Protective effects of melatonin against low- and high-LET irradiation

To investigate the protective effects of melatonin against high-LET ionizing radiation, V79 Chinese hamster cells were irradiated with 100 keV/mu m carbon beam. Parallel experiments were performed with 200 kV X-rays. To avoid the impact from extra solvents, melatonin was dissolved directly in culture medium. Cells were cultured in melatonin medium for 1 hr before irradiation. Cell inactivation was measured with conventional colony forming assay, medium containing 6-thioguanine was used for the selection of mutants at hprt locus, and the cell cycle was monitored by flow cytometry. Both carbon beam and X-rays induced cell inactivation, hprt gene mutation and cell cycle G2 block dose-dependently. But carbon beam showed stronger effects as indicated by all three endpoints and the relative biological effectiveness (RBE) was 3.5 for cell killing (at 10% survival level) and 2.9 for mutation induction (at 5 x 10(-5) mutants/ cell level). Melatonin showed protective effects against ionizing radiation in a dose-dependent manner. In terms of cell killing, melatonin only increased the survival level of those samples exposed to 8Gy or larger of X-rays or 6 Gy or larger of carbon beam. In the induction of hprt mutation and G2 block, melatonin reduced such effects induced by carbon beam but not by X-rays. The results suggest that melatonin reduces the direct interaction of particles with cells rather than an indirect interaction. Further studies are required to disclose the underlying mechanisms.

Primmorphs from archaeocytes-dominant cell population of the sponge Hymeniacidon perleve: Improved cell proliferation and spiculogenesis

Marine sponges (Porifera) possess an extraordinary diversity of bioactive metabolites for new drug discovery and development. In vitro cultivation of sponge cells in a bioreactor system is very attractive for the sustainable production of sponge-derived bioactive metabolites; however, it is still a challenging task. The recent establishment of sponge primmorphs, multicellular aggregates from dissociated mixed-cell population (MCP), has been widely acknowledged to hold great promise for cultivation in vitro. Here we present a new method to establish an in vitro sponge primmorph culture from archaeocyte-dominant cell population (ADCP) enriched by a Ficoll gradient, rather than a mixed-cell population (MCP). Our rationale is based upon the totipotency (the ability of a cell to differentiate into other cell types) of archaeocyte cells and the different biological functions of various sponge cell types. A sponge, Hymeniacidon perleve collected from the China Yellow Sea was used as a model system for this investigation. Distinct dynamics of primmorph formation were observed while significant increases in DNA synthesis, cell proliferation (up to threefold), and cell growth (up to fourfold) were achieved. Furthermore, a time-dependent spiculogenesis was clearly demonstrated in our longterm culture, indicating high metabolic activity of primmorphs from the ADCP. This new method represents an important step forward to advance sponge cell culture in vitro that may lead to commercial exploitation of sponge-derived drugs. (C) 2003 Wiley Periodicals, Inc.

Towards manipulation of post-biosynthetic events in secondary metabolism of plant cell cultures

Plant cell cultures have been suggested as a feasible technology for the production of a myriad of plant-derived metabolites. However, commercial application of plant cell culture has met limited success with only a handful of metabolites produced at the pilot- and commercial-scales. To improve the production of secondary metabolites in plant cell cultures, efforts have been devoted predominantly to the optimization of biosynthetic pathways by both process and genetic engineering approaches. Given that secondary metabolism includes-the synthesis. metabolism and catabolism of endogenous compounds by the specialized proteins, this review intends to draw attention to the manipulation and optimization of post-biosynthetic events that follow the formation of core metabolite structures in biosynthetic pathways. These post-biosynthetic events-the chemical and enzymatic modifications, transport, storage/secretion and catabolism/degradation have been largely unexplored in the past. Potential areas are identified where further research is needed to answer fundamental questions that have implications for advanced bioprocess design. Anthocyanin production by plant cell cultures is used as a case study for this discussion, as it presents a good example of compounds for which there are extensive research publications but still no commercial bioprocess. It is perceived that research on post-biosynthetic processes may lead to future opportunities for significant advances in commercial plant cell cultures. (C) 2002 Elsevier Science Inc. All rights reserved.

Layer-by-layer assembly of viral capsid for cell adhesion

Cowpea mosaic virus (CPMV)-based thin films are biologically active for cell culture. Using layer-by-layer assembly of CPMV and poly(diallyldimethylammonium chloride), quantitatively scalable biomolecular surfaces were constructed, which were well characterized using quartz crystal microbalance, UV-vis and atomic force microscopy. The surface coverage of CPMV nanoparticles depended on the adsorption time and pH of the virus solution, with a greater amount of CPMV adsorption occurring near its isoelectric point. It was found that the adhesion and proliferation of NIH-3T3 fibroblasts can be controlled by the coverage of viral particles using this multilayer technique.

Capillary liquid chromatographic-high-resolution mass spectrometric analysis of ribonucleotides

A method of capillary HPLC-high-resolution MS was developed for the trace analysis of ATP, GTP, dATP and dGTP Dimethylhexylamine (DMHA) was used as ion-pairing agent for the HPLC retention and separation of the nucleotides and positive ion electrospray time-of-flight MS was used for the detection. The application of capillary HPLC allowed minimal usage of DMHA while providing excellent peak retention and resolution, which significantly reduced the ion suppression in electrospray ionization-MS analysis and thus increased the sensitivity. Adduct ions of nucleotides and DMHA were used as quantitative ions in order to achieve the best sensitivity. DMHA concentration at 5 mM in the aqueous mobile phase at pH 7 was found to be the optimal conditions for the C Is capillary column. The method was applied to determine ATP level in cultured C6 glioma cells that were treated with toxic concentrations of Zn. The results showed that the cellular ATP level decreased from 2.7 pmol/cell (<10% cell death) in average control cell samples to 0.36 pmol/cell as the concentration of Zn increased to 120 mg/l (>35% cell death) in culture medium.

Study on impact of dinoflagellate Alexandrium tamarense on life activities of marine bivalves

The effects of a PSP producing dinoflagellate Alexandrium tamarense on marine bivalves at their several important life,stages: egg, D - shape larva, eyespot larva, juvenile and adult, were studied! The results show that the hitching survival, activity, filtration and! growth were adversely affected by the alga and the impact was significantly increased with the increase of algal density. The inhibitory effect on egg hatching was most significant, which the hatching rate was only 30% of the control when exposed to the alga at 100 cell/cm(3) after 36 h. Further experiments show that the algal culture, re-suspended cells and cell fragments had the inhibitory effect, while no such effect was from the cell-free medium, cell contents and standard STX. The results indicate that the alga could produce unknown toxins, rather than PSP, associated with the cell surface.

Interactions between the bloom-forming dinoflagellates Prorocentrum donghaiense and Alexandrium tamarense in laboratory cultures

Interactions between Prorocentrum donghaiense and Alexandrium tamarens, two bloom-forming dinoflagellates, were investigated using bi-algal cultures. All R donghaiense died, but A. tamarense was hardly affected by the end of the experiment when the initial cell density was set at 1.0 X 10(4) cells mL(-1) for P. donghaiense and 0.28 x 10(4) cells mL(-1) for A. tamarense. However, significant growth suppression occurred in either species when the initial cell density of P donghaiense increased to I. 0 X 105 Cells mL(-1) in the bi-algal culture, but no out-competement was observed. The simultaneous assay on the culture filtrates showed that P donghaiense filtrate prepared at a lower initial density (1.0 X 10(4) cells mL(-1)) stimulated growth of the co-cultured A. tanzarense (0.28 x 10(4) cells mL(-1)), but filtrate at a higher initial density (1.0 x 10(5) cells mL(-1)) depressed its growth. The filtrate of A. tamarense at a density of 0.28 x 10(4) cells mL(-1) killed all R donghaiense at a lower density (1.0 x 10(4) cells mL(-1)), but only exhibited an inhibitory effect on it at a higher density (1.0 x 10(5) cells mL(-1)). It is likely that these two species of microalgae interfere with each other mainly by releasing allelochemical substance(s) into the culture medium, and a direct cell-to-cell contact was not necessary for their mutual interaction. The allelopathic test further proved that A. tamarense could affect the growth of co-cultured P. donghaiense by producing allelochemical(s); moreover, A. tamarense culture filtrate at the stationary growth phase (SP) had a strongly inhibitory effect on P donghaiense compared to that at the exponential phase (EP). Results also demonstrated a dose-dependent relationship between the microalgal initial cell density and the degree of the allelopathic effect. The growth of R donghaiense and A. tamarense in the bi-algal cultures was simulated using a mathematical model to quantify the interaction. The estimated parameters from the model showed that the inhibition exerted by A. tamarense on P. donghaiense was about 17 and 8 times stronger than the inhibition P. donghaiense exerted on A. tamarense, when the initial cell density was set at 1.0 X 10(4) and 1.0 X 10(5) cells mL(-1) for P donghaiense, respectively. and 0.28 x 10(4) cells mL(-1) for A. tamarense in the bi-algal cultures. A. tamarense seems to have a survival strategy that is superior to that of P. donghaiense in bi-algal cultures under controlled laboratory conditions. (c) 2006 Elsevier B.V. All rights reserved.

Substrate Strain and Proliferation & Differentiation of Bone Marrow Mesenchymal Stem Cells

New methods of surface modification of transparent silicone substrate were developed, and a new set of cell culture devices that provide homogeneous substrate strain was designed. Using the new device, effects of cyclic substrate strain on bone marrow mesenchymal stem cells(MSCs) were studied. It was found that cyclic strain influenced proliferation and differentiation of bone marrow MSCs in different ways.

七叶树组织及细胞培养产生生育酚的研究

从七叶树（Aesculus Chinensis Bunge）的未成熟的果实的子叶中诱导出愈伤组织;愈伤组织在pH5.8，温度26 ± 1 ℃，加有NAA，TBA，K，CA的MS培养基上生长良好。光对愈伤组织的生长有促进作用，植醇对生长有抑制作用;进行了发酵罐培养。 利用TLC、质谱分离鉴定了γ-生育酚的存在，并利用HPLC、荧光法测定了生育酚的含量。结果表明，愈伤组织中生育酚的含量在3.2～6.6mg/100g干重;光对生育酚的合成有促进作用;植醇是生育酚合成的可能前体;悬浮培养不利于生育酚的合成，培养液中没有发现生育酚的存在。生育酚的合成与组织的生长速率成正相关。

新疆紫草细胞悬浮培养生产紫草宁衍生物的动态测定机理化因子调控

本文在本实验室提供的新疆紫草愈伤组织高产系A1的基础上，采用二步培养法，进行摇瓶悬浮培养，分别在生长及生产培养基中测定了细胞生长，次生产物合成，培养基的C源（蔗糖）消耗，溶氧，电导率和pH值的动态变化曲线，确定了各动态曲线之间的关系，为进一步的放大培养提供了参考依据。同时，还测定了与细胞生长密切相关的过氧化物酶及与产物合成密切相关的苯丙氨酸解氨酶(PAL)的活性的动态变化曲线，进一步将宏观参数的动态变化与微观参数的动态变化联系起来。 本文还对不同理化因子对生产培养基中悬浮培养的细胞的生长及紫草宁衍生物合成的影响进行了研究。结果表明：过高或过低的供氧水平均不利于细胞的生长及产物的合成;C源及N源有较好的协同作用，适当地提高C源及N源的水平能明显提高紫草宁衍生物的产量：接种前往培养基里加入一定量的前体苯丙氨酸( Phe)，能明显提高紫草宁衍生物的产量，而在培养中期添加则有一定的负致应;一定量的拜土及琼脂(agar)的添加，对产物的合成均具有正效应，并且作用大小和细胞的生理状态有关。高密度培养的研究表明，在合适的接种量和培养基浓度下，适当提高溶氧，较大幅度地提高产量是有可能的，这还有待于进一步的研究验证。

水母雪莲高产细胞系的筛选及黄酮类化合物合成的调控

水母雪莲属菊科植物，为名贵中药材其主要活性成分为黄酮类化合物。为解决雪莲资源其中匮乏，本文开展了利用水母雪莲细胞培养生产黄酮类活性成分的可行性研究。 采用目视法从水母雪莲原始愈伤组织中筛选得到白色、黄色和红色3种细胞系，其中黄色系的黄酮含量最高。利用γ射线辐射处理从红色系得高产黄酮细胞系。 证明了多种理化因子对水母雪莲培养细胞中黄酮类合成的调控作用。研究发现，温度和光照对水母雪莲愈伤组织黄酮合成影响较大。25℃是最佳培养温度;红光促进愈伤组织生长，蓝光促进黄酮的合成;进一步研究表明：光调节黄酮代谢途径第一步所需酶苯丙氨酸裂解酶（PAL）活性，PAL活性被红光所抑制，被蓝光所促进。碳源、氮源、植物激素对愈伤组织生长和黄酮形成影响较为显著。对MS培养基成分进行调整得到M－13培养基，在M－13培养基上培养的愈伤组织生长量和黄酮产量比MS培养基分别提高33％和82％。 首次建立水母雪莲细胞悬浮培养体系。确定了水母雪莲细胞悬浮培养的最适培养条件和最适培养基成分组成。摇床转速 90～120 r/min、接种量 2.5～4.0gDW／L、接种物种龄 8～10 d 对黄酮合成有利。调整MS培养基成分得到适合于培养水母雪莲悬浮细胞的生长培养基G和黄酮合成培养基MP，从而使细胞生长量与黄酮产量分别长比MS培养基提高32％和70％。 应用2－L搅拌式生物反应器对水母雪莲细胞进行了悬浮培养。TLC和HPLC分析表明，水母雪莲细胞培养物能够形成2种黄酮活性成分金合欢素和高车前素。本论文对水母雪莲细胞培养生产药用活性成分作了基础性工作。

雪莲细胞培养物中黄酮类物质代谢调控及其生物活性成份分析

水母雪莲（Saussurea medusa Maxim.）和新疆雪莲（Saussurea involucrata Karel. et Kir.）是我国珍稀的药用植物资源，具有清热解毒、止痉镇痛、敛伤、消肿及治疗热病、风湿等多种功效。雪莲的主要药用成份为紫丁香甙（Syringin）、芦丁(Rutin)、高车前素（Hispidulin）和Jaceosidin等苯基丙酸类（phenylpropanoid）和黄酮类（flavonoids）物质。最新的药理研究表明，上述物质还具有抗菌消炎、保肝降压、延缓衰老和抑制癌细胞增殖等重要的研发价值。 雪莲生境恶劣，生长缓慢，人工引种困难，加上长期掠夺性采挖，已使雪莲处于灭绝的边缘。为了保存国家珍稀植物品种，保护生态环境，满足临床上对雪莲药物的需求，本研究在雪莲组织培养的基础上，应用诱导子添加技术和毛状根培养技术对雪莲中具有重要药用价值的次生代谢物质进行调控，并对雪莲MYB类转录因子的功能进行了初步探索，为保护珍稀植物资源、维护生态环境、开发野生雪莲替代产品、缩短雪莲药用成份的生产周期奠定了基础。另外，分析了野生雪莲和雪莲培养物中主要生物活性成份的种类及含量，为今后雪莲药理药效研究及品质评价奠定了基础。 为了提高雪莲黄酮的产量，满足工业化生产的需要，在细胞培养水平上，通过添加茉莉酸甲酯（MJ），对雪莲黄酮类物质的代谢进行调控。研究了诱导子的添加时间、添加浓度对水母雪莲红色系悬浮细胞的生物量和总黄酮产量的影响。发现在细胞培养的指数期（第9天）添加5.0 µmol/L的MJ，可以使总黄酮产量提高2.4倍（1134.5 ± 63.86 mg/L），而雪莲细胞干重（dw）仅比对照提高23.8 %（20.4 ±0.27 g/L）。另外，细胞中苯丙氨酸裂解酶（PAL）的活性分析表明，MJ添加后PAL活性的增加与雪莲总黄酮含量增长之间存在相关性。 在器官培养水平上，对雪莲毛状根的诱导频率及其培养条件进行了研究。结果表明，选择发根农杆菌R1601侵染预培养2天的新疆雪莲根段外植体，毛状根的诱导效率可达到83 %。毛状根的冠瘿碱检测、PCR和Southern分析表明，Ri质粒中的T-DNA已整合到植物基因组中并稳定表达。以新疆雪莲毛状根为外植体，能够容易地获得再生芽。在含有1.0 mg/L 6-BA的MS固体培养基上，其再生频率高达91 ± 5.9 %，是其正常根的2.4倍。而水母雪莲在该培养条件下，仅有少量的畸形芽出现。进而对毛状根的培养条件进行初步研究，结果表明在无激素附加的MS液体培养基中，新疆雪莲的HR1601根系在一个培养周期内（32 天），其生物量能够达到接种量的16倍，而紫丁香甙含量（43.5 ± 1.13 mg/g dw）能够达到野生雪莲的83倍。从而显示了雪莲毛状根培养体系的优良特性。 在基因水平上，对雪莲黄酮类物质代谢调控的研究已经展开。玉米P基因编码的Myb类转录因子能够调节黄酮类物质代谢途径关键酶基因的表达。根据P基因的保守序列设计引物，从雪莲细胞培养物中获得了SmP基因。核酸序列分析表明，SmP基因与烟草中涉及苯丙素类物质代谢途径的LBM 1、LBM 3和MybAS 1基因具有较高的一致性，分别为66 %、60 %和61 %。因此为了研究雪莲SmP基因的功能，构建了正义表达载体，并与先前构建好的反义表达载体分别导入烟草，分析了转基因植株的形态特征及黄酮类物质的含量变化。其中，约有30 %转反义SmP基因的株系表现叶片皱缩、叶脉紊乱、主侧脉角度缩小、叶片、花瓣失去对称性以及花粉败育等性状。 另外，通过正交试验设计优化了雪莲提取工艺的条件，并对雪莲细胞提取物进行了分离纯化。正交试验设计结果表明，温度对雪莲黄酮提取效率的影响极为显著，而分批多次提取比一次性浸提，能够收到较好的提取效果。考虑到工业生产中的实际问题，推荐在60 ℃水浴条件下，采用50 %乙醇对雪莲样品连续浸提2次的方案。对雪莲提取物的纯化研究表明，雪莲成份复杂，仅依靠单一的分离手段，往往难以奏效。另外，野生雪莲及雪莲培养物中生物活性成份的比色法、HPLC（High Performance Liquid Chromatography）、LC-ESI-MS（Liquid Chromotagraphy Electrospray Ionization Mass Spectrometry）分析表明，传统的NaNO2-AlCl3 法测定雪莲总黄酮的含量，结果偏高，不利于雪莲黄酮的实验室研究分析与今后工业化生产的质量监控。而AlCl3 法的显色反应较为特异，今后有望取代NaNO2-AlCl3 法，作为雪莲类药材品质评价的标准。而HPLC-DAD结合LC-ESI-MS可以对雪莲中的主要生物活性成份进行较为准确的定性分析，从而解决了由于缺乏相应的雪莲化合物标准品而难以对雪莲中的成份进行定性定量分析及比较的难题。最后综合利用上述分析方法，对雪莲细胞培养物中的花素类物质进行了分析。结果表明，雪莲细胞中至少含有7种花色素类物质，分别为矢车菊素-3-O-葡萄糖甙及其衍生物、天竺葵素糖甙衍生物和芍药色素糖甙衍生物。

Methylation of tumor associated genes in tissue and plasma samples from liver disease patients

To investigate whether aberrant hypermethylation in plasma DNA could be used as diagnosis makers for hepatocellular carcinoma (HCC), we performed methylation-specific PCR (MSP) to check the methylation status of five tumor associated genes in 36 cases of

THE POSSIBLE INVOLVEMENT OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR IN LUTEOLYSIS OF RHESUS-MONKEY

Changes of plasminogen activators (PA) during different stages of development of the corpus luteum, and their possible physiological role in luteolysis were studied in rhesus monkeys. It was demonstrated for the first time that monkey corpus luteal cells not only produce PA, but that the function of the corpus luteum is also closely related to the activity of this enzyme system. Generally, the life span for a corpus luteum in monkey is approximately 14-16 days, its demise beginning thereafter. In the present study, we found that urokinase in the corpus luteum is higher on day 5 and day 10 after human chorionic gonadotrophin injection, while the tissue type (t) PA is mainly produced on day 13 when luteolysis may take place. Progesterone production remained high on day 5 and day 10 and decreased dramatically from day 13, indicating the important role of tPA but not urokinase (u) PA in suppressing luteal function. When purified tPA (but not uPA) monoclonal antibody was added to luteal cell culture to neutralize endogenously produced tPA activity, progesterone production in the cells was increased significantly. Interestingly, prolactin alone was capable of increasing PA production by luteal cells; prolactin together with luteinizing hormone, however, had a synergistic luteotrophic effect.

Medaka vasa is required for migration but not survival of primordial germ cells

Vasa is essential for germline development. However, the precise processes in which vasa involves vary considerably in diverse animal phyla. Here we show that vasa is required for primordial germ cell (PGC) migration in the medakafish. vasa knockdown by two morpholinos led to the PGC migration defect that was rescued by coinjection of Vasa RNA. Interestingly, Vasa knockdown did not alter the PGC number, identity, proliferation and motility even at ectopic locations. We established a cell culture system for tracing PGCs at the single cell level in vitro. In this culture system, control and morpholino-injected gastrulae produced the same PGC number and the same time course of PGC survival. importantly, vasa-depleted PGCs in culture had similar motility and locomotion to normal PGCs. Expression patterns of wt1a, sdf1b and cxcT4b in migratory tissues remained unchanged by Vasa knockdown. By chimera formation we show that PGCs from vasa-depleted blastulae failed to migrate properly in the normal environment, whereas control PGCs migrated normally in vasa-disrupted embryos. Furthermore, ectopic PGCs in vasa-depleted embryos also retained all the PGC properties examined. Taken together, medaka vasa is cell-autonomously required for PGC migration, but dispensable to PGC proliferation, motility, identity and survival. (C) 2009 Elsevier Ireland Ltd. All rights reserved.