拟南芥AST基因的精细作图

拟南芥ast (anthocyanin spotted testa) 突变体是由碳离子束诱导产生的与花青苷生物合成有关的突变体，受单隐性核基因控制。由于花青苷的异常积累，突变体未成熟种子的种皮呈现紫红色的斑点;野生型植株幼嫩的种皮没有花青苷的异常积累，呈淡绿色。初步作图分析表明，AST基因定位于拟南芥第I号染色体上，并且位于SSLP分子标记nga280和CAPS分子标记PAB5之间。 AST基因与SSLP分子标记nga280紧密连锁，遗传距离为3.2cM;与CAPS分子标记PAB5相距较远，遗传距离为21.1cM。 采用DDRT-PCR的策略，分析野生型与突变型植株未成熟角果中基因表达的差异。通过调整DDRT-PCR中总RNA、锚定引物、随机引物、cDNA和dNTP等关键试剂的用量，优化了适用于银染检测的DDRT-PCR方法。PCR扩增产物经6%变性聚丙烯酰胺凝胶垂直电泳分离后，银染能检测到多而清晰的条带。泳道中的条带数最少为40个，最多达80个，平均为60个，条带大小分布在100bp-900bp范围，银染的灵敏度为5pg/mm2。此方法操作简便快速，灵敏度高，重复性好。采用这个改良的的方法，分析了拟南芥野生型和ast突变型植株未成熟角果中16,000个cDNA扩增产物条带，从中筛选出28个差异条带。二次PCR扩增后，进一步筛选出10个差异表达的cDNA条带，其中6个是野生型特异表达的，4个是突变型特异表达的。对这10个差异片段进行测序。BLASTN分析表明，这10个差异表达的cDNA片段与数据库中花青苷生物合成途径中的结构基因和调节基因序列没有同源性，表明用DDRT-PCR的方法克隆特定的AST基因有一定的局限性。 利用图位克隆（map-based cloning）的策略，对拟南芥AST 基因进行克隆。根据拟南芥数据库中的SNPs (simple nucleotide polymophisms) 序列和插入/缺失多态性（insertion/deletion polymorphisms）序列，设计了一系列分子标记。利用这些分子标记，对600个F2代有突变表型的植株进行重组子筛选，完成了对拟南芥AST基因的精细作图，成功地将AST 基因定位到BAC克隆T13M11上。初步确定该BAC克隆中的基因T13M11.8 可能是AST基因。该基因的DNA序列长1432bp，含有6个外显子和5个内含子，编码的蛋白与花青苷生物合成途径中的二氢黄酮醇4-还原酶有较高的同源性。功能互补实验正在进行当中。

鱼肉结合态MCLR的亚慢性毒性研究

为了揭示含有微囊藻毒素水产品的食用安全性,以雄性BALB/C小鼠为模式生物,通过为期13周的灌胃染毒实验,研究了鱼肉结合态MCLR对小鼠的亚慢性毒性作用,并与水溶态MCLR的毒性进行了对比.结果表明,68.75μg/kg(以单位体重计)剂量水溶态MCLR可引起小鼠肝脏显著系数增加(p<0.05),ALT、AST活性增强(p<0.01),引起肝细胞出现有空泡状病变;而肾脏酶学指标BUN、Cr及肾脏组织病理学观察均未发现明显异常;与之相比,同剂量鱼肉结合态MCLR对小鼠各项指标的影响并不明显,仅在实验第1周出

Plasma biochemical responses of the planktivorous filter-feeding silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis) to prolonged toxic cyanobacterial blooms in natural waters

The planktivorous filter-feeding silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis) are the attractive candidates for bio-control of plankton communities to eliminate odorous populations of cyanobacteria. However, few studies focused on the health of such fishes in natural water body with vigorous toxic blooms. Blood parameters are useful and sensitive for diagnosis of diseases and monitoring of the physiological status of fish exposed to toxicants. To evaluate the impact of toxic cyanobacterial blooms on the planktivorous fish, 12 serum chemistry variables were investigated in silver carp and bighead carp for 9 months, in a large net cage in Meiliang Bay, a hypereutrophic region of Lake Taihu. The results confirmed adverse effects of cyanobacterial blooms on two phytoplanktivorous fish, which mainly characterized with potential toxicogenomic effects and metabolism disorders in liver, and kidney dysfunction. In addition, cholestasis was intensively implied by distinct elevation of all four related biomarkers (ALP, GGT, DBIL, TBIL) in bighead carp. The combination of LDH, AST activities and DBIL, URIC contents for silver carp, and the combination of ALT. ALP activities and TBIL, DBIL. URIC concentrations for bighead carps were found to most strongly indicate toxic effects from cyanobacterial blooms in such fishes by a multivariate discriminant analysis. (C) 2009 Elsevier B.V. All rights reserved.

Mouse Toxicity of Anabaena flos-aquae from Lake Dianchi, China

Some species of the genera Anabaena can produce various kinds of cyanotoxins, which may pose risks to environment and human health. Anabaena has frequently been observed in eutrophic freshwater of China in recent years, but its toxicity has been reported only in a few studies. In the present study, the toxicity of an Anabaena flos-aquae strain isolated from Lake Dianchi was investigated. Acute toxicity testing was performed by mouse bioassay using crude extracts from the lyophilized cultures. The mice exposed to crude extracts showed visible symptoms of toxicity and died within 10-24 h of the injection. Serum biochemical parameters were evaluated by the use of commercial diagnostic kits. Significant alterations were found in the serum biochemical parameters: alkaline phosphatase (AKP), gamma-glutamyl transpepticlase (gamma-GT), aspartate amino transferase (AST), alanine amino transferase (ALT), AST/ALT ratio, total protein content, albumin content, albumin/globulin (A/G) ratio, blood urea nitrogen (BUN), serum creatinine (Ssr), and total antioxidative capacity (T-AOC). Histopathological observations were carried out with hematoxylin and eosin (HE) stain under light microscope. Severe lesions were seen in the livers, kidneys, and lungs of the mice injected with crude extracts. The alterations of biochemical parameters were in a dose-dependent manner, and the severities of histological lesions were in the same manner. Based on biochemical and histological studies, this research firstly shows the presence of toxin-producing Anabaena species in Lake Dianchi and the toxic effects of its crude extracts on mammals. (C) 2008 Wiley Periodicals, Inc. Environ Toxicol 24: 10-18, 2009.

Plasma biochemical responses of the omnivorous crucian carp (Carassius auratus) to crude cyanobacterial extracts

Healthy crucian carp (Carassius auratus) were treated by intraperitoneal (i.p.) injection of crude cyanobacterial extracts at two doses, 50 and 200 mu g MC-LR equiv kg(-1) BW. High mortality (100%) was observed within 60 h post injection in the high-dose group. In the treated fish, activities of four plasma enzymes, alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), all showed substantial increases, with both dose and time-dependent effects. These increases of enzyme activity indicate severe impairment occurred in the liver of crucian carp over time. Plasma concentrations of energy-related biomolecules including glucose (GLU), cholesterol (CHO), triglyceride (TG), and total protein (TP) showed marked changes in the high-dose group, possibly a nutritional imbalance correlated with the liver injury caused by intraperitoneal exposure to crude cyanobacterial extracts.

Sequential ultrastructural and biochemical changes induced in vivo by the hepatotoxic microcystins in liver of the phytoplanktivorous silver carp Hypophthalmichthys molitrix

Up to now, in vivo studies on the toxic effects of microcystins (MCs) on the ultrastructures of fish liver have been very limited. The phytoplanktivorous silver carp was injected i.p. with extracted hepatotoxic microcystins (mainly MC-RR and -LR) at a dose of 1000 mu g MC-LReq. kg(-1) body weight, showing a time-dependent ultrastructural change in liver as well as significant increases in enzyme activity of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). We observed for the first time the occurrence of a large amount of activated secondary lysosomes, which might be an adaptive mechanism to eliminate or lessen cell damage caused by MCs through lysosome activation. Quantitative and qualitative determinations of MCs in the liver were conducted by HPLC and LC-MS2, respectively. MCs concentration in the liver reached the maximum (114.20 mu g g(-1) dry weight) after 3 h post-injection, and then rapidly dropped to 7.57 mu g g(-1) dry weight at 48 h, indicating a deputation of 99% accumulated MC-LReq. On the other hand, a decrease trend in glutathione (GSH) concentration was observed in the liver of silver carp while the activity of glutathione S-transferase (GST) increased significantly after injection. The high tolerance of silver carp to MCs might be due to the high basic GSH level in their liver, and/or an increased GSH synthesis. (C) 2007 Elsevier Inc. All rights reserved.

automated test program generation for an industrial optimizing compiler

Association for Computing Machinery, ACM; IEEE; IEEE Computer Society; SIGSOFT

内蒙古鄂温克族牧民砖茶型氟铝联合中毒调查

目的调查鄂温克族牧民砖茶型氟铝联合中毒病情。方法随机对鄂温克族16~55岁男女均衡62名牧民进行发、血清、尿氟铝水平及血清电解质、生物化学检查,同时检测饮水、牛奶、砖茶、奶茶等氟铝水平,并进行临床检诊和拍摄前臂、骨盆正位X线片,不饮用砖茶的16~55岁男女均衡41名居民为对照组。结果发、血清、尿铝水平牧民组分别为(24.60±9.82)mg/Kg、(0.78±0.25)、(2.84±0.65)mg/L,对照组分别为(12.50±4.55)mg/kg、(0.18±0.11)、(2.09±0.64)mg/L;发、血清、尿氟水平牧民组分别为(1.00±0.52)mg/kg、(0.10±0.10)、(2.74±1.42)mg/L,对照组分别为(1.39±1.34)mg/kg、(0.04±0.03)、(0.87±0.43)mg/L;牧民发、血、尿铝及血、尿氟显著高于对照组(P<0.01)。牧民组血清AST、LDH、HBDH、ALP显著高于对照组(P<0.01)。X线摄片牧民组氟骨症检出率50.00%,关节退行性变检出率61.29%,骨间膜骨化33.87%,疏松型改变检出率29.03%,硬化型改变检出率8.06%;对照组氟骨症检出率2.44%,退行性变检出率34.15%,疏松型改变检出率21.95%,骨间膜骨化检出率2.44%,硬化型改变2.44%,X线氟骨症、关节退行性变、骨间膜骨化牧民组均高于对照组(P<0.01)。结论内蒙古鄂温克族牧民饮茶型氟中毒病情较为严重,其发病机理较为复杂,并导致多组织、器官损伤,可能为砖茶型氟铝联合中毒。

Capillary electrophoresis with electrochemiluminescence detection for measurement of aspartate aminotransferase and alanine aminotransferase activities in biofluids

A new sensitive assay for aspartate aminotransterase (AST) and alanine aminotransferase (ALT) activities in biofluids was developed, based on the separation and detection of alanine, glutamate, and aspartate using capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection. The three amino acids were separated in 5 mM phosphate of pH 2.1 as background electrolyte, and detected on a 500 mu m platinum disk electrode at 1.2 V (versus Ag/AgCl) in the presence of 10 mM tris(2,2'-bipyridyl)ruthenium(II) dissolved in 80 mM phosphate of pH 10.5. A mass detection limit of 37.3 fmol (or 81.5 fmol) for glutamate, corresponding to the product in the enzyme reaction catalyzed by 1.24 x 10(-9) U AST (or 2.72 x 10(-9) U ALT) in a 30 min reaction period, was achieved. This assay was applied to investigate the cytotoxicity effect of ethanol on HepG2 cells and differentiating nonalcoholic steatohepatitis (NASH) from alcoholic liver disease, indicating that the technique is promising for the application in the cell biological and clinical fields.

混合稀土对大鼠肝脏中7种酶的小剂量刺激作用

研究了常乐(用于肥料添加剂的一种混合稀土硝酸盐)对Wistar大鼠肝脏中7种常见酶(ALT,ICD,AST,LAP,ALP,γGT ,CHE)活性的变化,并用电感耦合等离子体质谱(ICP MS)测定了稀土在肝脏中的累积情况。结果表明,稀土对多种酶表现出低剂量激活、高剂量抑制的小剂量刺激作用 (Hormesis),但产生刺激作用的剂量随酶的种类不同而不同 ,并表现出明显的性别差异

藻蓝蛋白复合物对酒精性肝损伤的保护作用

本文对藻蓝蛋白复合物保护化学性肝损伤的功效学进行了研究。以Wistar大鼠建立酒精性肝损伤模型，将藻蓝蛋白复合物分为高、中、低三个剂量组，以联苯双酯作为阳性对照组，灌胃给药42d，分别测定血清谷丙转氨酶（ALT）、谷草转氨酶（AST）、血清丙二醛（MDA）、谷胱甘肽（GSH）、肝匀浆丙二醛（MDA）和谷胱甘肽（GSH）的含量，并对肝脏切片进行病理检查。结果表明：藻蓝蛋白复合物对血清ALT、AST具有显著的抑制作用，显著拮抗肝脏MDA的升高，显著提高肝脏GSH-Px含量。出现肝细胞浊肿、脂肪变性、点状坏死的大鼠数目极少。从而表明藻蓝蛋白复合物具有显著的保护酒精性肝损伤的功效。

柴达木盆地唐古特白刺籽油保护肝损伤作用研究

对柴达木盆地唐古特白刺种籽油保护化学性肝损伤作用进行了研究.将小鼠按体重随机分为白刺籽油高、中、低剂量组,另设空白对照组和联苯双酯、藏茵陈阳性对照组.小鼠连续灌胃给药15 d后,用CCl4进行肝损伤,测定小鼠血清谷丙转氨酶(ALT)、谷草转氨酶(AST);肝脏过氧化脂质(LPO)降解产物丙二醛(MDA)和谷胱甘肽过氧化物酶(GSH-Px)的含量.结果显示,白刺籽油对血清ALT、AST具有显著的抑制作用,显著拮抗肝脏MDA的升高,显著提高肝脏GSH-Px含量.表明白刺籽油具有明显的保护化学性肝损伤的保健作用.

青海黄芪属新分类群

青海黄芪属新分类群:贵南黄芪Astragalus guinanicus ; 都兰黄芪Ast ragalusclulanensis ;白花松潘黄芪Astragalus polyclandous ;大花多枝黄芪Ast ragaluspolyclaclus ;长苞东俄洛黄芪Ast ragalus tongolensis ; 少毛格尔木黄芪Astragalus goluensis ;多毛多花黄芪Astragalus floridus ;白花丛生黄芪Astragalus confertus