水稻花药绒毡层特异基因及减数分裂相关基因的分离与功能分析

　利用RNA减法杂交、差异筛选和5’-RACE等方法从水稻分离到了一花药绒毡层特异表达的基因RA39。Southern 杂交表明，RA39在水稻基因组中是以单拷贝的形式存在的。RT-PCR 结果初步表明，RA39是一水稻花药特异表达的基因。RNA原位杂交进一步表明，RA39主要在水稻花药的绒毡层中表达，而且在小孢子母细胞减数分裂期和四分体时期表达量最高。RA39 cDNA全长1013bp，编码298个氨基酸残基。 RA39 cDNA与数据库中的已知序列没有明显的相似性，由其推测的多肽与核糖体失活蛋白（ribosome-inactivating protein, RIP）的序列相似在19-34%之间。多重序列排列分析结果表明构成RIPs活性位点的5个关键氨基酸残基在RA39中是保守的，在蓖麻毒蛋白中分别为Tyr80、 Tyr123、 Glu177、 Arg180 and Trp211 。利用原核表达系统，通过蛋白质分离和纯化获得了在SDS电泳图谱上为单一条带的纯的RA39蛋白，用兔rRNA作底物进行的酶活性分析证明该蛋白有N-糖基化作用，是一种类型I的核糖体失活蛋白。反义转基因植株的花粉用TTC进行活性染色结果显示其活性明显减弱，成熟的T0代反义转基因植株的结实率明显降低，只有对照的20-60%。这说明，RA39蛋白可能和小孢子母细胞的发育相关。 　　酵母DMC1是减数分裂过程中同源染色体配对和重组修复所必需的减数分裂特异基因。根据酵母Dmc1和拟南芥AtDmc1的保守区设计简并性引物，通过RT-PCR和RACE等方法，从水稻中分离出了酵母DMC1的同源基因OsDMC1。RT-PCR分析表明，OsDMC1在花中表达量最高，在根中表达量较低，在叶片和幼芽几乎不表达。水稻基因组中有两个拷贝的OsDMC1。OsDmc1蛋白与酵母Dmc1和拟南芥AtDmc1氨基酸一致性分别为53%和81%。 　　酵母Spo11在减数分裂过程中具有催化DNA双链断裂从而起始同源重组的功能。以酵母Spo11氨基酸序列为探针和现有的数据库通过数据分析，结合RACE技术，克隆了水稻SPO11同源基因OsSPO11-1， OsSPO11-1是一个单拷贝基因，有3个外显子和2个内含子，在转录过程中通过内含子的可变剪切产生4个不同的转录本（OsSPO11-1A、OsSPO11-1B、OsSPO11-1C和OsSPO11-1），其中，OsSPO11-1A是一个未剪切的转录本，OsSPO11-1B包含内含子2，OsSPO11-1C包含内含子1，OsSPO11-1D是一个完全剪切的转录本。这些转录本编码的蛋白有一致的246氨基酸残基的C-端，包含了Spo11/TopVIA家族蛋白共有的5个功能基元，是该家族的新成员。OsSPO11-1A和 OsSPO11-1C在花中优势积累，OsSPO11-1B是花特异的，而OsSPO11-1D在营养器官中优势积累。在花中该基因主要在减数分裂的花粉母细胞和胚曩中表达，在减数分裂期的绒毡层细胞和不同花器官的微管束细胞中也表达。这些结果说明内含子涉及到了OsSPO11-1表达的器官特异性调节，该基因除了参与减数分裂的调节外，在体细胞的发育中可能起重要作用。

Exposure of spermatozoa to duroquinone may impair reproduction of the common carp (Cyprinus carpio) through oxidative stress

Toxicity of many waterborne organic contaminants to aquatic organisms is mediated through oxidative damages resulting from the production of reactive oxygen species (ROS). Using duroquinone as a model ROS inducer, we carried out in vitro and in vivo experiments to test the hypothesis that reproduction in common carp (Cyprinus carpio) can be impaired through oxidative damage of their spermatozoa. In vitro exposure of fish spermatozoa to 0, 12.5, 25, 50, 100 and 200 mu M duroquinone for 2 h showed a significant increase in the level of ROS in a dose-dependant manner. Sperm motility was significantly reduced in all exposure groups, but lipid peroxidation (LPO) and DNA strand break (measured by comet assay) were only enhanced at 50 mu M and above. A significant decrease in subsequent hatching rate was recorded in all the exposure groups, despite fertilization rate was not affected. In the in vivo experiment, spermatozoa were collected 24 and 72 h after fish received intra-peritoneal injections of 1.0 and 10 mg kg(-1) body weight duroquinone. DNA damage was clearly evident in spermatozoa of all treatment groups after 72 h exposure, and ROS was significantly enhanced in the high concentration group. LPO however, remained unchanged in both treatment groups. The overall results of both our in vitro and in vivo experiments demonstrated that duroquinone can induce ROS production in spermatozoa, which may impair sperm quality and subsequently reproductive success through oxidative stress. (c) 2006 Elsevier B.V. All rights reserved.

Antioxidant N-acetylcysteine attenuates the acute liver injury caused by X-ray in mice

The aim of this study was to evaluate the protective effects of different doses and administration modes of N-acetylcysteine (NAC) against X-ray-induced liver damage in mice. Kun-Ming mice were divided into four groups, each composed of six animals: two control groups and two NAC-treated groups. An acute study was carried out to determine alterations in lipid peroxidation (determined by measuring malondiadehyde (MDA) level), glutathione (GSH) content and superoxide dismutase (SOD) activity (assayed by colorimetric method), and DNA damage (characterized by DNA-single strand break using with comet assay) as well as cell apoptosis (measured by flow cytometry) at 12 h after irradiation. The results showed that there were dose-related decreases in MDA level, DNA damage and cell apoptosis, and dose-dependent increases in GSH content and SOD activity in all NAC-treated groups compared to control groups, indicating that pre-treatment or post-treatment with NAC significantly attenuates the acute liver damage caused by X-ray. In addition, significant positive correlations were observed between MDA level and DNA damage or cell apoptosis, implying that lipid peroxidation plays a major role in X-ray-induced liver injury. The data suggest that NAC exerts its radioprotective effect by counteracting accumulated reactive oxygen species in the liver through its properties as a direct antioxidant and a GSH precursor, when administered before or after X-ray irradiation.

Melatonin modulates acute testicular damage induced by carbon ions in mice

The present study was performed to obtain evidence of the radioprotective function of melatonin at different administration levels on carbon ion-induced mouse testicular damage. Outbred Kun-Ming strain mice were divided into six groups, each composed of eight animals: control group, melatonin alone group, irradiation group and three melatonin plus irradiation-treated groups. An acute study was carried out to determine alterations in DNA-single strand break, cell apoptosis, and oxidative stress parameters as well as histopathology in mouse testis 24 h after whole-body irradiation with a single dose of 4 Gy Tie results showed that pre-treatment and post-treatment with high-dose melatonin (10 mg/kg) both significantly alleviated carbon ion-induced acute testicular damage, a greater radioprotective effect being observed in the pre-treatment group. On the other hand, low-dose melatonin (1 mg/kg) had a limited radioprotective effect on irradiation-induced degeneration and DNA lesions in mouse testis. Taken together, the data suggest that prophylactic treatment with a higher dose of melatonin is probably advisable to protect against the effects of heavy-ion irradiation.

Loss of p15/Ink4b accompanies tumorigenesis triggered by complex DNA double-strand breaks

DNA double-strand breaks (DSBs) are the most deleterious lesion inflicted by ionizing radiation. Although DSBs are potentially carcinogenic, it is not clear whether complex DSBs that are refractory to repair are more potently tumorigenic compared with simple breaks that can be rapidly repaired, correctly or incorrectly, by mammalian cells. We previously demonstrated that complex DSBs induced by high-linear energy transfer (LET) Fe ions are repaired slowly and incompletely, whereas those induced by low-LET gamma rays are repaired efficiently by mammalian cells. To determine whether Fe-induced DSBs are more potently tumorigenic than gamma ray-induced breaks, we irradiated 'sensitized' murine astrocytes that were deficient in Ink4a and Arf tumor suppressors and injected the surviving cells subcutaneously into nude mice. Using this model system, we find that Fe ions are potently tumorigenic, generating tumors with significantly higher frequency and shorter latency compared with tumors generated by gamma rays. Tumor formation by Fe-irradiated cells is accompanied by rampant genomic instability and multiple genomic changes, the most interesting of which is loss of the p15/Ink4b tumor suppressor due to deletion of a chromosomal region harboring the CDKN2A and CDKN2B loci. The additional loss of p15/Ink4b in tumors derived from cells that are already deficient in p16/Ink4a bolsters the hypothesis that p15 plays an important role in tumor suppression, especially in the absence of p16. Indeed, we find that reexpression of p15 in tumor-derived cells significantly attenuates the tumorigenic potential of these cells, indicating that p15 loss may be a critical event in tumorigenesis triggered by complex DSBs.

Experimental relaxation behavior of chopped-strand mat glass-fiber reinforced polyester beams in 3 point bending

A dimensionless relation of the form for collating fatigue crack starting growth data is proposed in which Δkth represents the stress intensity factor range at the threshold. Based on experimental results, this relation attains the value of 0.6 for a fatigue crack to start growth in the Austenitic stainless steel investigated in this work. Metallurgical examinations were also carried out to show a transgranular shear mode of cyclic cleavage and plastic shear.

A de novo originated gene depresses budding yeast mating pathway and is repressed by the protein encoded by its antisense strand

Recent transcription profiling studies have revealed an unexpectedly large proportion of antisense transcripts in eukaryotic genomes. These antisense genes seem to regulate gene expression by interacting with sense genes. Previous studies have focused on the non-coding antisense genes, but the possible regulatory role of the antisense protein is poorly understood. In this study, we found that a protein encoded by the antisense gene ADF1 acts as a transcription suppressor, regulating the expression of sense gene MDF1 in Saccharomyces cerevisiae. Based on the evolutionary, genetic, cytological and biochemical evidence, we show that the protein-coding sense gene MDF1 most likely originated de novo from a previously non-coding sequence and can significantly suppress the mating efficiency of baker's yeast in rich medium by binding MAT alpha 2 and thus promote vegetative growth. These results shed new light on several important issues, including a new sense-antisense interaction mechanism, the de novo origination of a functional gene, and the regulation of yeast mating pathway.

Ultraviolet-B exposure induces photo-oxidative damage and subsequent repair strategies in a desert cyanobacterium Microcoleus vaginatus Gom.

Biological soil crusts are important in reversing desertification. Ultraviolet radiation, however, may be detrimental for the development of soil crusts. The cyanobacterium Microcoleus vaginatus can be a dominant species occurring in desert soil crusts all over the world. To investigate the physico-chemical consequences of ultraviolet-B radiation on M. vaginatus, eight parameters including the contents of chlorophyll a, reactive oxygen species, malondialdehyde and proline, as well as the activities of photosynthesis, superoxide dismutase (EC 1.15.1.1), peroxiclase (EC 1.11.1.7) and catalase (EC 1.11.1.6) were determined. As shown by the results of determinations, ultraviolet-B radiation caused decreases both in contents of chlorophyll a and in ratios of variable fluorescence over maximum fluorescence that indicate the growth and photosynthesis of M. vaginatus, besides, increases both in levels of reactive oxygen species and in contents of malondialdehyde and proline, while intensified activities of superoxide dismutase, peroxiclase and catalase reflecting the abilities of enzymatic preventive substances to oxidative stress of the treated cells. Therefore, ultraviolet-B radiation affects the growth of M. vaginatus and leads to oxidative stress in cells. Under ultraviolet-B radiation, the treated cells can improve their antioxidant abilities to alleviate oxidative injury. The change trends of reactive oxygen species, superoxide dismutase, peroxiclase and catalase are synchronous. These results suggest that a balance between the antioxidant system and the reactive oxygen species content may be one part of a complex stress response pathway in which multiple environmental factors including ultraviolet-B radiation affect the Survival of M. vaginatus. (C) 2009 Elsevier Masson SAS. All rights reserved.

RecA-mediated, targeted mutagenesis in zebrafish

We have evaluated the efficacy of RecA, a prokaryotic protein involved with homologous recombination, to direct site-specific mutagenesis in zebrafish embryos. For this we coinjected a vector containing a mutated enhanced green fluorescent protein (EGFP) gene plus 236-nucleotide corrective single-stranded DNAs coated with RecA into I-cell zebrafish embryos. Twenty-hours after fertilization, about 5% to 20% of injected embryos showed EGFP expression in I or more cells when RecA-coated corrective DNAs were used, but not when RecA was omitted. Mutated EGFP genes with 1-bp insertions or deletions were inefficiently activated, whereas those with 7-bp insertions were activated about 4-fold more efficiently. RecA-coated template strand had a higher efficiency than its complementary strand in activation of EGFP expression. Prior irradiation of the embryos with UV light enhanced RecA-mediated restoration of gene activity, suggesting that the effects we observed were augmented by one or more factors of zebrafish DNA repair systems.

Effects of Naotan Pill on repair of neural cells and cognitive disorders in juvenile rats following hypoxia and ischemia

BACKGROUND: Hypoxia and ischemia induce neuronal damage, decreased neuronal numbers and synaptophysin levels, and deficits in learning and memory functions. Previous studies have shown that lycium barbarum polysaccharide, the most effective component of barbary wolfberry fruit, has protective effects on neural cells in hypoxia-ischemia. OBJECTIVE: To investigate the effects of Naotan Pill on glutamate-treated neural cells and on cognitive function in juvenile rats following hypoxia-ischemia. DESIGN, TIME AND SETTING: The randomized, controlled, in vivo study was performed at the Cell Laboratory of Lanzhou University, Lanzhou Institute of Modern Physics of Chinese Academy of Sciences, and Department of Traditional Chinese Medicine of Gansu Provincial Rehabilitation Center Hospital, China from December 2005 to August 2006. The cellular neurobiology, in vitro experiment was conducted at the Institute of Human Anatomy, Histology, Embryology and Neuroscience, School of Basic Medical Sciences, Lanzhou University, and Department of Traditional Chinese Medicine of Gansu Provincial Rehabilitation Center Hospital, China from March 2007 to January 2008. MATERIALS: Naotan Pill, composed of barbary wolfberry fruit, danshen root, grassleaf sweetflag rhizome, and glossy privet fruit, was prepared by Gansu Provincial Rehabilitation Center, China. Rabbit anti-synaptophysin, choline acetyl transferase polyclonal antibody, streptavidin-biotin complex kit and diaminobenzidine kit (Boster, Wuhan, China), as well as glutamate (Hualian, Shanghai, China) were used in this study. METHODS: Cortical neural cells were isolated from neonatal Wistar rats. Neural cell damage models were induced using glutamate, and administered Naotan Pill prior to and following damage. A total of 54 juvenile Wistar rats were equally and randomly assigned into model, Naotan Pill, and sham operation groups. The left common carotid artery was ligated, and then rat models of hypoxic-ischemic injury were assigned to the model and Naotan Pill groups. At 2 days following model induction, rats in the Naotan Pill group were administered Naotan Pill suspension for 21 days. In the model and sham operation groups, rats received an equal volume of saline. MAIN OUTCOME MEASURES: Neural cell morphology was observed using an inverted phase contrast microscope. Survival rate of neural cells was measured by MTT assay. Synaptophysin and choline acetyl transferase expression was observed in the hippocampal CA1 region of juvenile rats using immunohistochemistry. Cognitive function was tested by the Morris water maze. RESULTS: Pathological changes were detected in glutamate-treated neural cells. Neural cell morphology remained normal after Naotan Pill intervention. Absorbance and survival rate of neural cells were significantly greater following Naotan Pill intervention, compared to glutamate-treated neural cells (P < 0.05). Synaptophysin and choline acetyl transferase expression was lowest in the hippocampal CA1 region in the model group and highest in the sham operation group. Significant differences among groups were observed (P < 0.05). Escape latency and swimming distance were significantly longer in the model group compared to the Naotan Pill group (P < 0.05). CONCLUSION: Naotan Pill exhibited protective and repair effects on glutamate-treated neural cells. Naotan Pill upregulated synaptophysin and choline acetyl transferase expression in the hippocampus and improved cognitive function in rats following hypoxia-ischemia.

3D quench simulation using finite element method for CR superconducting magnet's strand

The CR superconducting magnet is a dipole of the FAIR project of GSI in Germany. The quench of the strand is simulated using FEM software ANSYS. From the simulation, the quench propagation can be visualized. Programming with APDL, the value of propagation velocity of normal zone is calculated. Also the voltage increasing over time of the strand is computed and pictured. Furthermore, the Minimum Propagation Zone (MPZ) is studied. At last, the relation between the current and the propagation velocity of normal zone, and the influence of initial temperature on quench propagation are studied.

MEASUREMENT OF TWO-PROTON CORRELATION FROM THE BREAK-UP OF Al-23

Experiments of Al-23 and Mg-22 radioactive beams bombarding a C-12 target at an energy of 60 similar to 70 A MeV have been performed at the projectile fragment separator beamline (RIPS) in the RIKEN Ring Cyclotron Facility to study the two-proton emission from Al-23 and Mg-22 excited states, respectively. The trajectorie of the decay products, namely Na-21 + p + p from Al-23 and Ne-20 + p + p from Mg-22, are clean identified. The relative momentum and opening angle between two protons in the rest frame of three body decay channels are obtained by relativistic-kinematics reconstruction. The results demonstrate that there are some di-proton emission components from He-2 cluster for the excited Al-23 and Mg-22.