红豆杉细胞继代培养防褐变措施的研究

本研究针对红豆杉组织培养中经常遇到的褐变问题,主要从继代培养的各个方面进行研究,力求最大限度地避免或减轻褐变。结果表明,较低的PH值,液体悬浮培养,较低浓度的无机盐、适宜浓度的金属离子可以减轻褐变,同时,良好的抗氧化剂吸附剂如活性碳与聚乙烯吡咯烷酮的加入均能明显抑制或减轻褐变。     

红豆杉细胞稳定生产紫杉醇中的同步化协同诱导作用

同步化协同诱导可以稳定提高曼地亚红豆杉细胞培养物中紫杉醇含量。细胞培养周期第8d的低温同步化处理可促使细胞达到最高同步化率（20.4%）,而茉莉酸甲酯（MJ）的协同诱导可提高紫杉醇产量,使紫杉醇产量最高值达到54.7mg·L^-1。在细胞生长周期第8d,未经低温同步化的红豆杉细胞中的关键酶基因DXR、HMGR、GGPPS和DBAT的表达量在MJ诱导24h后均迅速下降,但在低温同步化的细胞中紫杉醇表达量下降缓慢,60h后仍维持较高的水平。低温同步化和MJ协同诱导的红豆杉细胞中,紫杉醇合成关键酶基因高效稳定的表达可能是引起紫杉醇产量稳定提高的原因之一。     

云南红豆杉细胞发酵培养的研究

利用云南红豆杉(Taxus yunnanensis)细胞悬浮培养的最佳培养条件,进行了细胞10L规模的发酵培养研究。对细胞发酵培养过程中的pH值变化、糖利用率以及细胞生长周期等实验参数进行了测定。已进行的3次发酵培养的实验结果表明:培养细胞的生长率已达0.4g/L.d(即12.0g/L),培养细胞中紫杉酵的含量为0.119％(其中培养液中的紫杉醇含量约占42％)。初步建立了优化的云南红豆杉细胞大量培养系统。     

云南红豆杉细胞的悬浮培养

在云南红豆杉细胞悬浮培养中,适宜的培养基为B5,接种量为0．5～0．8g干重细胞／100ml培养基,2,4－D浓度为1．0mg／L;培养细胞的生长周期约30d;培养基中较高浓度的蔗糖（40g／L）可提高紫杉醇含量;添加的椰子汁（CM）、酪蛋白氨基酸（C）和水解乳蛋白（LH）3种有机添加剂均能提高培养细胞中紫杉醇的含量,但只有CM和CA能促进细胞的生长。于B5培养基中添加不同浓度的NH4NO3对培养细胞无明显影响。     

红豆杉细胞两相培养生产紫杉醇的研究

研究了两相培养系统中红豆杉细胞的生长与代谢规律,经４０天培养,细胞生物量为１７．８５ｇ／Ｌ（干重）,紫杉醇产量为３０．１９ｍｇ／Ｌ。     

红豆杉细胞悬浮培养结构化数学模型的探讨

用１０Ｌ机械搅拌式生物反应器悬浮培养红豆杉细胞,得到细胞生长、基质消耗和紫杉醇合成动力学曲线。经过代谢动力学分析建立了结构化数学模型。并将模型值与实验值进行比较,结果表明模型预测值与实验值较吻合。     

红豆杉细胞悬浮培养生产紫杉醇研究进展

介绍了近年来由红豆杉细胞培养生产紫杉醇领域取得的进展,其中特别介绍了由紫杉醇生物合成途径及其代谢酶类的研究发展而来的用基因工程方法改良细胞系,为提高紫杉醇产量而采取添加前体物质,诱导子,抑制子等方面的研究。     

红豆杉悬浮细胞放大培养的细胞生长与紫杉醇合成动力学

研究了在Murashige&skoog s(MS)和 6 2号两种不同的培养基中 ,红豆杉细胞悬浮细胞从摇瓶到 1 0L机械通气搅拌式反应器放大培养过程中细胞生长与紫杉醇合成动力学 .结果表明 :尽管在不同的培养条件下 ,细胞生长曲线均呈现“S”型 .紫杉醇在延迟期与指数生长期中基本上没有积累 ,而且随着培养规模的增大 ,紫杉醇的含量逐渐降低 .进一步对各级放大培养的细胞生长 ,比生长率与胞内外紫杉醇合成量进行分析 ,发现MS利于细胞生长但不利于紫杉醇合成 ,而 6 2号则相反 .根据此文的结果 ,提出了红豆杉细胞培养条件的优化和大规模细胞培养生产紫杉醇应采取的策略     

南方红豆杉细胞悬浮培养条件优化研究

针对红豆杉细胞生长相对缓慢和培养褐化问题,本文通过正交实验和均匀实验方法并采用人工神经网络技术对南方红豆杉细胞悬浮培养条件进行优化,使细胞的生长速率和比生长速率有较大提高,并考察了细胞活性随时间的动态变化.     

中国红豆杉悬浮培养细胞聚集体异质性的初步研究

通过梯度浓度的蔗糖溶液（0－1mol/L）的筛选,分拣出7种不同物理学密度的红豆杉细胞聚集体,并对它们进行了木质素含量及紫杉醇含量测定。结果显示：不同的细胞聚集体在物理学密度、木质素含量、紫杉醇含量、生长速度等方面存在着差异,其中密度最小的细胞聚集体木质素及紫杉醇含量分别是密度最大的细胞聚集体的5倍和8倍,并且在一定密度范围内（大于0.2mol／L蔗糖溶液密度）,细胞聚集体的木质素含量与紫杉醇的含量呈平行关系,表明不同聚集体紫杉醇含量一细胞分化有一定的关系,首次提出了红豆杉悬浮细胞聚集体培养中存在着异质现象,并对其可能机理及意义给予阐述。     

强恒磁场对红豆杉细胞分裂和紫杉醇含量的影响

研究了磁场对红豆杉细胞分裂和紫杉醇含量的影响。结果表明,0.4T磁场处理3,4d对红豆杉细胞活力无显著性影响;各实验组中,预培养3d,磁场处理3d,细胞分裂指数提高了162％;加入诱导子后,紫杉醇含量较对照提高115％。     

Persistence of synchronization of the daily rhythms of body temperature and sleep‐wake in college students

Abstract

Bedtime, risetime, and body temperature of college students exposed to a regular 24‐h life schedule were recorded for 22–25 consecutive days. Average bedtime was 1:37 am, average risetime was 8:53 am, and average temperature acrophase was 5:01 pm. There was no evidence of desynchronization of the body temperature and activity rhythms.     

Further Studies on a Modification of the Gram Stain

In perfecting the modification of the Gram-stain previously proposed, the following points are of interest:

1. Acetone is too strong a decolorizer for Gram-positive organisms and alcohol too weak for Gram-negative organisms. Consequently, it is now recommended that equal parts of acetone (100% c.p.) and ethyl alcohol (95%) be used as a decolorizing agent. The time of application should not ordinarily exceed 10 seconds.

2. Aqueous basic fuchsin (0.1%) serves as a strongly contrasting counterstain. Prolonged application renders Gram-positive organisms doubtful or Gram-negative, while short application renders Gram-negative organisms doubtful or Gram-positive. Twenty (20) seconds is therefore recommended as the time of application of the counterstain.

3. The method here described, with due regard for its limitations, is of value in Gram-staining pure or mixed cultures as well as for organic materials, such as Acidophilus milk, feces, etc., either for research purposes or classroom use. The method is as follows:

Air-dry film and fix with least amount of heat necessary.

Flood with dye for 5 minutes. Previously mix 30 drops of a 1% aqueous solution of crystal violet or methyl violet 6B with 8 drops of a 5% solution of sodium bicarbonate. Allow the mixture to remain for 5 minutes or more.

Flush with iodine solution for 2 minutes. Two grams iodine dissolved in 10 cc. normal sodium hydroxide solution and 90 cc. water added.

Drain without blotting but do not allow film to dry.

Add a mixture of equal parts of acetone and alcohol drop by drop until the drippings are colorless. (10 seconds or less.)

Air-dry slide.

Counterstain for 20 seconds with 0.1% aqueous solution of basic fuchsin.

Wash off excess stain by short exposure to tap water and air-dry. If slide is not clear immersion in xylol is recommended.     

Chemical Studies on Taxus canadensis

A series of new taxanes, 1 – 93 , have been isolated, together with 37 known taxoids including Taxol^® (paclitaxel) and cephalomannine, from the Canadian yew, Taxus canadensis (Taxaceae) in the past 30 years. These new taxoids possess various skeletons containing 5/7/6, 6/10/6, 6/5/5/6, 6/8/6, and 6/12 ring systems and six new taxanes with four novel skeletons, i.e., a taxane with a 6/6/8/6 ring system, a taxane with a [3.3.3] propellane skeleton, three taxanes with [3.3.3] [3.4.5] dipropellane sytems, as well as a novel taxane with a unique 5/5/4/6/6/6 hexacyclic skeleton, containing a unique [3.3.2] propellane, were isolated for the first time from natural sources. It should be emphasized that 13‐acetyl‐9‐dihydrobaccatin III, a very useful starting material for the semisynthesis of Taxol^® and Taxotere^®, represents the most abundant taxane in the needles of this yew tree. These findings establish the above mentioned yew tree as significantly different from the remaining species. On the other hand, some chemical modifications on the taxanes isolated from this plant were carried out.     

Studies on low-level MDR cells

Acquired or spontaneous resistance is a major clinical problem in the treatment of cancer. Low levels of MDR gene expression or P-glycoprotein have been correlated with a high level of drug resistance in vitro and a poor response to chemotherapy in some tumors. A strong correlation between MDR mRNA, P-glycoprotein levels and degree of drug resistance has not been found in several resistant model tumor cell lines. In some cell lines at low and high level of resistance different mechanisms seem to be involved.     

Modeling of the influence of gap-junction coupling on synchronization of central and peripheral sinoatrial node cells

Mathematical modeling of the electric activity of pairs of true (central) and latent (peripheral) sinoatrial pacemaker cells coupled through gap junctions revealed that attenuation of coupling conductance increases the beat phase shift; below a critical conductance there is no synchronization. The phase difference also depends on the type of interacting cells, being maximal for a “central”-“peripheral” cell pair.     

Biosynthesis of copper nanoparticles and its effect on actively dividing cells of mitosis in Allium cepa

Nanobiotechnological application of copper nanoparticles has paved the way for advancement in agriculture owing to its bactericidal and fungicidal activities. Recently, researchers have focussed on bioinspired synthesis of copper nanoparticles as a viable alternative to existing physicochemical techniques. For the commercialization of nanocopper, the toxicity evaluation is a major issue. In this context, Citrus medica (L.) fruit extract‐mediated copper nanoparticles were synthesized and its different concentrations (10, 20, 40, 60, 80, and 100 µg mL^?1) were evaluated for its effect on actively dividing cells of Allium cepa. The study clearly revealed that copper nanoparticles increased mitotic index up to the concentration of 20 µg mL^?1. In addition, a gradual decline in mitotic index and increase in abnormality index was observed as the concentration of copper nanoparticles and treatment duration were increased. Aberrations in chromosomal behavior such as sticky and disturbed chromosomes in metaphase and anaphase, c‐metaphase, bridges, laggard, disturbed telophase, and vacuolated nucleus were also observed. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:557–565, 2015     

DNA endoreduplication in maize endosperm cells: the effect of exposure to short-term high temperature

DNA endoreduplication in Zea mays L. (cv. A619 × W64A) endosperm peaks between 16 and 18 d after pollination (DAP). The physiological function of DNA endoreduplication is not known but it is believed to be important in maize kernel development. In the present study, we investigated how 2, 4 or 6 d of high temperature (35 °C) affected DNA endoreduplication and maize kernel development in comparison with control kernels grown at 25 °C. Data were collected on fresh weight (FW), nuclei number, mitotic index, and DNA endoreduplication. Maize endosperm FW and nuclei number were reduced by exposure to 4 or 6 d of high temperature. At 18 DAP, the 2 d high temperature treatment (HTT) caused a reduction in FW and nuclei number, but had no effect on DNA endoreduplication and average DNA content per endosperm. However, when the exposure to high temperature was increased to 4 or 6 d, FW, nuclei number and the magnitude of DNA endoreduplication were progressively reduced, and the peak mitotic index was delayed compared with the control endosperm. At 18 DAP, the 4 d treatment showed 54·7% of the cells were 3 or 6 C, whereas only 41·2% were 12 C or higher. Six days of high temperature also resulted in a reduction in endosperm FW, nuclei number and a delay in the peak of mitotic index. DNA endoreduplication occurred in the kernels exposed to this treatment, although the magnitude was severely reduced compared with the control kernels. Nuclear DNA content was highly correlated (r = 0·93) with kernel FW, suggesting an important role of DNA endoreduplication in determining endosperm FW. The data suggest that high temperature during endosperm cell division exerted negative effects on DNA endoreduplication by dramatically reducing the nuclei number, leaving fewer nuclei available for DNA endoreduplication. However, the data also suggest that prolonged exposure to high temperature restricts entry of mitotic cells into the endoreduplication phase of the cell cycle.     

茉莉酸甲酯诱导下红豆杉细胞产生紫杉烷类物质群代谢轮廓分析

红豆杉细胞培养物经甲醇提取和固相萃取粗分离,进行反相高效液相色谱分析,获得了约含13个与紫杉醇极性相关的化合物色谱图,通过质谱联用和对照品参照,归属了这些化合物组成。进一步利用茉莉酸甲酯（MJ）诱导细胞,比较这些组分在诱导前后的相对色谱峰面积变化,结果表明,所有紫杉烷组分的浓度在诱导后都提高,其中以taxchinin M及其结构类似物提高最显著,紫杉醇,B-Ⅲ和B-Ⅵ等比C14位取代的taxuyunnanine C及其衍生物的浓度增加幅度要大,提示MJ对紫杉醇合成中非有效乙酰化旁路代谢有促进作用,而对C14位取代物生成的旁路促进作用不明显。本文为紫杉醇的生物合成研究提供了新的思路和方法。