同。     

匹美克莫司使线粒体受损并激活AMPK信号通路与线粒体自噬

自噬是真核生物中普遍存在的现象,它可以降解细胞中堆积的错误折叠蛋白和衰老蛋白或者破损细胞器,从而维持细胞稳态平衡。研究表明,钙调神经磷酸酶能调节自噬,但其具体分子机制未阐明,尚有待研究。研究发现,钙调神经磷酸酶的抑制剂匹美克莫司通过腺苷酸活化蛋白激酶[adenosine 5′-monophosphate(AMP)-activated protein kinase,AMPK]信号通路诱导自噬。进一步的研究表明,匹美克莫司使线粒体受损,并使线粒体内膜移位酶复合物23(mitochondrial inner membrane translocase complex,subunit 23,Tim23)下调。自噬特异抑制剂3-甲基腺嘌呤(3-methyladenine,3MA)和sh RNA稳定敲低AMPK基因表达能抑制匹美克莫司引起Tim23的下调。由此可见,匹美克莫司通过AMPK信号通路诱导线粒体自噬发生。该研究阐明了钙调神经磷酸酶调节线粒体自噬的机制。     

Lactofen induces isoflavone accumulation and glyceollin elicitation competency in soybean

Lactofen, the active ingredient of the soybean disease resistance-inducing herbicide, Cobra, induces large accumulations of isoflavone conjugates and aglycones in soybean tissues. The predominant isoflavones induced in cotyledon tissues are daidzein (and its conjugates) and formononetin and glycitein aglycones. The latter two isoflavones are usually present only at very low levels in soybean seedling tissues. In leaves, the predominant lactofen-induced isoflavones are daidzein and formononetin aglycones and the malonyl-glucosyl conjugate of genistein. Isoflavone induction also occurs in cells distal to the point of treatment, but is only weakly systemic. Lactofen also induces elicitation competency, the capacity of soybean cells to accumulate the pterocarpan phytoalexin glyceollin in response to glucan elicitors from the cell wall of the pathogen Phytophthora sojae. Comparison of the activity of a series of diphenyl ether herbicides demonstrated that while all diphenyl ethers tested induced some degree of elicitation competency, only certain ones induced isoflavone accumulation in the absence of glucan elicitor. As a group the diphenyl ethers are thought to inhibit protoporhyrinogen oxidase, eventually leading to singlet oxygen generation. Another singlet oxygen generator, rose bengal, also induced elicitation competency, but little isoflavone accumulation. It is hypothesized that diphenyl ether-induced activated oxygen species mimic some aspects of hypersensitive cell death, which leads to elicitation competency in infected tissues.     

Isoflavonoid accumulation in soybean hairy roots upon treatment with Fusarium solani.

Hairy roots were initiated from two soybean [Glycine max (L.) Merr.] genotypes with different susceptibility (susceptible 'Spencer' and partially resistant 'PI567.374') to the disease sudden death syndrome (SDS) caused by the soil-borne fungal pathogen Fusarium solani f. sp. glycines (FSG) to study the role of isoflavonoids in the plant response to FSG infection. Hairy root cultures obtained by transformation with Agrobacterium rhizogenes allows normal root growth that can be visually monitored. The principal isoflavones (genistin, daidzin, glycitin and their malonyl conjugates and aglycones) and also isoflavonoid phytoalexins (coumestrol and glyceollin) were measured by HPLC in extracts of the FSG-inoculated and non-inoculated hairy roots. FSG mycelia grew more slowly on inoculated PI567.374 hairy roots than on Spencer hairy roots. The glyceollin content was higher in FSG-inoculated PI567.374 hairy roots than in Spencer hairy roots even though the glyceollin precursor, the isoflavone daidzein, was higher in Spencer. The de novo synthesis of isoflavones and glyceollin was confirmed by [(14)C]Phe incorporation into glyceollin, which was higher both in the FSG-inoculated roots and surrounding medium of the cv. PI567.374 than that of Spencer. Glyceollin was the most inhibitory to FSG growth among eight isoflavonoids tested. The levels of coumestrol, a putative phytoalexin, did not change upon FSG inoculation. The defense response was also elicited by FSG culture filtrates in hairy roots grown in liquid culture. The data obtained indicate that the ability of soybean roots to rapidly produce sufficient amounts of glyceollin in response to FSG infection might be important in providing partial resistance to this fungus.     

Protein phosphatase inhibitors activate anti-fungal defence responses of soybean cotyledons and cell cultures

The application of a variety of structurally different protein phosphatase inhibitors (okadaic acid, acanthifolicin, microcystins, nodularin, tautomycin, calyculin A, cantharidin and endothall) to cut surfaces of soybean cotyledons (Glycine max L.) resulted in the production of isoflavonoid phytoalexins (plant defence compounds). Daidzein was the predominant isoflavonoid produced by soybean cotyledons in response to protein phosphatase inhibitors. In contrast, several isoflavonoid phytoalexins were seen after application of either an elicitor β-glucan fraction isolated from yeast extract or hepta-(1→3, 1→6)-β-glucoside which is the most potent elicitor-active component isolated from the soybean pathogen Phytophthora megasperma f. sp. glycinea. Isoflavonoid production in response to either protein phosphatase inhibitors or elicitors reached a maximum after 20–24 h. The addition of protein phosphatase inhibitors to a soybean cell suspension culture induced the expression of phenylalanine ammonia-lyase (PAL), the first enzyme in the isoflavonoid biosynthetic pathway. Induction of PAL activity was blocked by protein synthesis inhibitors, cycloheximide or anisomysin, and largely prevented by a protein kinase inhibitor, K252a. Another common response of plant cells to fungal elicitation, alkalinization of the soybean cell culture media, was induced within minutes in response to protein phosphatase inhibitors and was largely prevented by K252a. These studies suggest a direct role for phosphorylation in activation of plasma membrane ion flux(es), whereas the longer-term effects of protein phosphatase inhibitors on isoflavonoid production and PAL expression could be due to either direct effects of increased protein phosphorylation, or the secondary consequences of other phosphorylation-induced cellular changes. They also indicate that protein phosphatase inhibitors are likely to be of general use in investigating mechanisms of plant responses to environmental stimuli.     

Signaling in Soybean Phenylpropanoid Responses (Dissection of Primary,Secondary, and Conditioning Effects of Light,Wounding, and Elicitor Treatments)

The spatial and temporal deployment of plant defense responses involves a complex interplay of signal events, often resulting in superimposition of signaling processes. We have employed a minimal-wound protocol to clearly separate and characterize the specific contributions of light, wounding, and a wall glucan elicitor preparation (PWG) from Phytophthora sojae (Kauf. and Gerde.) to the regulation of phenylpropanoid defense responses in soybean (Glycine max L. [Merr.]) cotyledon tissues. The assay also allowed us to clearly reconstitute responses to combinations of these primary signals and to examine the effects of other pathogenesis-related molecules on the responses in a defined manner. Light specifically triggers accumulation of malonylglucosyl conjugates of the 5-hydroxy-isoflavone, genistein, which is normally found in epidermal cells. PWG selectively induces accumulation of conjugates of the 5-deoxy-isoflavone daidzein, the first committed precursor of the phytoalexin glyceollin. Wounding initiates phenolic polymer deposition, a process greatly potentiated by PWG and light. Whereas glutathione selectively enhances light induction of genistein conjugates, methyl jasmonate enhances both light and PWG-induced isoflavone conjugate accumulations. Wound exudate fully activates the cell's capacity (competency) for the phenolic polymer and glyceollin responses to PWG, whereas glutathione partially restores competency, favoring coumestrol and phenolic polymer responses to PWG. Abscisic acid inhibits all induced phenylpropanoid responses.     

Effects of Ca2+ on phytoalexin induction by fungal elicitor in soybean cells

A glucan elicitor from the cell walls of the fungus Phytophthora megasperma f.sp. glycinea caused increases in the activities of the phytoalexin biosynthetic enzymes, phenylalanine ammonia-lyase and chalcone synthase, and induced the production of the phytoalexin, glyceollin, in soybean (Glycine max) cell suspension cultures when tested in culture medium containing 1.2 mmol/liter Ca2+. Removal of extracellular Ca2+ by treatment with ethylene glycol bis(beta-aminoethyl ether)-N, N'-tetraacetic acid followed by washing the cells with Ca2+-free culture medium abolished the elicitor-mediated phytoalexin response. This suppression was largely reversed on readdition of Ca2+. Elicitor-mediated enhancement of biosynthetic enzyme activities and accumulation of glyceollin was strongly inhibited by La3+; effective concentrations for 50% inhibition were (mumol/liter) 40 for phenylalanine ammonia-lyase, 100 for chalcone synthase, and 30 for glyceollin. Verapamil caused similar effects only at concentrations higher than 0.1 mmol/liter, whereas trifluoperazine and 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate did not affect enzyme induction by the elicitor in the concentration range tested. Uptake of alpha-amino isobutyric acid into soybean cells, which was rapidly inhibited in the presence of the glucan elicitor, was not affected by La3+ nor was uptake inhibition by the elicitor relieved by La3+. The Ca2+ ionophore, A23187, enhanced phytoalexin biosynthetic enzyme activities and glyceollin accumulation in a dose-dependent manner, with 50% stimulation (relative to the elicitor) occurring at about 5 mumol/liter. The results suggest that the glucan elicitor causes changes in metabolite fluxes across the plasma membrane of soybean cells, among which changes in Ca2+ fluxes appear to be important for the stimulation of the phytoalexin response.     

Metabolic Regulation during Glyceollin Biosynthesis in Green Soybean Hypocotyls

The accumulation of the isoflavonoid phytoalexin, glyceollin, occurs in hypocotyls of green soybean seedlings (Glycine max L. Merr. cv Harosoy 63) in response to the injection of a glucan elicitor isolated from the mycelial walls of the fungus, Phytophthora megasperma f. sp. glycinea. This accumulation, which levels off after 24 hours, is preceded by a dramatic, transient rise in extractable activities of two early enzymes in the biosynthetic pathway, phenylalanine ammonia-lyase (PAL) and p-coumaryl CoA ligase (pCL). The maximum amount of extractable activity occurs 12 to 16 hours after elicitor treatment and is coincident with the most rapid period of glyceollin accumulation. These results suggest a regulatory role for these early enzymes in the biosynthesis of this secondary metabolite. High performance liquid chromatography analysis of the early intermediates in the pathway further corroborates this hypothesis. The relative pool size and rate of turnover of p-coumaric acid, an early intermediate in glyceollin production, increase during the period of rapid increases in enzyme activities. Removal of cotyledons from elicitor-treated seedlings reduces glyceollin accumulation approximately 70%. This limitation of phytoalexin accumulation by cotyledon removal is correlated with a similar cotyledon effect on reduction of extractable activities of both PAL and pCL as well as a decrease in the flux of carbon through the p-coumaric acid pool. This research further supports the hypothesis that early enzymic steps in a biosynthetic pathway diverting carbon from primary to secondary metabolites function as regulatory control points.     

Isoflavonoid-inducible resistance to the phytoalexin glyceollin in soybean rhizobia.

The antibacterial effect of the soybean phytoalexin glyceollin was assayed using a liquid microculture technique. Log-phase cells of Bradyrhizobium japonicum and Sinorhizobium fredii were sensitive to glyceollin. As revealed by growth rates and survival tests, these species were able to tolerate glyceollin after adaptation. Incubation in low concentrations of the isoflavones genistein and daidzein induced resistance to potentially bactericidal concentrations of glyceollin. This inducible resistance is not due to degradation or detoxification of the phytoalexin. The inducible resistance could be detected in B. japonicum 110spc4 and 61A101, representing the two taxonomically divergent groups of this species, as well as in S. fredii HH103, suggesting that this trait is a feature of all soybean-nodulating rhizobia. Glyceollin resistance was also inducible in a nodD1D2YABC deletion mutant of B. japonicum 110spc4, suggesting that there exists another recognition site for flavonoids besides the nodD genes identified so far. Exudate preparations from roots infected with Phytophthora megasperma f. sp. glycinea exhibited a strong bactericidal effect toward glyceollin-sensitive cells of B. japonicum. This killing effect was not solely due to glyceollin since purified glyceollin at concentrations similar to those present in exudate preparations had a much lower toxicity. However, glyceollin-resistant cells were also more resistant to exudate preparations than glyceollin-sensitive cells. Isoflavonoid-inducible resistance must therefore be ascribed an important role for survival of rhizobia in the rhizosphere of soybean roots.     

Ozone injury in soybeans: isoflavonoid accumulation is related to necrosis

Fumigation of soybean leaves (Glycine max [L.] Merr. with ozone caused stippling and silvering at the same time that large accumulations of the isoflavonoid compounds daidzein, coumestrol, and sojagol occurred. Nitrogen dioxide and sulfur dioxide caused lesser accumulation of the isoflavonoids, and peroxyacetyl nitrate did not result in significant accumulation. Visible toxicity and chemical changes in ozone-fumigated leaves were similar to the hypersensitive disease defense reaction of soybean leaves to the pathogen Pseudomonas glycinea, except that the phytoalexin hydroxyphaseollin was not produced in the ozone-treated leaves.