Oligonucleotide ligation assay-based DNA chip for multiplex detection of single nucleotide polymorphism

An oligonucleotide ligation assay-based DNA chip has been developed to detect single nucleotide polymorphism. Synthesized nonamers, complementary to the flanking sequences of the mutation sites in target DNA, were immobilized onto glass slides through disulfide bonds on their 5' terminus. Allele-specific pentamers annealed adjacent to the nonamers on the complementary target DNA, containing 5'-phosphate groups and biotin labeled 3'-ends, were mixed with the target DNA in tube. Ligation reactions between nonamers and pentamers were carried out on chips in the presence of T4 DNA ligase. Ligation products were directly visualized on chips through enzyme-linked assay. The effect of G:T mismatch at different positions of pentamers on the ligation were evaluated. The results showed that any mismatch between pentamer and the target DNA could lead to the decrease of ligation, which can be detected easily. The established approach was further used for multiplex detection of mutations in rpoB gene of rifampin-resistant Mycobacterium tuberculosis clinical isolates. (C) 2003 Elsevier B.V. All rights reserved.

Introduction of the human lactoferrin gene into grass carp (Ctenopharyngodon idellus) to increase resistance against GCH virus

Haemorrhage can be an epidemic and fatal condition in grass carp. It is known now that the Grass Carp Haemorrhage Virus (GCHV) triggers haemorrhage. Human lactoferrin (hLF) plays an important role in the non-specific immune system, making some organisms more resistant to some viruses. Sperm of grass carp was mixed with linearized pCAhLFc, which is a DNA construct containing an hLF cDNA and the promoter of common carp beta-actin gene, and then electroporated. Then, mature eggs were fertilized in vitro with the treated sperm cells. The fry were sampled and analyzed by polymerase chain reaction (PCR). Results indicated that the foreign gene had been transferred successfully into the cells of some fry. Under optimal electroporation conditions, the efficiency of gene transfer was as high as 46.8%. About 35.7% of treated 5-month-old grass carp contained foreign genes. Most transgenic fry demonstrated significant delays in onset of symptoms of haemerrhage after injection of GCHV, suggesting a significant positive relationship between hLF cDNA and levels of disease resistance (P < 0.01). Results suggest that transgenic grass carp could be bred for increased resistance to haemorrhage. (C) 2002 Elsevier Science B.V. All rights reserved.

东亚人群 LMBR1 基因 Intron 5的遗传多样性研究

人LMBR1(Limb region 1 homolog (mouse)) 基因位于染色体7q36区域，全长约210.2 kb，含17个外显子，编码一个由490个氨基酸构成的跨膜蛋白。研究表明，LMBR1 基因的表达活性与脊椎动物四肢的手指或脚趾数目变化有关；另外，发生在其重要元件——intron 5 内的许多变异与多种表型的轴前多指症((PPD, Preaxial polydactyly)存在相关性，这主要是因为LMBR1 intron 5 内含有一个与骨骼系统发育有关的基因（SHH(Sonic hedgehog)基因）的远程顺式调控元件。本研究旨在探究LMBR1基因 intron 5 内的遗传多样性，进而评估HapMap计划的样本选择策略，并检测该区域是否受自然选择的作用。 国际人类基因组单体型图计划(HapMap Project，The International Haplotype Map Project) 于2002年10月正式启动，该计划旨在构建人类基因组中常见变异的遗传图式。自其数据发布以来，广泛应用于生物医学、群体遗传学等领域，在复杂疾病的遗传机理研究、自然选择的检测等方面做出了前所未有的贡献；但是HapMap计划中样本的代表性有待评估。 本研究中，我们综合考虑地理来源信息及线粒体单倍型类群 (Haplogroup)信息选择了41个东亚人作为样本（以保证样本的代表性），测定位于LMBR1 基因intron 5 内的目的片段中存在的单核苷酸多态性(SNP, Singe nucleotide polymorphism)位点，通过所得数据与HapMap数据的比较，发现二者之间差异显著且HapMap数据不能覆盖所有我们得到的常见变异，因而我们认为：HapMap计划中国部分的样本选择策略有待进一步完善。 关于自然选择的研究不仅可以使我们了解生物的进化机制，同时还对复杂疾病的遗传机理研究具有重要的提示作用，因而，对于自然选择的检测，一直以来都是生物学研究的重点。平衡选择是一种维持遗传多态性的自然选择方式，现已发现很多与特定疾病或性状相关的基因或调控序列受平衡选择的作用，如 G6PD 基因、PTC 基因、FMO3 基因、FSHB 基因及 CCR5 基因5’端顺式调控区等我们对41个东亚样本中LMBR1 intron 5 内一段长为9256 bp (Chr7: 156280954-156271699 (Build36))的序列进行以 Tajima’s D 检验为主的群体遗传学分析，发现该区域在进化历程中受到平衡选择的作用。LMBR1 intron 5 内的多态位点与多种表型的多指症存在相关性，受其调控的 SHH 基因在骨骼系统发育中具有重要作用，人类骨骼系统的适应性进化等三方面的因素为该区域受平衡选择的作用提供了进一步的佐证。 总之，本研究对HapMap计划的样本选择策略和数据应用提供了一定的参考；同时还发现一个与骨骼系统发育有关的基因调控元件受平衡选择的作用。

青藏高原栽培青稞遗传多样性研究

青稞，是我国藏区居民对裸大麦的称谓，它不仅是藏民的主要食粮、燃料和牲畜饲料，而且也是啤酒、医药和保健品生产的原料；青稞不仅为藏区人民的健康和经济发展做出了很大的贡献，而且对人类健康和社会经济的可持续发展都有重要的意义。青藏高原是我国及世界上青稞分布和种植面积最大的地区，资源极其丰富。虽然从经典遗传直到分子标记对我国大麦遗传多样性都有研究，但研究手段、数量仍然不够深入，对我国大麦资源遗传多样性研究的信息非常有限，不能很好地满足大麦遗传研究和育种应用的需要，尤其是对西藏栽培大麦的遗传多样性的研究还只是刚刚开始，关于栽培青稞多态性的研究报道很少。本研究采用SSR标记和蛋白质电泳两类技术，从SSR标记位点、单体醇溶蛋白、B组醇溶蛋白和淀粉粒结合蛋白（SGP）等四个方面对我国青藏高原栽培青稞的遗传多样性进行了综合评价。 SSR标记具有基因组分布广泛、数量丰富、多态性高、容易检测、共显性、结果稳定可靠、实验重现性好、操作简单、经济、易于高通量分析等许多优点，被认为是用于遗传多样性、品种鉴定、物种的系统发育、亲缘关系及起源等研究的非常有效的分子标记。本研究采用SSR标记分析了64份青藏高原栽培青稞的遗传多样性，同时评估SSR标记在我国大麦育种和品种鉴定中的应用潜力。选择了30个已知作图位点SSR标记，其中25个标记与重要性状的控制位点连锁紧密。选择的30个SSR标记，5个未得到很好的扩增产物，3个无多态性。22个多态性SSR标记位点中，每位点检测出等位基因2~15个，共检测出等位基因132个，平均每位点6.0 个。各多态位点检测出基因型为2~11种，位点HVM33的基因型最多。各多态位点的多态信息指数为0.16~0.91, 平均为0.65。根据PIC值选择了13个SSR标记用于我国青藏高原栽培青稞基因型鉴定，这些标记的PIC值为0.6以上。结合PIC值和基因型差异，选择了8个多态信息含量高的SSR标记，构建了高效指纹图谱，此图谱能把64份材料完全区分。 贮藏蛋白电泳分析是研究相关编码蛋白基因多态性的非常有效的方法。大麦单体蛋白与小麦醇溶蛋白相对应，具有丰富的多态性，可用于大麦遗传多样性、品种鉴定和群体进化等研究。本研究通过A-PAGE电泳技术研究了84份青藏高原栽培青稞的单体醇溶蛋白多态性。大麦单体醇溶蛋白图谱与小麦醇溶蛋白电泳图谱类似，所分离的蛋白清晰地分为ω－，γ－，β－和α－四个部分。青藏高原栽培青稞单体醇溶蛋白具有丰富的多态性，84份青稞材料中存在43条不同的蛋白带，75种组合带谱；其中67种为单一材料所独有，另8种则分别包含了2-3份材料。每份材料中拥有醇溶蛋白带为6－16条，含有6-10条单体醇溶蛋白带材料较多。西藏和四川材料群体单体醇溶蛋白多态性不同，具有区域特异性。西藏材料中发现了40条不同蛋白带，3条特异带，46 种蛋白组合；四川材料中出现了40种不同蛋白带，26种条带组合, 3条特异带。基于单体蛋白多态性的聚类与材料的来源有一定的相关性。A-PAGE单体蛋白具有丰富的多态性，可作为遗传研究和品种鉴定的标记。 大麦醇溶蛋白（hordein）是大麦籽粒的主要贮藏蛋白，与大麦的营养品质和加工品质密切相关，而且具有丰富的多态性，广泛用于品种鉴定、种质筛选、遗传多样性和亲缘关系研究。B组醇溶蛋白是主要的醇溶蛋白组份，约占总醇溶蛋白的80%，而且具有丰富的多态性。本研究采用SDS-PAGE分析了72份青藏高原栽培青稞B组醇溶蛋白的遗传多样性。青藏高原栽培青稞B组醇溶蛋白具有丰富的多态性，72份青稞材料中存在15种蛋白带，30种组合带谱，其中15种为单一材料所独有，另15种则分别包含了2-10份材料。每份材料中B组醇溶蛋白条带数为4-8条，含5、6条的材料较常见。不同来源的群体材料间B组醇溶蛋白组成存在差异，西藏青稞含有26种蛋白组合带谱，其中有19种特异带谱；四川群体中共发现11种蛋白组合带型，其中有4种特有带谱。两群体中都存在稀有条带。聚类分析将材料分成三组，材料聚类与材料来源地没有明显的相关性。 淀粉粒蛋白（Starch granule proteins, SGPs）是一类与淀粉粒结合的微量蛋白，一些淀粉粒蛋白具有淀粉生化合成中主要的酶蛋白功能，其变异会影响淀粉含量和特性，从而影响淀粉的应用。关于我国大麦淀粉粒组成研究还未见报道。本实验首次开创了我国大麦淀粉粒结合蛋白的研究工作。采用SDS-PAGE电泳技术研究了青藏高原栽培青稞的SGP组成，并分析了不同SGP组合间淀粉含量的差异，初步探索了所分离的SGP蛋白与淀粉合成的关系。66份青稞材料中分离了10种主要的SGP，其表观分子量为40-100KD，低于60KD的SGP带有7条，共有16种组合带谱；各SGP蛋白和组合带谱出现的频率存在差异，青藏高原青稞的SGP组成存在多态性。西藏青稞和四川青稞的SGP组成有很大差异，SGP组成具有地域差异性，西藏青稞含有12种蛋白组合带谱，其中有9种特异带谱；四川群体中共发现7种蛋白组合带型，其中有4种特有带谱；两群体中仅有3种共同的蛋白组合带谱。SGP蛋白特性将66份青稞分为三组, 即Ⅰ、Ⅱ、Ⅲ，材料聚类与材料来源具有一定的相关性。不同组合带谱材料间淀粉含量差异显著性检验结果显示，不同带谱间材料的总淀粉含量、直链淀粉含量和支链淀粉含量有差异，带谱2（SGP1+3+7+9+10）和8（SGP1+2+4+6+8）的总淀粉含量及支链淀粉含量显著大于组合带谱3（SGP1+3+7+10）的总淀粉含量。组合带谱7（SGP1+2+6+8）的直链淀粉含量显著低于带谱11（SGP1+5+8）的直链淀粉。带谱SGP2、3、4、5、6、7、8、9、10可能参与淀粉合成，SGP9可能与高支链淀粉的合成相关。 SSR标记位点、单体醇溶蛋白、B组醇溶蛋白、淀粉结合蛋白等四个方面的研究结果表明青藏高原SSR标记多态性、单体醇溶蛋白多态性、B组醇溶蛋白多态性和SGP多态性都非常丰富，与青藏高原是栽培青稞的多样性分布中心的观点一致。 青藏高原栽培青稞的SSR标记、单体醇溶蛋白、B组醇溶蛋白和SGP多态性表现出很大差异。SSR标记覆盖了整个基因组，多态性非常高。单体蛋白、B组醇溶蛋白、SGP蛋白是育种中非常关注的性状，他们只是代表基因组中的某一区域或位点，多态性相对较低。但单体蛋白多态性很高，84份材料中检测出43条不同蛋白带，75种不同的组合带谱。SSR标记技术和单体蛋白技术都是遗传多样性研究的有力工具，但单体蛋白技术不仅多态性高，而且经济、操作简便，是种质鉴定的理想方法。 对不同标记的多态性材料数据进行聚类，聚类图能为我们提供各材料间的遗传相似信息，为材料选择提供参考。但材料聚类与材料来源的地理区域的相关性表现不一致。SSR聚类和B组醇溶蛋白聚类与材料的来源地无相关性，而单体醇溶蛋白和SGP聚类与材料来源地有一定相关性，即西藏群体和四川群体分别有集中类群，这可能是人为选择的附加效应。 不同来源的群体材料的遗传多样性不同，具有区域特异稀有基因，加强不同地区间资源的交换和配合使用，有利于增加群体遗传多样性和新品种培育。 青藏高原栽培青稞的麦芽浸提性状、淀粉性状、病虫及裸粒等重要农艺性状控制位点存在丰富的变异，遗传基础宽广，可能蕴藏着多种不同的等位基因，是研究重要性状遗传特性、基因资源挖掘和遗传育种的宝贵资源库。 Hulless barley, due to its favorable attributes such as high feed value, good human nutrition，rich dietary fiber and ease processing, attracts people,s attention . Hulless barley plays a very important role in Tibetan life, used as essential food crop, main animal feed and important fuel. In addition to tsampa (roasted barley flour), a main food for Tibetan, hulless barley is also made into cake, soup, porridge, recent naked barley liquor and cornmeal. Qinghai-Tibet Plateau is one of a few areas which plant naked barley widely in the world and also has a long growing history. Genetic diversity of the cultivated hulless barley in this region , however, has not been documented. The study of genetic diversity existing within this population is of particular interest in germplasm identification, preservation, and new cultivar development. This study analyzed the genetic diversity of the cultivated naked barley from Qinghai-Tibet plateau through the study of SSR marker loci and monomeric prolamins, B-horden and starch granule proteins. SSRs are present abundantly in genomes of higher organisms and have become a popular marker system in plant studies. SSRs offer a number of advantages, such as the high level of polymorphisms, locus specificity, co-dominance, reproducibility, ease of use through PCRand random distribution throughout the genome. In barley, several hundred SSRs have been developed and genetically mapped and can therefore be selected from specific genomic regions. The genetic diversity of 64 cultivated naked barley from Tibet and Sichuan was studied with 30 SSRs of known map location.Among the selected SSR markers, PCR products of 5 SSR markers were not obtained and 3 SSR marker loci were monomeric. A total of 132 alleles were identified at 22 polyomeric SSR loci. The number of alleles per locus ranged from 2 to 15, with an average of 6.0. The polymorphism information content values for the SSRs ranged from 0.08 to 0.94, with an average of 0.65. 13 SSR markers with the PIC value >0.6 have been selected for discrimination of Qinghai-Tibet naked barley genotypews. A finger Print map was developed through 7 SSR markers with the high PIC value. It could be used as an efficient tool for gene discovery and identification of gernplasm. Hordeins, the main storage proteins of the barley seed, are composed of momomeric and polymeric prolamins and divided into -A, B, C and D groups in order of decreasing electrophoretic mobility. Hordeins show high inter-genotypic variation and have been extensively used as markers for cultivar identification and analyzing the genetic diversity. This study analyzed the genetic diversity of B-hordein in 72 naked barley from Qinqhai-Tibet Plateau. Extensive diversity was observed. A total of 15 different bands and 30 distinct patterns were found. Jaccard's coefficient of similarity was calculated, and the accessions were divided into three　main groups by cluster analysis using UPGMA. Differentiation among the populations from different collecting regions based on the polymorphism of B-hordein was investigated. Monomeric prolamins show high inter-genotypic variation and have been used as molecular markers for cultivar identification, analyzing the genetic diversity in collections and investigating the evolution processes and structure of populations However, the cultivated hulless accessions from Qinghai-Tibet Pateau in China have never been examined with respect to monomeric prolamins. This study analyzed the genetic diversity of monomeric prolamins (protein fraction corresponding to wheat gliadins) using the Acid -PAGE technique in eighty-four cultivated hulless barley from Qinqhai-Tibet Plateau in China. Extensive diversity was observed. A total of 43 different bands were found, of which 21 different bands were in the region of ω group, 8 in the region of γ, 8 in the region of β, and 6 in the region of α group. Among the 86 accessions, 75 distinct patterns were identified. The number of bands ranged from 6 to 16, depending on the variety. Jaccard’s coefficient of similarity was calculated, and the lines were grouped by cluster analysis using UPGMA. A dendrogram was obtained from the analysis of the groups and five main clusters were identified. No relationship between the distribution in the dendrogram and growth habits and origins of the cultivars could be detected. Starch is the major constituent of the cereal endosperm, comprising approximately 65% of the dry weight of the mature wheat grain. The starch formed in all organs of plants is packaged into starch granules, which vary widely between species and cultivars in size and shape. Wheat endosperm starch granules contain about corresponding to the main biosynthase of starch. This report firstly dealed with intraspecific variation of the major SGPs in cultivated naked barley from Qinghai-Tibet plateau. A total of 10 major SGPs were observed in the range of 40KD-100KD and 16 types of patterns were found. Based on the variation of SGPs, accessions studied were classified into 3 groups. A geographical cline of electrophoregram was observed. In addition, significance test of the difference of starch content among groups and types of patterns were done, and the results indicated those SGPs could be related to the content of starch. Diagram obtained through cluster analysis exhibited a structuration of diversity and genetic relationship among cultivated hulless accessions. In breeding program, parents with genetically distant relationship for hybridization will increase genetic diversity of progenies. In conclusion, cultivated naked barley from Qinghai-Tibet Plateau in China presents a high variability with respect to monomeric prolamins，SSR markers , B- hordeins and SGPs. The result of this study supports Qinghai-Tibet Plateau is the center of cultivated hulless barley and the cultivated naked barley is considered to be a gene pool with large diversity and could be applied to breeding for cereal.

Radiosensitivity of hepatoma cell lines and human normal liver cell lines exposed in vitro to carbon ions and argon ions at the HIRFL

Human hepatoma (SMMC-7721) and normal liver (L02) cells were irradiated with c-rays, 12C6+ and 36Ar18+ ion beams at the Heavy Ion Research Facility in Lanzhou (HIRFL). By using the Calyculin-A induced premature chromosome condensation technique, chromatid-type breaks and isochromatid-type breaks were scored separately. Tumor cells irradiated with heavy ions produced a majority of isochromatid break, while chromatid breaks were dominant when cells were exposed to c-rays. The relative biological effectiveness (RBE) for irradiation-induced chromatid breaks were 3.6 for L02 and 3.5 for SMMC-7721 cell lines at the LET peak of 96 keVlm 1 12C6+ ions, and 2.9 for both of the two cell lines of 512 keVlm 1 36Ar18+ ions. It suggested that the RBE of isochromatid-type breaks was pretty high when high-LET radiations were induced. Thus we concluded that the high production of isochromatid-type breaks, induced by the densely ionizing track structure, could be regarded as a signature of high-LET radiation exposure.

Prematurely condensed chromosome fragments in human lymphocytes induced by high doses of high-linear-energy-transfer irradiation

This study provides a useful biodosimetry protocol for radiation accidents that involve high doses of heavy particle radiation. Human peripheral blood lymphocytes (PBLs) were irradiated in vitro with high doses (5–50 Gy) of charged heavy-ion particles (carbon ions, at an effective linear-energy-transfer (LET) of 34.6 keV/ m), and were then stimulated to obtain dividing cells. PBLs were treated with 100nMcalyculin A to force chromosomes to condense prematurely, and chromosome spreads were obtained and stained with Giemsa. The G2 prematurely condensed chromosome (G2-PCC) index and the number of G2-PCC including fragments (G2-PCC-Fs) per cell for each radiation dose point were scored. Dose-effect relationships were obtained by plotting the G2-PCC indices or G2-PCC-Fs numbers against radiation doses. The G2-PCC index was greater than 5% up to doses of 15 Gy; even after a 30Gy radiation dose, the index was 1 to 2%. At doses higher than 30 Gy, however, the G2-PCC indices were close to zero. The number of G2-PCC-Fs increased steeply for radiation doses up to 30 Gy at a rate of 1.07 Gy−1. At doses higher than 30 Gy, the numbers of G2-PCC-Fs could not be accurately indexed because of the limited numbers of cells for analysis. Therefore, the number of G2-PCC-Fs could be used to estimate radiation doses up to 30 Gy. In addition, a G2-PCC index close to zero could be used as an indicator for radiation doses greater than 40 Gy.

Effect of 80.55 MeV/u(12)C(6+) ions on radiosensitivity and cell cycle of human hepatoma cell lines

In this paper, the relationship between radiosensitivity, cell cycle alteration and the change of apoptosis in different human hepatoma cell lines irradiated by heavy ions were studied with the aim of building up the base data for clinical therapy. Exponentially growing hepatoma cell lines were irradiated by 80.55 MeV/u(12)C(6+) ions at a dose of 0 Gy, 0.5 Gy, 1 Gy, 2 Gy, 4 Gy and 8 Gy. The radiosensitivity was assessed by means of the colony-forming assay. The DNA content, the percentage of each cell-cycle phase and the apoptosis rate were obtained with flow cytometry methods. After the irradiation, the SF2 (survival fraction at 2 gray) of SMMC-7721 cells were evidently lower than that of HepG2 cells. The S phase arrest, G2/M phase arrest delay and the apoptosis in the two hepatoma cell lines varied with the increase of the dose and repair time. The heavy ions could obviously kill the human hepatoma cell lines. Compared to HepG2 cells, SMMC-7721 cells were more radiosensitive to C-12(6+) ions.

Radiobiological response of human hepatoma and normal liver cells exposed to carbon ions generated by Heavy Ion Research Facility in Lanzhou

Human hepatoma and normal liver cells were irradiated with C-12(6+), ion beams (LET= 96.05 keV/mu m) and gamma-rays at Heavy Ion Research Facility in Lanzhou (HIRFL). The chromatid breaks and break types were detected using the premature chromosome condensation technique. Our experimental results showed that chromatid breaks seem to have a good relation with C-12(6+) absorbed dose and C-12(6+) are more effective to induce chromatid breaks as compared to they-rays. For C-12(6+) ion irradiation the major break was isochromatid break, while chromatid breaks were dominant for gamma-ray irradiation. We also observed that the Relative Biology Effectiveness (RBE) of C-12(6+) ion is about 2.5 times higher than that of gamma-rays.

High-efficiency transfer and expression of AdCMV-p53 in human hepatocellular carcinoma cells induced by low-dose carbon-ion radiation

Objective To investigate whether the irradiation with C-beam could enhance adenovirus-mediated transfer and expression of p53 in human hepatocellular carcinoma. Materials and methods HepG2 cells were exposed to C-beam or gamma-ray and then infected with replicationdeficient adenovirus recombinant vectors containing human wild-type p53 or green fluorescent protein, respectively. The transfer efficiency and expression level of the exogenous gene were detected by flow cytometric analysis. Cell survival fraction was detected by clonogenic assay. Results The transfer frequency in C-beam or gamma-irradiated groups increased by 50-83% and 5.7-38.0% compared with the control, respectively (P < 0.05). Compared with C-beam alone, p53 alone, and gamma-ray with p53, the percentages of p53 positive cells for 1 Gy C-beam with p53 increased by 56.0-72.0%, 63.5-82.0%, and 31.3-72.5% on first and third day after the treatments, respectively (P < 0.05). The survival fractions for the 2Gy C-bearn and AdCMV-p53 infection groups decreased to similar to 2%. Conclusion C-beam irradiation could significantly promote AdCMV-green fluorescent protein transfer and expression of p53.

Radio-resistance induced by nitric oxide to heavy ion irradiation in A172 human glioma cells

To investigate effects of nitric oxide on cellular radio-sensitivity, three human glioma cell lines, i.e. A172, A172 transfected green fluorescence protein (EGFP) gene (EA172) and A172 transfected inducible nitric oxide synthesis (iNOS) gene (iA72), were irradiated by C-12(6+) ions to 0, 1 or My. Productions of nitric oxide and glutathione (GSH) in A172, EA172 and iA172 were determined by chemical methods, cell cycle was analyzed by flow cytometry at the 24th hour after irradiation, and survival fraction of the cells was measured by colorimetric MTT assay at the 5th day after irradiation. The results showed that the concentrations of nitric oxide and GSH in iA172 were significantly higher than in A172 and EA172; the G(2)/M stage arrest induced by the C-12(6+) ion irradiation was observed in A172 and EA172 but not in iA172 at the 24th hour after exposure; and the survival fraction of iA172 was higher than that of EA172 and iA172. Data suggest that the radio-sensitivity of the A172 was reduced after the iNOS gene transfection. The increase of GSH production and the change of cellular signals such as the cell cycle control induced by nitric oxide may be involved in this radio-resistance.

Early effects of low dose C-12(6+) ion or X-ray irradiation on human peripheral blood lymphocytes

The aim of this study was to estimate the acute effects of low dose C-12(6+) ions or X-ray radiation on human immune function. The human peripheral blood lymphocytes (HPBL) of seven healthy donors were exposed to 0.05 Gy C-12(6+) ions or X-ray radiation and cell responses were measured at 24 h after exposure. The cytotoxic activities of HPBL were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT); the percentages of T and NK cells subsets were detected by flow cytometry; mRNA expression of interleukin (IL)-2, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were examined by real time quantitative RT-PCR (qRT-PCR); and these cytokines protein levels in supematant of cultured cells were assayed by enzyme-linked immunosorbent assays (ELISA). The results showed that the cytotoxic activity of HPBL, mRNA expression of IL-2, IFN-gamma and TNF-alpha in HPBL and their protein levels in supernatant were significantly increased at 24 h after exposure to 0.05 Gy C-12(6+) ions radiation and the effects were stronger than observed for X-ray exposure. However, there was no significant change in the percentage of T and NK cells subsets of HPBL. These results suggested that 0.05 Gy high linear energy transfer (LET) C-12(6+) radiation was a more effective approach to host immune enhancement than that of low LET X-ray. We conclude that cytokines production might be used as sensitive indicators of acute response to LDL (C) 2009 COSPAR. Published by Elsevier Ltd. All rights reserved.

A single-strand conformation polymorphism method by capillary electrophoresis with laser-induced fluorescence for detection of the T1151A mutation in hMLH1 gene

Mutation of hMLH1 gene plays an important role in human tumorigenesis. A highly sensitive single-strand conformation polymorphism (SSCP) method for detection of the T1151A mutation in exon 12 of the hMLH1 gene was for the first time developed employing laser-induced fluorescence capillary electrophoresis (LIF-CE). Effects of the concentration of linear polyacrylamide solution, running temperature, running voltage and the addition of glycerol on SSCP analysis were investigated, and the optimum separation conditions were defined. Thirty colorectal cancer patients and eight lung cancer patients were screened and the T1151A mutation was found in four of them. Based on CE-sequencing the mutation was further confirmed. To our knowledge, this is for the first time that the T1151A mutation is found in lung cancer. Our method is simple, rapid, and highly sensitive and is well suited to the analysis of large numbers of clinical samples.

Study on the multiple sites binding of human serum albumin and porphyrin by affinity capillary electrophoresis

Equations to describe the two sites binding between proteins and ligands were deduced. According to these equations, not only the binding constants, but also the mole fraction of proteins in different forms could be obtained. Using the published data on the interaction between human serum albumin (HSA) and three kinds of porphyrin (coproporphyrin (CP), uroporphyrin I (UP) and protoporphyrin (PP)), a further study on their binding was carried out. It was concluded that there may exist two binding sites with the binding constants at the first site. proved to be the preferential one, being 6.50 x 10(5) 1.94 x 10(6) and 8.94 x 10(5). respectively. In addition. it was also demonstrated that the two binding sites of HSA with CP and UP might be of different kinds, though those of HSA and PP were of the same kind but at different positions. (C) 2002 Elsevier Science B.V. All rights reserved.