Expression pattern of dmrt4 from olive flounder (Paralichthys olivaceus) in adult gonads and during embryogenesis

The dmrt (doublesex and mab-3 related transcription factor) gene family comprises several transcription factors that share a conserved DM domain. Dmrt1 is considered to be involved in sexual development, but the precise function of other family members is unclear. In this study, we isolated genomic DNA and cDNA sequences of dmrt4, a member of the dmrt gene family, from olive flounder, Paralichthys olivaceus, through genome walking and real-time reverse transcriptase (RT)-PCR. Sequence analysis indicated that its genomic DNA contains two exons and one intron. A transcriptional factor binding sites prediction program identified a sexual development-related protein, Sox9 (Sry-like HMG box containing 9) in its 5' promoter. Protein alignment and phylogenetic analysis suggested that flounder Dmrt4 is closely related to tilapia Dmo (DM domain gene in ovary). The expression of dmrt4 in adult flounder was sexually dimorphic, as shown by real-time RT-PCR analysis, with strong expression in the testis but very weak expression in the ovary. Its expression was also strong in the brain and gill, but there was only weak or no expression at all in some of the other tissues tested of both sexes. During embryogenesis, its expression was detected in most developmental stages, although the level of expression was distinctive of the various stages. Whole mount in situ hybridization revealed that the dmrt4 was expressed in the otic placodes, forebrain, telencephalon and olfactory placodes of embryos at different developmental stages. These results will improve our understanding of the possible role of flounder dmrt4 in the development of the gonads, nervous system and sense organs.

Hybrids between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus: karyotype, allozyme and RAPD analyses

The hybrid between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus was produced by artificial insemination of olive flounder eggs with stone flounder sperm. Sinistral and dextral are two types of hybrid progeny after metamorphosis. Karyotypes of both hybrid flounders are the same as those of the two parental species. Of the 22 loci examined from 12 allozymes, 12 confirmed hybridization of the paternal and maternal loci in hybrids and no difference was found in allozyme patterns of sinistral and dextral hybrid fishes. RAPD patterns of these specimens were also studied with 38 primers selected from 104 tested. Among them, the PCR products of 30 primers showed hybridization of the paternal and maternal bands. Genetic variation between hybrids and their parental stocks was analyzed by RAPD using 10 of the above 38 primers. The average heterozygosity and genetic distance were calculated. The results suggested that the filial generation could inherit a little more genetic materials from paternal fish than that from maternal fish.

Effects of hydrostatic pressure on microtubule organization and cell cycle in gynogenetically activated eggs of olive flounder (Paralichthys olivaceus)

Indirect immunofluorescence staining was used to detect cytological changes of isolated blastodisks during mitosis of flounder haploid eggs treated with hydrostatic pressure. Changes in microtubule structure and expected cleavage suppression were observed from blastodisk formation to the third cell cycle, with obvious differences between treated and control eggs. In most eggs, microtubules were disassembled and the nucleation capacity of the centrosome was temporarily inhibited after pressure treatment. Within 15-20 min after treatment, the nucleation capacity of the centrosome began to gradually recover, with slow regeneration of microtubules; approximately 25 min after treatment, the nucleation capacity of the centrosome recovered completely, regenerated distinct bipolar spindles, and the first mitosis ensued. During the second cell cycle, approximately 61% of the embryos were at the two-cell stage, with a monopolar spindle in each blastomere; that treatment was effective was based on second cleavage blockage. Approximately 15% of the eggs still remained at the one-cell stage and had a monopolar spindle (treatment was effective, according to the general model of first cleavage blockage). However, treatment was ineffective in approximately 15% of the embryos (bipolar spindle in each blastomeres) and in another 8% (bipolar spindle in one of the two blastomeres and a monopolar spindle in the other; both mechanisms operating in different parts of the embryo). This is the first report elucidating mitotic gynogenetic diploid induction by hydrostatic pressure in marine fishes and provides a cytological basis for developing an efficient method of inducing mitotic gynogenesis in olive flounder. (C) 2007 Elsevier Inc. All rights reserved.

Effects of cold shock on microtubule organization and cell cycle in gynogenetically activated eggs of olive flounder (Paralichthys olivaceus)

Cytological changes and subsequent mitotic processes were studied in gynogenetically activated eggs of olive flounder subjected to cold-shock treatment using indirect immunofluorescence staining of isolated blastodisks. Obvious differences between controls and treated eggs were detected during early cell division. The developmental process of haploid control was similar to that of the diploid control except several minutes delayed. Spindles disassembled by the cold-shock treatment regenerated soon after treatment, resulting in the occurrence of the first mitosis. The immature daughter centriole was easily depolymerized by cold-shock treatment, leading to the formation of the bipolar spindle in the first cell cycle and the formation of the monopolar spindle in the second cell cycle, resulting in chromosome set doubling. Some two-cell stage eggs had a monopolar spindle in one blastomere and a bipolar spindle in another during the second mitosis. These eggs had a high potency developing into haploid-diploid mosaics. To the best of our knowledge, this study is the first to clarify the mechanism of chromosome set doubling in marine fishes and provides a preliminary cytological basis for developing a reliable and efficient protocol for mitotic gynogenesis induction by cold-shock treatment in olive flounder.

单细胞凝胶电泳技术检测碳离子束和X射线照射对Lewis肺癌细胞的DNA损伤

比较研究了89.63MeV/u的碳离子束和6MeV的X射线照射Lewis肺癌细胞所致的细胞克隆存活和DNA损伤效应,以探讨单细胞凝胶电泳检测细胞辐射敏感性及重离子束治疗肿瘤的优势。结果表明,在10%细胞存活水平上碳离子束的相对生物学效应(Relative biological effectiveness,RBE)值达到1.77。单细胞凝胶电泳检测损伤DNA尾部百分含量(Tail DNA,TD)和Olive���矩(Olive tail moment,OTM)的剂量效应曲线表明,X射线的剂量效应曲线为线性,而碳离子束诱导出一个包含线性和指数项的双阶段效应曲线。碳离子束辐照剂量大于8Gy后TD和OTM都存在饱和效应。在2Gy的剂量点,高传能线密度(LET)碳离子束比X射线产生更低的存活分数和更高的初始OTM。本研究提示:在Lewis肺癌细胞中,碳离子束照射比X射线产生更为强烈的细胞致死和DNA损伤效应,可使肿瘤治疗具有更高效率。

彗星电泳检测~(12)C~(6+)离子束辐照人类肝L02细胞的DNA损伤效应

以传能线密度为30keV/μm的12C6+离子束辐照人类肝L02细胞,利用彗星电泳技术检测了以DNA链断裂为生物终点的DNA辐射损伤效应。CASP软件分析彗星图像,主要检测尾部DNA(TDNA%)、彗星全长(CL)、尾长(TL)、尾矩(TM)和Olive���矩(OTM)等指标,SPSS11.5软件进行统计学分析,绘制并拟合TM-剂量曲线。结果显示,辐照以剂量依赖的方式引起L02细胞彗星图像各指标的增大,且TM值与剂量线性正相关。说明12C6+离子束对DNA有较强的致损伤效应,且与剂量正相关。研究为正确评价重离子对人体正常组织的辐射风险及危害提供了一定的基础数据和依据。

Assessment of DNA damage of Lewis lung carcinoma cells irradiated by carbon ions and X-rays using alkaline comet assay

DNA damage and cell reproductive death determined by alkaline comet and clonogenic survival assays were examined in Lewis lung carcinoma cells after exposure to 89.63 MeV/u carbon ion and 6 MV X-ray irradiations, respectively. Based on the survival data, Lewis lung carcinoma cells were verified to be more radiosensitive to the carbon ion beam than to the X-ray irradiation. The relative biological effectiveness (RBE) value, which was up to 1.77 at 10% survival level, showed that the DNA damage induced by the high-LET carbon ion beam was more remarkable than that induced by the low-LET X-ray irradiation. The dose response curves of '' Tail DNA (%)'' (TD) and "Olive tail moment" (OTM) for the carbon ion irradiation showed saturation beyond about 8 Gy. This behavior was not found in the X-ray curves. Additionally, the carbon ion beam produced a lower survival fraction at 2 Gy (SF2) value and a higher initial Olive tail moment 2 Gy (OTM2) than those for the X-ray irradiation. These results suggest that carbon ion beams having high-LET values produced more severe cell reproductive death and DNA damage in Lewis lung carcinoma cells in comparison with X-rays and comet assay might be an effective predictive test even combining with clonogenic assay to assess cellular radio sensitivity

重离子诱导正常组织细胞的突变及损伤效应

对于重离子束辐照诱导的人类肝L02细胞hprt基因突变和DNA损伤效应目前还很少有报导。在重离子治癌和载人空间飞行当中，评价重离子辐照对病灶周围正常组织的辐射危害和空间重离子射线对宇航员的辐射风险越来越重要。本论文通过研究hprt基因突变频率、突变谱以及DNA损伤情况，为正确评价重离子对人体正常组织细胞的辐射风险及危害提供基础数据和依据。利用兰州重离子研究装置（HIRFL）提供的12C6+离子束(到达细胞样品处的能量为83.6MeV/n，对应的LET值约为30keV/m)和医用电子直线加速器提供的X射线（8MV，对应的LET值约为0.2keV/m）对体外培养的L02细胞进行0~6Gy照射后，在含有6-TG的培养基中克隆、6-TG筛选hprt突变细胞株，细胞克隆法测定hprt基因突变频率。各照射剂量分别随意挑取9~10个hprt基因突变株扩大培养，分别提取DNA后用多重PCR法扩增hprt基因的九个外显子，利用琼脂糖凝胶电泳观察其缺失突变谱。利用常规彗星电泳方法检测12C6+离子束（LET为30 keV/μm）辐照后人类肝L02细胞的DNA损伤情况，以CASP软件逐个分析彗星图像，主要检测头部DNA（HDNA％）尾部DNA（TDNA％）、彗星全长（CL）、尾长（TL）、尾矩（TM）和Olive���矩（OTM）等指标的剂量-效应关系，并用SPSS11.5软件进行统计学分析。最后绘制出并线性拟合TM-剂量曲线。实验结果显示，人类肝细胞L02细胞系hprt基因对12C6+离子束和X射线辐照是敏感的。L02细胞对12C6+离子的存活分数明显低于对X射线的存活分数，即12C6+离子对L02细胞的致死效应强于X射线。两种射线照射后，每106个存活细胞中突变克隆的个数随照射剂量增大而增大。受致死效应影响，受照细胞的突变频率先增大后减小，都在1Gy处达到最大值，与文献报道的其他重离子辐照细胞诱导突变的结果相似。在所分析的突变细胞克隆中，发生缺失突变的概率最大，且大多数为大片段缺失突变。文献报道X射线多诱导微小突变和小片段缺失突，说明高LET的重离子辐射比低LET的X射线所引起的细胞损伤更大。另外，随着照射剂量的增加，完全缺失突变几率呈逐渐增大的趋势。但由于能够分析的细胞克隆数有限，进一步的研究是必要的。彗星电泳实验结果发现辐照以剂量依赖的方式引起L02细胞彗星图像TDNA％、CL、TL、TM和OTM等指标的增大，且TM值与剂量成线性正相关。说明该LET的12C6+离子束对DNA有较强的致损伤效应，且与剂量成正相关。本文实验所用碳离子束的LET(30keV/m)恰处于重离子治癌当中离子束贯穿健康组织到达肿瘤靶区之前入射通道上所具有的LET范围之内。本文的突变频率实验结果显示重离子束诱导正常组织细胞hprt基因突变的频率要高于X射线，也就是重离子束辐射对正常组织的远后效应及辐射风险与危害要大于X射线。突变谱实验结果表明单次大剂量碳离子照射对于hprt基因乃至细胞和组织都会造成很大的损伤。彗星电泳的结果也显示重离子对正常细胞一定的致DNA损伤效应。因此，应在充分考虑重离子束对正常组织辐射风险的前提条件下合理地制订治疗计划，在治疗当中应尽量避免对正常细胞组织的单次大剂量照射，使得重离子治癌成为一种最有效且安全的放射治疗模式。另外，在宇宙空间中，碳离子虽然在空间重离子中所占的比例较小，但从本文结果可以看到其也存在一定的辐射风险。因此，在载人空间飞行当中也应充分考虑其对宇航员的辐射风险及危害，并制定相应的辐射防护计

Sonochemical synthesis and luminescence properties of single-crystalline BaF2:Eu3+ nanospheres

BaF2 nanocrystals doped with 5.0 mol% Eu3+ has been successfully synthesized via a facile, quick and efficient ultrasonic solution route employing the reactions between Ba(NO3)(2), Eu(NO3)(3) and KBF4 under ambient conditions. The product was characterized via X-ray powder diffraction (XRD), scanning electron micrographs (SEM), transmission electron microscopy (TEM), high-resolution transmission electron micrographs (HRTEM), selected area electron diffraction (SAED) and photoluminescence (PL) spectra. The ultrasonic irradiation has a strong effect on the morphology of the BaF2:Eu3+ particles. The caddice-sphere-like particles with an average diameter of 250 nm could be obtained with ultrasonic irradiation, whereas only olive-like particles were produced without ultrasonic irradiation. The results of XRD indicate that the obtained BaF2:Eu3+ nanospheres crystallized well with a cubic structure. The PL spectrum shows that the BaF2:Eu3+ nanospheres has the characteristic emission of Eu3+ D-5(0)-F-7(J) (J = 1-4) transitions, with the magnetic dipole D-5(0)-F-7(1) allowed transition (590 nm) being the most prominent emission line.

褐牙鲆和夏牙鲆杂交的遗传学研究

在进行褐牙鲆(Paralichthys olivaceus)和夏牙鲆(P. dentatus)的杂交及回交的基础上，利用染色体计数、AFLP、线粒体DNA和核基因部分序列等分析方法对褐牙鲆和夏牙鲆正反交和回交子代进行遗传学研究，探讨了褐牙鲆和夏牙鲆正反交不对称的遗传学基础及其生殖隔离现象，主要结果如下： 1. 褐牙鲆和夏牙鲆正反交的活力是不对称的，褐牙鲆♀×夏牙鲆♂的正交杂种活力正常，能够正常存活、生长和发育，而反交夏牙鲆♀×褐牙鲆♂的杂种体态畸形，孵出后不久死亡。染色体计数发现正交个体的染色体核型与父母本一致，均为48条端部着丝粒染色体；而反交杂种比亲本缺失了两条染色体，仅为46条端部着丝粒染色体，这表明反交杂种为非整倍体。进一步利用AFLP方法对遗传物质从亲本到子代的传递进行了分析，结果显示正反交遗传物质的传承方式存在很大差异。几乎所有亲本的AFLP位点(97.71%)均传递到正交子代。然而，仅有86.64%的AFLP位点从亲本传递到反交子代，反交子代中亲本位点的丢失比例显著高于正交子代和亲本种内交配子代的比例 ( P < 0.05)，这可能与反交杂种染色体丢失有关。进一步分析发现，杂交组中的偏分离标记高于对照组，尽管经2检验发现其差异并不显著 (P > 0.05)。 2. 对于可以成活的正交杂种进行培育达到性成熟后，利用褐牙鲆和夏牙鲆的精液分别与雌性杂交鲆的卵子进行母本回交实验。通过统计受精率、孵化率及杂交适合度值（CFM，受精率和孵化率相乘获得的结果）评估褐牙鲆和夏牙鲆的杂交可适度，结果表明正交及各回交组中的CFM值均显著低于褐牙鲆自交(P < 0.05)。同时，利用AFLP对回交子代基因组的变化进行了分析，发现回交中不仅存在亲本位点的丢失(褐牙鲆回交子代-回交1, 3.96%; 夏牙鲆回交子代-回交2, 6.03%)的现象，也存在非亲位点(回交1, 5.63%; 回交2, 3.28%)的现象。而且，两回交组合分别有27.40%和31.18%的AFLP标记偏离孟德尔遗传。 3. 利用线粒体DNA 16S rDNA、COⅠ基因及核基因rag1的部分序列对正反交及回交子代的线粒体及核DNA的传承进行分析，发现正反交子代的16S rDNA和线粒体DNA片段的同源性和母本一致，各回交组中16S rDNA和COⅠ基因片段与褐牙鲆的同源性较高 (98%)，这表明褐牙鲆和夏牙鲆杂交及回交遵循母性遗传规律。但在回交子代中发现16S rDNA和COⅠ基因具有多种单倍型。褐牙鲆和夏牙鲆的rag1基因具有高度的保守性，但在正交子代中发现rag1多种单倍型。 4. 进一步利用线粒体DNA的16S rDNA、COⅠ基因的部分序列对8种重要海水养殖鱼类的系统进化分析，计算了其种间的遗传距离。根据这几种鲆鲽鱼的杂交是否可行的试验结果，评价种间遗传距离与杂交可适度的关系，结果表明，这8种鲆鲽鱼类的种间遗传距离与杂交可适度呈显著的负相关 (r2 = 0.805，P < 0.01)，即种间遗传分化越大，杂交成功的可能性越小，这表明鲆鲽鱼类中可能存在物种进化的不亲和钟 (Incompatibility clock)。