P、Zn在小麦细胞内的积累、分布及交互作用的研究

报道了小麦无性系细胞在含有3个P水平(0.5、1.5、3.5mmol·L^-1)和2个Zn水平(0、60μmol·L^-1)的M.S培养液中,细胞对P、Zn的积累、分布特性及交互作用的研究.P和Zn在细胞内的积累分别随外施P、Zn浓 度的提高而提高.与缺Zn比较,加Zn处理能抑制培养7d细胞中P的含量.营养液中0.5～1.5mmol·L^-1 P能促进细胞的含Zn量,但高P处理即抑制细胞的含Zn量.小麦细胞壁固定了细胞83.9%～88.3%的P,而外施P浓度越高,则细胞壁中P分配的比例越大.在供Zn条件下,细胞壁截留了细胞中一半以上的Zn(52.0～60.5%).小麦液泡中P和Zn的含量较少,分别为2.2%～3.8%和10.6%～30%.     

营养胁迫对雨生红球藻虾青素累积的影响

通过改变营养条件可诱导雨生红球藻积累虾青素.氮限制实验表明,色素的累积速率与原初氮浓度成反比,也与细胞分裂速率负相关,当BBM培养基中的NaNO3浓度减半时(0.13g@L-1),对细胞增殖及色素累积相对都有利.在高光强下,进一步进行氮、磷饥饿,红球藻细胞分裂明显受抑,但色素的累积作用增强,培养9d,细胞内次生类胡萝卜素的含量分别比对照组提高141.0%和60.5%,色素的累积高峰也比对照组提前2-4d.提高NaCl浓度至0.8%时的盐胁迫,不能诱导虾青素的形成.实验结果还表明,色素的累积与厚壁孢子的形成并不完全相关,游动细胞也能大量积累红色色素.     

代谢工程在微生物法产辅酶Q_(10)中的应用

辅酶Q10作为细胞呼吸链上的重要组成部分在电子传递过程中发挥着重要的作用。辅酶Q10的抗氧化、抗衰老功能使其广泛应用于医药、食品和化妆品等行业。CoQ10市场需求不断增加,这使得大规模提高CoQ10工业化生产的产量显得十分必要。目前,主要依靠从自然界中筛选到的各种微生物作为生产菌种发酵生产CoQ10,但这些原始生产菌种由于产量低、营养要求高等各种原因很难实现大规模发酵生产。随着对CoQ10生物合成途径以及代谢调控机制的了解清楚,通过对易于商业化生产的优良宿主细胞(如大肠杆菌)进行代谢工程的改造,有助于促进代谢工程菌的CoQ10工业化生产发展。     

米酒乳杆菌胞外多糖BXR-5-3生物合成条件的研究

主要针对来源于内蒙古锡林郭勒牧区马奶酒中的米酒乳杆菌BXR-5-3进行胞外多糖生物合成能力进行研究,分别改变基础培养基的碳源、氮源以及发酵温度、时间、pH等条件,探讨其对BXR-5-3胞外多糖生物合成能力的影响.优化的培养基MRSB的组成为蛋白胨1.0%,胰蛋白胨1.0%,葡萄糖1.5%,乳糖1.5%,K2HPO4 0.2%,MnSO4*4H2O 0.02%,MgSO4*7H2O 0.02%,醋酸钠0.5%,酵母粉0.5%,Tween80 1 mL/L,确定其胞外多糖的最佳生物合成条件为初始pH 6.5,发酵温度 35 ℃,发酵时间 26 h.优化的条件显著提高了EPS的合成量.     

温度胁迫对白桦悬浮细胞中三萜积累及防御酶活性的影响

以白桦悬浮培养细胞为试材,悬浮培养第7d进行高温(50℃热处理1h)和低温(4℃冷胁迫3h)胁迫处理,此后不同时间(0h～7d)收获白桦细胞,研究细胞干重、三萜积累及防御酶活性的变化。研究结果表明,高温和低温胁迫后均表现在24h～3d收获细胞显著促进了白桦总三萜的合成,且以高温处理效果最佳,48h收获细胞时分别比对照(25℃)和冷胁迫处理提高36.4%和12.87%;而在冷胁迫处理后4d～7d表现显著刺激了总三萜的合成,其中在第7d总三萜的含量比对照增加了22.1%,且两温度胁迫处理后15h～4d显著促进了齐墩果酸三萜的积累,24h收获时,热处理和冷胁迫均比对照提高38.1%和39.65%。两温度胁迫后不同程度促进了超氧化物岐化酶(SOD)、过氧化氢酶(CAT)、苯丙氨酸裂解酶(PAL)、过氧化物酶(POD)活性提高及丙二醛的积累,但各酶活性出现高峰及变化规律存在差异,明确各酶相互协调、补充是保证细胞适应温度胁迫,减轻自由基伤害,并促进次生产物合成的原因。     

辅酶Q10的生物合成及其高表达研究进展

辅酶Q10(CoQ10)不仅是呼吸链上的电子传递体,同时也具有抗氧化功能。目前全球市场上的CoQ10正处于一种供不应求的状态。我们简要论述了CoQ10的结构、性质、功能及其生物合成过程,同时概括总结了现阶段为提高CoQ10产量而采用的新型技术手段。     

几种营养物质对烟草细胞生长和CoQ_(10)含量的影响

研究了蔗糖、KH2PO4、NH 4/NO3比对烟草细胞生长和CoQ10含量的影响,结果表明,当蔗糖浓度为30g/L时,CoQ10总量最高,此时细胞产量、CoQ10含量和总量分别为198g/L、4147μg/g(dwt)、8212μg/L。在上述蔗糖浓度下,当KH2PO4起始浓度为340mg/L、NH 4/NO-3为12时,细胞产量、CoQ10含量和总量分别最高,高于此比例时有利于CoQ10形成,但不利于细胞生长。     

荣莉酸甲酯对丹参培养细胞中迷迭香酸生物合成及相关酶活性的影响

茉莉酸甲酯是植物细胞响应外界刺激产生的重要信号分子,与植物次生代谢物的生物合成有关。本研究考察了茉莉酸甲酯（methyl jasmonate,MeJA）对丹参培养细胞中迷迭香酸（rosmarinic acid,RA）生物合成的影响。结果显示,诱导24h后可显著提高丹参愈伤细胞中RA的积累量及其相关酶（PAL、TAT）的活性,在48h时RA积累量和酶活性达到最大。布洛芬（IBu）处理可抑制MeJA对RA积累量和相关酶活性的促进作用,外源施加MeJA可部分解除IBU对RA合成及其相关酶活性的抑制作用。说明MeJA可以显著促进丹参培养细胞中RA的生物合成,IBU抑制了MeJA合成、PAL和TAT活性,从而导致了RA合成受阻。     

自养和兼养条件下蛋白核小球藻昼夜节律的响应

【目的】研究自养和兼养两种培养方式对蛋白核小球藻(Chlorella pyrenoidosa)生长、细胞分裂和生化组分积累的影响,探讨人工培养蛋白核小球藻的昼夜节律响应机制和优化技术。【方法】小球藻自养培养采用BG11培养基,兼养培养基在BG11培养基中添加4种不同浓度(1、5、10、20 g/L)的葡萄糖,培养周期为10 d。血球板计数法测定藻细胞浓度,干重法测定藻细胞生物量。显微观察藻细胞大小和分裂情况。脂染色法测定小球藻总脂的含量,藻细胞的叶绿素、蛋白和淀粉分别采用甲醇、氢氧化钠、硝酸钙浸提后通过紫外分光光度法定量测定。【结果】葡萄糖兼养培养对蛋白核小球藻具有显著的促生长效应,最适浓度为10 g/L。10 d收获时,兼养组(10 g/L葡萄糖)藻细胞浓度和干重分别是自养组的2.57倍和6.73倍。分析一昼夜中的藻细胞增殖规律可知,第2天和第5天时自养组中增殖的新生子细胞约有76.00%在黑暗期分裂产生,而兼养组中第2天和第5天光照期的新细胞增殖量占比分别达到40.90%和67.50%。一昼夜内藻细胞大小的迁移动态监测表明,第2天自养组藻细胞的体积变化静息期为8 h,兼养组只有4 h;第5天两组藻细胞大小迁移动态的昼夜节律明显,但兼养组黑暗结束后较大细胞(D6μm)占比显著高于自养组。第8天时,兼养组藻细胞已处于稳定期,总脂和蛋白含量均显著高于自养组,藻细胞总脂和色素含量在一昼夜中相对稳定,但蛋白和淀粉含量分别在光照8 h和12 h左右达到峰值。从第2天开始,对兼养组细胞每天进行2 h光延长,收获时藻细胞浓度和干重分别比对照组提高13%和11%。【结论】葡萄糖兼养培养能大幅提高蛋白核小球藻的生物量。蛋白核小球藻生长增殖与生化组分积累均受昼夜节律调控,自养条件下藻细胞以光照期生长黑暗期增殖为主。兼养培养提高藻细胞生物量的机制在于缩短藻细胞生长静息期,在昼夜节律中加速藻细胞生长并显著提高通过细胞周期检查点的细胞比例,光照期效应尤其明显。藻细胞蛋白和淀粉含量昼夜节律明显,最佳收获时间分别在光照8 h和12 h后。     

植物细胞多倍体诱导实验方法的改进

从植物多倍体诱导实验材料处理的方法上,介绍了改进植物多倍体诱导实验的技术,提高了多倍体细胞的分裂指数。实验用0．1％和0．01％浓度的秋水仙素溶液分别处理大蒜根尖24h、36h、48h、72h,进行多倍体诱导,再在清水中分别培养0h、12h、24h、36h,结果表明经清水培养的多倍体细胞分裂指数高于对照：0．01％浓度的秋水仙素处理的多倍体细胞分裂指数高于0．1％多倍体细胞分裂指数。     

几种营养物质对烟草细胞生长和CoQ10含量的影响

研究了蔗糖、KH₂PO₄、NH₄NO₃比对烟草细胞生长和CoQ₁₀量的影响,结果表明,当蔗糖浓度为30g/L时,CoQ₁₀总量最高,此时细胞产量、CoQ₁₀含量和总量分别为19.8g/L、414.7μg/g(dwt)、8212μg/L。在上述蔗糖浓度下,当KH₂PO₄起始浓度为340mg/L、NH₄NO₃为12时,细胞产量、CoQ₁₀含量和总量分别最高,高于此比例时有利于CoQ₁₀形成 ,但不利于细胞生长。     

辅酶Q_(10)产生菌发酵条件的优化

对辅酶Q10生产菌株鞘氨醇单胞菌YZ0803的发酵条件进行优化,确定发酵时间为90 h,250 mL摇瓶装液量为30 mL。培养基组成(质量分数,下同):葡萄糖1.5%,淀粉2.5%,黄豆饼粉2.5%,(NH4)2SO40.5%,NaCl0.03%,K2HPO40.02%,MgSO40.005%。优化后的辅酶Q10产量达到192 mg/L,比采用基础培养基的产量(138mg/L)提高了39.13%。     

Effect of coenzyme Q10 and vitamin E on brain energy metabolism in the animal model of Huntington's disease

The neuropathological and clinical symptoms of Huntington's disease (HD) can be simulated in animal model with systemic administration of 3-nitropropionic acid (3-NP). Energy defects in HD could be ameliorated by administration of coenzyme Q(10) (CoQ(10)), creatine, or nicotinamid. We studied the activity of creatine kinase (CK) and the function of mitochondrial respiratory chain in the brain of aged rats administered with 3-NP with and without previous application of antioxidants CoQ(10)+vitamin E. We used dynamic and steady-state methods of in vivo phosphorus magnetic resonance spectroscopy ((31)P MRS) for determination of the pseudo-first order rate constant (k(for)) of the forward CK reaction, the phosphocreatine (PCr) to adenosinetriphosphate (ATP) ratio, intracellular pH(i) and Mg(i)(2+) content in the brain. The respiratory chain function of isolated mitochondria was assessed polarographically; the concentration of CoQ(10) and alpha-tocopherol by HPLC. We found significant elevation of k(for) in brains of 3-NP rats, reflecting increased rate of CK reaction in cytosol. The function of respiratory chain in the presence of succinate was severely diminished. The activity of cytochromeoxidase and mitochondrial concentration of CoQ(10) was unaltered; tissue content of CoQ(10) was decreased in 3-NP rats. Antioxidants CoQ(10)+vitamin E prevented increase of k(for) and the decrease of CoQ(10) content in brain tissue, but were ineffective to prevent the decline of respiratory chain function. We suppose that increased activity of CK system could be compensatory to decreased mitochondrial ATP production, and CoQ(10)+vitamin E could prevent the increase of k(for) after 3-NP treatment likely by activity of CoQ(10) outside the mitochondria. Results of our experiments contributed to elucidation of mechanism of beneficial effect of CoQ(10) administration in HD and showed that the rate constant of CK is a sensitive indicator of brain energy disorder reflecting therapeutic effect of drugs that could be used as a new in vivo biomarker of neurodegenerative diseases.     

Effects of strong static magnetic fields used in magnetic resonance imaging on insulin-secreting cells

The magnetic flux density of MRI for clinical diagnosis has been steadily increasing. However, there remains very little biological data regarding the effect of strong static magnetic fields (SMFs) on human health. To evaluate the effects of strong SMFs on biological systems, we cultured insulin-secreting cells under exposure to sham and SMF conditions (3-10 T of magnetic flux density, and 0-41.7 T/m of magnetic field gradient) for 0.5 or 1 h, and analyzed insulin secretion, mRNA expression, glucose-stimulated insulin secretion, insulin content, cell proliferation and cell number. Exposure to SMF with a high magnetic field gradient for 1 h significantly increased insulin secretion and insulin 1 mRNA expression. Exposure to SMF with a high magnetic flux density for 0.5 h significantly enhanced responsiveness to glucose stimulation. Exposure to SMF did not affect the insulin content, cell proliferation or cell number. Our results suggested that MRI systems with a higher magnetic flux density might not cause cell proliferative or functional damages on insulin-secreting cells, and that SMF with a high magnetic field gradient might be used clinically after thorough in vivo investigations are conducted.     

Coenzyme Q10 concentration in plasma and blood cells: what about diurnal changes?

Coenzyme Q10 (CoQ10) is used by the body as an endogenous antioxidant and performs essential functions in mitochondrial energy production. The value of CoQ10 as a biomarker for oxidative stress will be severely restricted if there are huge individual daily variations in its concentration. For analysis of diurnal changes in CoQ10 plasma and blood cell concentrations, blood was collected from nine healthy adults (at two- or three-hour intervals for plasma, and three times a day for blood cells). CoQ10 was analysed by HPLC using electrochemical detection and internal standardisation. Daytime variations in CoQ10 concentration in plasma are maintained within narrow limits and show no statistically significant difference (Kruskal-Wallis). However, a drop at night-time (0300 h) is accompanied by a drop in total cholesterol concentration. Remarkable inter-individual differences in blood cell (erythrocytes, platelets, white blood cells) content of CoQ10 occur with only slight intra-individual daily variations. A correlation (Spearman) is found for cholesterol and CoQ10 content in circulation which may be explained by the carrier capacity of blood for this highly lipophilic substance. Moreover, a diurnal change in hepatic HMG-CoA reductase activity may suggest a common diurnal regulation of synthesis of both CoQ10 and cholesterol.     

Coenzyme Q10 reduces ethanol-induced apoptosis in corneal fibroblasts

Dilute ethanol (EtOH) is a widely used agent to remove the corneal epithelium during the modern refractive surgery. The application of EtOH may cause the underlying corneal fibroblasts to undergo apoptosis. This study was designed to investigate the protective effect and potential mechanism of the respiratory chain coenzyme Q(10) (CoQ(10)), an electron transporter of the mitochondrial respiratory chain and a ubiquitous free radical scavenger, against EtOH-induced apoptosis of corneal fibroblasts. Corneal fibroblasts were pretreated with CoQ(10) (10 μM) for 2 h, followed by exposure to different concentrations of EtOH (0.4, 2, 4, and 20%) for 20 s. After indicated incubation period (2-12 h), MTT assay was used to examine cell viability. Treated cells were further assessed by flow cytometry to identify apoptosis. Reactive oxygen species (ROS) and the change in mitochondrial membrane potential were assessed using dichlorodihydrofluorescein diacetate/2',7'-dichlorofluorescein (DCFH-DA/DCF) assays and flow-cytometric analysis of JC-1 staining, respectively. The activity and expression of caspases 2, 3, 8, and 9 were evaluated with a colorimetric assay and western blot analysis. We found that EtOH treatment significantly decreased the viability of corneal fibroblasts characterized by a higher percentage of apoptotic cells. CoQ(10) could antagonize the apoptosis inducing effect of EtOH. The inhibition of cell apoptosis by CoQ(10) was significant at 8 and 12 h after EtOH exposure. In EtOH-exposed corneal fibroblasts, CoQ(10) pretreatment significantly reduced mitochondrial depolarization and ROS production at 30, 60, 90, and 120 min and inhibited the activation and expression of caspases 2 and 3 at 2 h after EtOH exposure. In summary, pretreatment with CoQ(10) can inhibit mitochondrial depolarization, caspase activation, and cell apoptosis. These findings support the proposition that CoQ(10) plays an antiapoptotic role in corneal fibroblasts after ethanol exposure.     

Coenzyme Q10 production in recombinant Escherichia coli strains engineered with a heterologous decaprenyl diphosphate synthase gene and foreign mevalonate pathway

In the present work, Escherichia coli DH5alpha was metabolically engineered for CoQ(10) production by the introduction of decaprenyl diphosphate synthase gene (ddsA) from Agrobacterium tumefaciens. Grown in 2YTG medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl, and 0.5% glycerol) with an initial pH of 7, the recombinant E. coli was capable of CoQ(10) production up to 470 microg/gDCW (dry cell weight). This value could be further elevated to 900 microg/gDCW simply by increasing the initial culture pH from 7 to 9. Supplementation of 4-hydroxy benzoate did not improve the productivity any further. However, engineering of a lower mevalonate semi-pathway so as to increase the isopentenyl diphosphate (IPP) supply of the recombinant strain using exogenous mevalonate efficiently increased the CoQ(10) production. Lower mevalonate semi-pathways of Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis, and Saccharomyces cerevisiae were tested. Among these, the pathway of Streptococcus pneumoniae proved to be superior, yielding CoQ(10) production of 2,700+/-115 microg/gDCW when supplemented with exogenous mevalonate of 3 mM. In order to construct a complete mevalonate pathway, the upper semi-pathway of the same bacterium, Streptococcus pneumoniae, was recruited. In a recombinant E. coli DH5alpha harboring three plasmids encoding for upper and lower mevalonate semi-pathways as well as DdsA enzyme, the heterologous mevalonate pathway could convert endogenous acetyl-CoA to IPP, resulting in CoQ(10) production of up to 2,428+/-75 microg/gDCW, without mevalonate supplementation. In contrast, a whole mevalonate pathway constructed in a single operon was found to be less efficient. However, it provided CoQ(10) production of up to 1,706+/-86 microg/gDCW, which was roughly 1.9 times higher than that obtained by ddsA alone.     

Coenzyme Q(10) in submicron-sized dispersion improves development, hatching, cell proliferation, and adenosine triphosphate content of in vitro-produced bovine embryos.

Coenzyme Q(10) (CoQ(10)) is an essential component of the plasma membrane ion transporter (PMIT) system and of the electron transport chain in the inner mitochondrial membrane. Because of its intrinsic functions in cell growth and energy metabolism (ATP synthesis), and its protective effects against oxidative stress, CoQ(10) is a good candidate for supporting growth of cells in culture. However, because of its quinone structure, CoQ(10) is extremely lipophilic and practically insoluble in water. We used a specific technology to prepare a submicron-sized dispersion of CoQ(10), inhibiting re-crystallization by a stabilizer. This dispersion, which exhibits a very large specific surface area for drug dissolution, was tested as a supplement for the in vitro culture of bovine embryos in a chemically defined system. The rate of early cleavage of embryos (5- to 8-cell stages) was evaluated 66 h postinsemination (hpi) and was highest in medium supplemented with 30 or 100 microM CoQ(10) (66.5 +/- 0.8% and 68.7 +/- 1.1%, respectively) and lowest in 10 microM CoQ(10) (55.3 +/- 0.8%). The proportions of oocytes developing to blastocysts by 186 hpi were 19.0 +/- 0.6% and 25.2 +/- 0.3% in medium supplemented with 10 microM and 30 microM CoQ(10), respectively, and were significantly (p < 0.001) higher than those obtained with the equivalent amounts of stabilizer (9.9 +/- 0.4% and 11.3 +/- 0.4%). In the presence of 30 microM CoQ(10), significantly (p < 0.001) more blastocysts hatched by 210 hpi than in the equivalent amount of stabilizer (31.8 +/- 1.3 vs. 8.4 +/- 2.2). Expanded blastocysts produced in the presence of 30 microM CoQ(10) had significantly (p < 0.01) more inner cell mass cells and trophectoderm cells, and a significantly (p < 0.001) increased ATP content as compared to expanded blastocysts produced in the presence of the corresponding amount of stabilizer. Our results show that noncrystalline CoQ(10) in submicron-sized dispersion supports the development and viability of bovine embryos produced in a chemically defined culture system.     

激光和磁场对滇紫草愈伤组织色素含量的影响

用He-Ne激光和一定强度的磁场处理滇紫草愈伤组织,发现2~3h激光辐照可提高色素含量;而1.0T磁场能促进细胞生长和色素形成。硅胶薄层层析比较两种物理因子对紫草色素成分无显著影响。     

Coenzyme Q(10) supplementation inhibits aortic lipid oxidation but fails to attenuate intimal thickening in balloon-injured New Zealand white rabbits

Oxidized lipoproteins are implicated in atherosclerosis, and some antioxidants attenuate the disease in animals. Coenzyme Q(10) (CoQ(10)) in its reduced form, ubiquinol-10, effectively inhibits lipoprotein oxidation in vitro and in vivo; CoQ(10) supplements also inhibit atherosclerosis in apolipoprotein E gene knockout (apoE-/-) mice. Here we tested the effect of dietary CoQ(10) supplements on intimal proliferation and lipoprotein lipid oxidation in balloon-injured, hypercholesterolemic rabbits. Compared to nonsupplemented chow, CoQ(10) supplementation (0.5% and 1.0%, w/w) significantly increased the plasma concentration of CoQ(10) and the resistance of plasma lipids to ex vivo oxidation. CoQ(10) supplements also increased the content of CoQ(10) in the aorta and liver, but not in the brain, skeletal muscle, kidney, and heart. Surprisingly, CoQ(10) supplementation at 1% increased the aortic concentrations of all lipids, particularly triacylglycerols, although it significantly inhibited the proportion of triacylglycerols present as hydroperoxides by > 80%. The observed increase in vessel wall lipid content was reflected in elevated plasma concentrations of cholesterol, cholesteryl esters and triacylglycerols, and hepatic levels of mRNA for 3-hydroxy-3-methylglutaryl-coenzyme A reductase. CoQ(10) supplements did not attenuate lesion formation, assessed by the intima-to-media ratio of injured aortic vessels. Thus, like in apoE-/- mice, a high dose of supplemented CoQ(10) inhibits lipid oxidation in the artery wall of balloon-injured, hypercholesterolemic rabbits. However, unlike its antiatherosclerosis activity in the mice, CoQ(10) does not inhibit intimal hyperplasia in rabbits, thereby dissociating this disease process from lipid oxidation in the vessel wall.