NO和茉莉酸甲酯对黄芩悬浮细胞生长及黄芩苷合成的影响

以硝普钠(sodium nitroprusside, SNP)为一氧化氮(nitric oxide, NO)的供体, 向黄芩(Scutellaria baicalensis)悬浮培养细胞系中添加SNP和茉莉酸甲酯(methyl jasmonate, MJ), 考察这两种诱导子在不同的添加时间、添加浓度及混合配比使用对黄芩悬浮细胞系生长和黄芩苷含量的影响。研究结果表明：低浓度的外源NO有利于细胞的生长, 但对黄芩苷积累无作用, 而MJ有利于黄芩苷的合成, 但抑制细胞生长,且两者的适用浓度范围和添加时间存在差异。在细胞培养初期(0天)添加0.05 mmol.L-1 SNP, 而在细胞生长对数中期(8天)添加10 μmol.L-1的MJ, 细胞鲜重可达到对照的1.2倍, 黄芩苷总量达到对照的2.96倍。     

几种影响黄芩愈伤组织中黄芩苷含量的因素

研究碳源、生长调节物质、氮源、磷酸盐以及有机添加物对黄芩愈伤组织中黄芩苷含量和产量影响的结果表明,在(25±1)℃暗培养条件下,黄芩苷积累的最佳培养基为MS基本培养基,其中氮源浓度为60mmol·L-1(NH4+:NO3-为1:1),KH2PO4浓度为1.5mmol·L-1,附加80g·L-1蔗糖、0.3mg·L-1IAA、2mg·L-16-BA和200mg·L-1蛋白胨。在上述培养基中,黄芩愈伤组织培养40d后的黄芩苷含量达到167.4mg·g-1,产量达到4.8g·L-1,分别是对照的5.54倍和18.2倍。     

光质对悬浮培养黄岑细胞生长及黄岑苷积累的影响

研究了不同光质、光强和光期对悬浮培养黄芩(Scutellaria baicalensis)细胞的生长、细胞中苯丙氨酸氨基裂解酶(PAL)活性及黄芩苷含量的影响。白光、紫外光、红光和蓝光对悬浮培养黄芩细胞PAL活性和黄芩苷含量有不同的影响。白光、紫外光和蓝光对PAL活性和黄芩苷的积累具有诱导促进作用,其中紫外光的促进作用最强,白光和蓝光的促进作用较弱,而红光照射对PAL活性和黄芩苷的积累有一定的抑制作用。在0-60μmolm-2s-1紫外光照射强度范围内,PAL活性和黄芩苷含量随着紫外光照射强度的增加而显著提高。实验结果同时表明,红光照射显著促进黄芩细胞的生长,紫外光抑制黄芩细胞的生长,而蓝光和白光对悬浮培养黄芩细胞生长的促进作用不明显。与黑暗(对照)相比,每天8h光照强度为60μmolm-2s-1的紫外光、16h光照强度为50μmolm-2s-1的红光交替处理,在培养12d后,细胞的鲜重和黄芩苷含量为对照的1.16和3.2倍,黄芩苷的产量达到439mgL-1,是对照的3.8倍。     

茉莉酸甲酯与水杨酸对肉苁蓉悬浮细胞中苯乙醇甙合成的影响

向肉苁蓉悬浮细胞培养系中添加茉莉酸甲酯(MJ)和水杨酸(SA) ,分别考察了这两种诱导子的添加浓度及添加时间对肉苁蓉悬浮细胞系中苯乙醇甙含量的影响。研究结果表明:MJ和SA能够促进肉苁蓉悬浮细胞系中苯乙醇甙(PeG)和松果菊甙(Echinacoside)的合成,但两者的适用的浓度范围和最佳添加时间存在差异。与未经诱导子处理的细胞培养结果相比,MJ在对数生长初期(培养14d) ,添加浓度为5 μmol L条件下,可使肉苁蓉悬浮细胞系中PeG含量提高2 5 9倍,Echin含量提高3 82倍;而SA在对数生长后期(培养2 8d) ,添加浓度为5 0 μmol L条件下,可使PeG含量提高2 71倍,Echin含量提高3 16倍。     

NO对银杏悬浮细胞生长及黄酮类物质合成的影响

以硝普钠(sodium nitroprusside,SNP)为一氧化氮(NO)的供体,向银杏悬浮细胞培养液中加入不同浓度的SNP,研究外源NO对银杏悬浮细胞生长状况、过氧化氢酶(CAT)活性、苯丙氨酸解氨酶(PAL)活性和黄酮类物质生物合成的影响.结果表明,低浓度SNP有利于银杏悬浮细胞生长,而高浓度SNP可以促进黄酮类物质的合成.银杏悬浮细胞在添加0.5和10 mmol/L SNP的培养基中培养16 d时,细胞干重分别为对照组的134%和73%;在添加10 mmol/L SNP的培养基中培养20 d时,细胞中黄酮类物质的含量为对照组的136%.同时,10 mmol/L SNP促进银杏悬浮细胞PAL和CAT活性显著升高.NO专一性淬灭剂c-PITO(carboxyl phenyltetramethylimidazoleoxide)抑制SNP对银杏悬浮细胞生长、CAT活性、PAL活性和黄酮类物质含量的促进作用,说明SNP是通过其分解产物NO影响细胞生长和黄酮类物质的合成.根据这些结果推测,NO可能通过触发银杏悬浮细胞的防卫反应,激活了细胞中黄酮类物质的生物合成途径.     

NO对金丝桃悬浮细胞生长及金丝桃素生物合成的促进作用研究

一氧化氮 (NO)是近年来发现的一种新型植物信号分子。以硝普钠 (Sodiumnitroprusside ,SNP)为一氧化氮 (NO)的供体 ,研究外源NO对金丝桃悬浮细胞生长及金丝桃素生物合成的影响。试验结果表明 ,金丝桃悬浮细胞在含 0 5和 15 0mmol LSNP的培养基中培养 2 0d后 ,细胞的干重分别为对照组的 140%和50% ;细胞中金丝桃素的含量分别为对照组的 98%和210%。试验结果表明 ,低浓度SNP处理有利于金丝桃悬浮细胞生长 ,而高浓度SNP可以促进金丝桃素的合成。在细胞培养初期 (0d)加入 0.5mmol LSNP并在指数生长后期 (14d)加入15.0mmol LSNP的金丝桃悬浮细胞在培养 2.5d后 ,细胞的干重和金丝桃素的含量分别为对照组的1.4和1.8倍 ,金丝桃素的产量达15.2mg/L ,比对照高3.2倍。SNP对金丝桃悬浮细胞生长及金丝桃素含量的影响可以被NO专一性淬灭剂CPITO(2-4-carboxyphenyl-4 ,4 ,5 ,5-tetramethylimidazoline-1-oxyl-3-oxide)所抑制,说明SNP是通过其分解产物NO影响细胞生长和金丝桃素的合成。试验结果同时表明,在15.0mmol/L的SNP处理下,金丝桃悬浮细胞中的苯丙氨酸解氨酶(PAL)的活性显著升高,推测NO可能通过触发金丝桃悬浮细胞的防卫反应,激活了细胞中金丝桃素的生物合成途径。     

野黄芩苷对内毒素抑制人牙周膜细胞碱性磷酸酶活性的影响

目的观察野黄芩苷对内毒素(LPS)抑制人牙周膜细胞的碱性磷酸酶活性的影响。方法原代培养人牙周膜细胞,采用酶动力学方法观察野黄芩苷对LPS抑制人牙周-膜细胞碱性磷酸酶活性的影响。结果100μg/mL LPS可显著抑制体外培养的人牙周膜细胞碱性磷酸酶活性。加入0.001-10μg/ml野黄芩苷干预后,对LPS抑制碱性磷酸酶活性有一定的拮抗作用,在1μg/ml时达到高峰。结果 野黄芩苷可能通过拮抗LPS抑制牙周膜细胞碱性磷酸酶的活性,促使牙周膜细胞向成骨细胞分化而利于牙周组织再生修复。     

黄芩苷对结肠癌细胞凋亡的影响及作用机制

黄芩苷在多种肿瘤中具有较高的抗肿瘤活性,然而,其在结肠癌中的抗肿瘤作用尚不清楚。本研究考察了黄芩苷对人结肠癌细胞系RKO的增殖和凋亡的影响,并探讨了其相关作用机制。研究发现,黄芩苷可按照剂量依赖性方式和时间依赖性方式抑制结肠癌细胞增殖及集落形成能力。流式细胞术显示,与对照细胞相比,黄芩苷处理后的RKO细胞的凋亡率显著增加(5.6%vs 33.6%)。RT-PCR和Western blotting检测显示,黄芩苷处理可显著增加结肠癌RKO细胞中DKK1 (Wnt信号通路的关键负调节因子) mRNA和蛋白表达水平。相反,黄芩苷处理则显著抑制结肠癌RKO细胞中c-Myc和β-catenin (DKK1的下游靶基因)的m RNA和蛋白表达。敲低DKK1后明显阻断了黄芩苷诱导的结肠癌细胞中DKK1蛋白的上调。此外,敲低DKK1逆转了黄芩苷对其下游基因c-Myc和β-catenin的抑制作用。黄芩苷处理后,结肠癌细胞中miR-217的表达显著下调。RT-PCR和Western blotting结果显示,下调miR-217显著促进DKK1的mRNA和蛋白表达。综上所述,本研究表明黄芩苷可通过抑制结肠癌细胞增殖并诱导细胞凋亡来发挥其抗肿瘤作用,黄芩苷通过激活结肠癌细胞中DKK1来抑制Wnt信号通路。此外,黄芩苷通过下调结肠癌细胞中miR-217的表达来上调DKK1的表达,从而起到抗癌作用。     

酶解法提取黄芩中黄芩素的工艺研究

本文对黄芩药材中黄芩苷的酶解条件和黄芩素的提取工艺进行研究,以样品中黄芩素的含量为指标,采用正交实验对影响酶解的温度、时间和药材粒度进行考察;以黄芩素的提取率为指标,经正交实验优选乙醇浓度、用量、回流次数和时间。得出如下较理想的酶解条件和提取工艺:黄芩药材粉碎至10~20目,加水6倍,于60℃保温10 h;酶解后的黄芩药材加8倍量70%乙醇回流提取2次,每次1 h。黄芩素的提取率大于70%,工艺稳定可行。     

L-丙氨酸对海南粗榧悬浮细胞合成三尖杉酯类碱的影响

该研究在海南粗榧悬浮细胞培养的不同阶段(5、10、15、20 d),分别添加不同剂量的L-丙氨酸(10、30、50、100 mg·L~(-1)),测定细胞生长、细胞活力及产物含量,确定L-丙氨酸最佳的添加时间及添加剂量。结果表明:添加L-丙氨酸对细胞生长和细胞活力均有抑制作用;在海南粗榧悬浮培养第15天、添加30 mg·L~(-1)L-丙氨酸时,产物含量最高(4.853 6 mg·L~(-1)),是对照(2.853 8 mg·L~(-1))的1.7倍。同时,为了探讨添加L-丙氨酸对海南粗榧悬浮细胞糖代谢的影响,对培养基糖耗程度、细胞内糖酵解途径(glycolytic pathway,EMP途径)关键酶丙酮酸激酶(Pyruvate kinase,PK)活力、磷酸戊糖途径(hexose monophosphate pathway,HMP途径)关键酶6-磷酸葡萄糖脱氢酶(glucose 6-phosphate dehydrogenase,G6PDH)活力进行了测定,结果显示添加L-丙氨酸后,植物细胞培养液中总耗糖速度与对照相比无明显差异,丙酮酸激酶(PK)活力与对照(25.37 U·g~(-1))相比下降了29.10%,G6DPH活力是对照组(53.49 U·g~(-1))的1.33倍。以上结果说明,糖代谢途径中碳通量在一定程度上由EMP途径转向了HMP途径,三尖杉酯类碱合成的前体物PEP积累,E4P合成量增加,均有利于产物三尖杉酯类碱含量的增加。     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

国内外蝗害治理技术现状与展望

本文首先概述了国内外蝗虫发生与为害的态势,总结了现阶段我国蝗虫发生与为害的主要特点:即农田飞蝗暴发频繁而且严重,草原土蝗的发生时常造成严重的经济损失,而且侵入城市干扰市民生活,我国与周边国家之间蝗虫过境迁移频繁,使用化学农药污染环境和农产品;分析了国内外蝗虫防治对策与技术的发展现状,重点介绍了应急防治和可持续治理对策、...     

Coiled-coil nanomechanics and uncoiling and unfolding of the superhelix and alpha-helices of myosin

The nanomechanical properties of the coiled-coils of myosin are fundamentally important in understanding muscle assembly and contraction. Force spectra of single molecules of double-headed myosin, single-headed myosin, and coiled-coil tail fragments were acquired with an atomic force microscope and displayed characteristic triphasic force-distance responses to stretch: a rise phase (R) and a plateau phase (P) and an exponential phase (E). The R and P phases arise mainly from the stretching of the coiled-coils, with the hinge region being the main contributor to the rise phase at low force. Only the E phase was analyzable by the worm-like chain model of polymer elasticity. Restrained molecular mechanics simulations on an existing x-ray structure of scallop S2 yielded force spectra with either two or three phases, depending on the mode of stretch. It revealed that coiled-coil chains separate completely near the end of the P phase and the stretching of the unfolded chains gives rise to the E phase. Extensive conformational searching yielded a P phase force near 40 pN that agreed well with the experimental value. We suggest that the flexible and elastic S2 region, particularly the hinge region, may undergo force-induced unfolding and extend reversibly during actomyosin powerstroke.     

放牧对牧草光合作用、呼吸作用和氮、碳吸收与转运的影响

研究放牧对草地植物生理活动的影响,对于揭示草地放牧演替的生理机制有重要意义.大量研究表明,家畜放牧对牧草光合作用、呼吸作用以及C和N吸收与转运的影响,可以分为生理伤害和生理恢复2个阶段.放牧通过改变草地冠层结构影响牧草光合作用,净光合作用速率短期内迅速下降,随着叶面积指数增加又逐渐上升,呼吸作用有相似的变化趋势.牧草放牧后再生长所需的C和N最初主要来自根系和留茬中的贮藏物质,此后随着牧草生长恢复逐渐由同化作用供给,C代谢与土壤N水平负相关.放牧后牧草生理活动变化与牧草遗传特性、种间竞争、家畜放牧特征、非生物环境等因素密切相关.     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000     

Synthesis of hapten and conjugates of coumestrol and development of immunoassay

3-O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized with potassium carbonate. 3-O-Ethoxycarbonylmethylcoumestrol was separated by column chromatography and finally was hydrolyzed with formic acid. 1H and 13C NMR data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective, as 3-O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The hapten was then conjugated to bovine serum albumin and used for immunization of rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal antiserum and a 125I-labeled hapten-tyrosine methyl ester conjugate as the radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept: 140 pg per tube, working range: 20-4000 pg per tube. The cross-reactivity of a panel isoflavonoid and lignan phytoestrogens was either negligible (e.g. formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major immunoreactive peak in HPLC fractions from an alfalfa extract had the same retention time as coumestrol standard and represented 94.8% of the signal. The remaining 5.2% of immunoreactivity was distributed between five minor peaks. We conclude that after the validation for particular matrices, the method will be a useful tool for analysis of coumestrol, especially in low volume and low concentration samples.     

白术同源四倍体的诱导和鉴定及其与二倍体过氧化物酶的比较

以白术（Atractylodes macrooephala Koidz.）二倍体组培苗为材料,对其四倍体诱导方法进行研究,共获得45个白术同源四倍体株系,为优良株系的选育提供了材料。此外,还分析比较了其中8个白术四倍体株系与二倍体的过氧化物酶同工酶（POD）的酶谱差异,发现四倍体各株系过氧化物酶同工酶谱比二倍体的均多了Rf0.310的谱带,且总过氧化物酶比活力也发生了很大改变,对探讨白术四倍体优良株系的生理生化机理具有一定的参考价值。     

Morphology and putative function of the colon and cloaca of marine and freshwater snakes

Among tetrapods, evidence for postrenal modification of the urine by the distal digestive tract (including the colon and cloaca) is highly variable. Birds and bladderless reptiles are of interest because the colon and cloaca represent the only sites from which water and ions can be reclaimed from the urine secreted by the kidney. For animals occupying desiccating environments (e.g., deserts and marine environments), postrenal modification of the urine may directly contribute to the maintenance of hypo‐osmotic body fluids. We compared the morphology and distribution of key proteins in the colon, cloaca, and urogenital ducts of watersnakes from marine (Nerodia clarkii clarkii) and freshwater (Nerodia fasciata) habitats. Specifically, we examined the epithelia of each tissue for evidence of mucus production by examining the distribution of mucopolysaccharides, and for evidence of water/ion regulation by examining the distribution of Na⁺/K⁺‐ATPase (NKA), Na⁺/K⁺/Cl^? cotransporter (NKCC), and aquaporin 3 (AQP3). NKCC localized to the basolateral epithelium of the colon, urodeal sphincter, and proctodeum, consistent with a role in secretion of Na⁺, Cl^?, and K⁺ from the tissue, but NKA was not detected in the colon or any compartment of the cloaca. Interestingly, NKA was detected in the basolateral epithelium of the ureters, suggesting the urothelium may play a role in active ion transport. AQP3 was detected in the ureters and coprodeal complex, consistent with a role in urinary and fecal dehydration or, potentially, in the production of the watery component of the mucus secreted by the coprodeal complex. Since no differences in general cloacal morphology, production of mucus, or the distribution of ion transporters/water channels were detected between the two species, cloacal osmoregulation may either be regulated by proteins not examined in this study or may not be responsible for the differential success of N. c. clarkii and N. fasciata in marine habitats. J. Morphol. 2011. © 2011 Wiley Periodicals, Inc.