Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono- and dicotyledonous plant cells.

The expression of the jellyfish green fluorescent protein (GFP) in plants was analyzed by transient expression in protoplasts from Nicotiana tabacum, Arabidopsis thaliana, Hordeum vulgare, and Zea mays. Expression of GFP was only observed with a mutated cDNA, from which a recently described cryptic splice site had been removed. However, detectable levels of green fluorescence were only emitted from a small number of protoplasts. Therefore, other mutations in the GFP cDNA leading to single-amino acid exchanges in the chromophore region, which had been previously studied in Escherichia coli, were tested in order to improve the sensitivity of this marker protein. Of the mutations tested so far, the exchange of GFP amino acid tyrosine 66 to histidine (Y66H) led to detection of blue fluorescence in plant protoplasts, while the exchange of amino acid serine 65 to cysteine (S65C) and threonine (S65T) increased the intensity of green fluorescence drastically, thereby significantly raising the detection level for GFP. For GFP S65C, the detectable number of green fluorescing tobacco (BY-2) protoplasts was raised up to 19-fold, while the fluorimetricly determined fluorescence was raised by at least 2 orders of magnitude.     

胰岛素样生长因子Ⅰ与肝纤维化

胰岛素样生长因子I(IGF-I)是体内普遍存在的多肽,循环系统中IGF-I主要来源于肝脏．在垂体生长激素的调控下,IGF-I对多种细胞如成纤维细胞、成骨细胞、平滑肌细胞等的有丝分裂均有调节作用．目前观点认为肝星状细胞(HSC)活化后可分泌大量胶原纤维,是肝纤维化时细胞外基质的主要来源．实验表明 IGF-I能够促进体外培养HSC增殖、活化并抑制其凋亡．而体内研究发现,肝硬化患者血清IGF-I浓度显著下降,外源性小剂量IGF-I 注射能够改善肝功能,为肝纤维化的治疗提供了新的理念．     

Detection of the hemoglobin E mutation using the color complementation assay: application to complex genotyping

The color complementation assay (CCA) is a method of allele-specific DNA amplification by which competitive priming and extension of fluorescently labeled oligonucleotide primers determine the color of DNA amplification product. This diagnostic method precludes the need for radioisotopes, electrophoresis, and multiple high-stringency reaction conditions. The multiplicity of mutant globin genes present in Southeast Asians complicates clinical diagnosis and underscores the importance of DNA-based diagnostic methods. We have applied CCA to distinguish beta A and beta E alleles. Competing 15mer primers were a fluorescein-labeled complement to beta A and a rhodamine-labeled complement to beta E, identical except for their central nucleotides. A common unlabeled primer was used to amplify DNA product, the color of which was determined by the perfectly complementary primer. Color photography and spectrofluorometry, as well as a method of black-white photography that we developed to distinguish fluorescein- and rhodamine- labeled DNA, were used to record results. We applied CCA to define the complex genotype of a Thai woman with thalassemia intermedia, 96% HbE, and 4% HbF whose possible genotypes included several permutations of alpha-thalassemia, beta-thalassemia, and beta E genes. zeta-Globin gene mapping of DNA doubly digested with Bg/II and Asp 718 showed the -alpha 3.7/--SEA genotype, and CCA confirmed homozygous beta E/beta E. The CCA is useful for diagnosing the compound hemoglobin genotypes of Southeast Asians and could be applied also to prenatal diagnosis in this population.     

Molecular analysis of relapse in chronic myeloid leukemia after allogeneic bone marrow transplantation

Four patients with Philadelphia (Ph') positive chronic myeloid leukemia (CML) were studied before, after, and on relapse following allogeneic bone marrow transplantation (BMT). Southern analysis of DNA from cells collected before and at relapse after BMT was performed in order to investigate the origin of the leukemia at relapse. Using minisatellite probes we showed that the relapse occurred in cells of host origin in all four patients and this was confirmed with a Y chromosome specific probe in two male patients who had a female donor. Furthermore, using two probes for the breakpoint cluster region (bcr) on chromosome 22, we showed that leukemic cells at relapse bore identical rearrangements to those in the disease at time of presentation of each patient. We conclude that relapse in all four patients is due to re-emergence of the original leukemic clone.     

四种中成药对气血双虚模型小鼠血象及免疫水平的影响

目的:为艾滋病抗病毒疗法所致的骨髓不良反应筛选疗效确切的中成药,观察分析参芪颗粒、复方阿胶浆、贞芪扶正颗粒、复方皂矾丸四种中成药对放血和注射环磷酰胺联合复制的气血双虚模型小鼠血象及免疫水平的影响。方法:实验于2005-08/09在河南中医学院药理实验室完成。①参芪颗粒(江西山高制药有限公司生产,批号040702);复方阿胶浆(山东东阿阿胶股份有限公司生产,批号050446);贞芪扶正颗粒(甘肃扶正药业科技股份有限公司生产,批号040803);复方皂矾丸(陕西郝其军制药有限责任公司生产,批号041014);当归补血口服液(郑州市协和制药厂生产,批号041122);环磷酰胺(上海华联制药有限公司生产,批号050101)。②选取清洁级昆明种小鼠150只,随机数字表法分为15组,10只/组:1～3组分别灌服参芪颗粒混悬液3,2,1g/kg;4～6组分别灌服复方阿胶浆30,20,10mL/kg;7～9组分别灌服贞芪扶正颗粒混悬液15,10,5g/kg;10～12组分别灌服复方皂矾丸混悬液2.4,1.6,0.8g/kg;第13组灌服当归补血口服液10g/kg;剩余2组为空白对照组和模型对照组,分别给于同体积生理盐水10g/kg。各组给药1次/d,连续给药10d。③除空白对照组外,其他各组从给药第1天开始建立气血双虚模型。每只鼠尾部放血0.25mL/10g,然后分别于第2,4,6,8天腹腔注射环磷酰胺80,40,40,40mg/kg。空白对照组同时间点仅腹腔注射等体积生理盐水。末次注射环磷酰胺后2h,眼眶取血,一部分用于血象测定,另一部分离心取血清,测定血细胞比容及血清中巨噬细胞集落刺激因子水平;解剖取胸腺和脾脏,检测胸腺皮质厚度、胸腺淋巴细胞数、脾小结大小、脾脏淋巴细胞数病理学指标的变化。结果:150只小鼠全部进入结果分析,放血和注射环磷酰胺并用可成功建立小鼠气血双虚模型。①与模型对照组比较,参芪颗粒3g/kg组、贞芪扶正颗粒10,5g/kg组、复方皂矾丸1.6g/kg组均可升高气血双虚模型小鼠白细胞水平(t=2.18～2.74,P<0.05),贞芪扶正颗粒15g/kg组作用更为显著(t=2.98,P<0.01);参芪颗粒1g/kg组、复方阿胶浆20mL/kg组、贞芪扶正颗粒15,10g/kg组均可升高红细胞水平(t=2.44～2.69,P<0.05),复方阿胶浆30mL/kg组、贞芪扶正颗粒5g/kg组、复方皂矾丸2.4,1.6g/kg组作用更为显著(t=2.91～3.66,P<0.01);当归补血口服液组、复方阿胶浆20mL/kg组、参芪颗粒3,1g/kg组、贞芪扶正颗粒15,10,5g/kg组均可升高血红蛋白水平(t=2.27～2.85,P<0.05),复方阿胶浆30mL/kg组、复方皂矾丸2.4,1.6g/kg组作用更为显著(t=3.07～4.04,P<0.01);当归补血口服液组、参芪颗粒3,2g/kg组均可升高血小板水平(t=2.20～2.41,P<0.05)。②与模型对照组比较,参芪颗粒2g/kg组、贞芪扶正颗粒5g/kg组均可升高气血双虚模型小鼠血细胞比容(t=2.01～2.62,P<0.05),参芪颗粒1g/kg组、复方阿胶浆30,20,10mL/kg组、贞芪扶正颗粒15,10,5g/kg组、复方皂矾丸2.4,1.6,0.8g/kg组作用更为显著(t=3.18～4.36,P<0.01);参芪颗粒2g/kg组、复方阿胶浆30,20,10mL/kg组、贞芪扶正颗粒15,10,5g/kg组、复方皂矾丸2.4,1.6g/kg组均可显著升高巨噬细胞集落刺激因子水平(t=3.60～6.80,P<0.01)。③与模型对照组比较,当归补血口服液组、参芪颗粒3,2,1g/kg组、复方阿胶浆30,20,10mL/kg组、贞芪扶正颗粒15,10,5g/kg组、复方皂矾丸2.4,1.6,0.8g/kg组均可显著增加气血双虚模型小鼠胸腺皮质厚度(t=3.71～9.34,P<0.01),增大脾小结(t=3.36～11.97,P<0.01),增加脾脏淋巴细胞数(t=4.29～10.44,P<0.01);复方阿胶浆30mL/kg组可明显增加小鼠胸腺淋巴细胞数(t=2.45,P<0.05),当归补血口服液组、参芪颗粒3,2,1g/kg组、复方阿胶浆20,10mL/kg组、贞芪扶正颗粒15,10,5g/kg组、复方皂矾丸2.4g/kg组作用更为显著(t=3.22～8.20,P<0.01)。结论:①四种中成药对气血双虚模型小鼠血红蛋白升高作用相近,以复方阿胶浆和贞芪扶正颗粒对白细胞和红细胞水平升高作用为强,以复方阿胶浆和贞芪扶正颗粒对血小板水平升高作用为优。②四种中成药对气血双虚模型小鼠血细胞比容的影响无差异,以贞芪扶正颗粒和复方皂矾丸对巨噬细胞集落刺激因子水平的升高作用为优。③以参芪颗粒、复方阿胶浆、贞芪扶正颗粒对胸腺皮质厚度和淋巴细胞数的促进作用为优,以参芪颗粒、贞芪扶正颗粒、复方皂矾丸对脾小结和脾脏淋巴细胞数的促进作用为优。     

Die physiologischen Grundlagen der Herzmechanik

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