冠脉内导入重组β-半乳糖苷酶腺病毒的实验研究

目的 :研究经冠状动脉内导入重组 β-gal腺病毒后心肌的转染效率 ,证明冠脉内导入途径是否为重组基因导入心脏的有效的可行的途径。方法 :用定向克隆的方法构建重组 β -gal腺病毒载体 ,将 1 .53× 1 0 1 2 pfu重组 β-gal腺病毒经冠状动脉造影导管由左冠状动脉分别导入到 7只雄性Yorkshire猪心脏中 ,于术后 1周、2周、4周及 8周处死动物 ,取左右冠状动脉、3～ 5mm厚的不同部位的心肌及各实质脏器标本 ,经 1 .2 5 %戊二醛固定 2 0min ,用PBS洗涤 3次 ,在室温下用X -gal染液染色 1 6～ 2 4h ,脱水、石蜡包埋、切片及HE复染 ,光镜下计算出表达 β -gal的心肌细胞百分数即为转染效率。结果 :经冠状动脉内导入重组基因后 ,于术后 1周即有 β-gal表达 ,2周达高峰 ,心肌细胞的转染效率高达 45 .57% ,全层左冠状动脉壁、心肌间质中均有 β-gal表达 ,4周 β-gal表达下降 ,8周 β -gal恢复至对照组的基线水平。结论 :经冠状动脉内导入重组腺病毒 ,心肌细胞及冠脉壁重组基因表达效率高 ,说明冠状动脉内导入重组目的基因将是冠心病基因治疗中有效的可行的方法     

Intrafamilial phenotypic variability in four families with Anderson‐Fabry disease

We analysed the clinical history of 16 hemizygous males affected by Anderson‐Fabry Disease, from four families, to verify their intrafamilial phenotypic variability. Seven male patients, ranging from 26 to 61 years of age, died, whereas nine (age range 23–55) are alive. Eleven patients have undergone enzyme replacement therapy (ERT) for a period of 5–10 years. We have found a wide range of intrafamilial phenotypic variability in these families, both in terms of target‐organs and severity of the disease. Overall, our findings confirm previous data from the literature showing a high degree of intrafamilial phenotypic variability in patients carrying the same mutation. Furthermore, our results underscore the difficulty in giving accurate prognostic information to patients during genetic counselling, both in terms of rate of disease progression and involvement of different organs, when such prognosis is solely based on the patient's family history.     

β-半乳糖苷酶的糖苷键作用特异性研究

研究了不同微生物来源乳糖酶的糖苷键键型作用特异性,研究表明米曲霉乳糖酶的水解优先顺序是β-1－6键＞β-1－4键＞β-1－3键,乳酸克鲁维酵母乳糖酶的水解顺序则是β-1－4键＞β-1－6键＞β-1－3键。并发现所实验所用的环状芽孢杆菌对β－1－4键表现出了高度的键专一性。乳糖水解合成低聚双糖的种类和有关动力学数据都表明不同酶源的乳糖酶在键特异性方面存在很大差异。     

S‐Gal®, A novel 1H MRI reporter for β‐galactosidase

Reporter genes and associated enzyme activity are becoming increasingly significant for research in vivo. The lacZ gene and β‐galactosidase (β‐gal) expression have long been exploited as reporters of biologic manipulation at the molecular level, and a noninvasive detection strategy based on proton MRI is particularly attractive. 3,4‐Cyclohexenoesculetin β‐D‐galactopyranoside (S‐Gal®) is a commercial histologic stain, which forms a black precipitate in the presence of β‐gal and ferric ions, suggesting potential detectability by MRI. Generation of the precipitate is now shown to cause strong T₂* relaxation, revealing β‐gal activity. A series of tests with the enzyme in vitro and with tumor cells shows that this approach can be used as an assay for β‐gal activity. Proof of principle is shown in human breast tumor xenografts in mice. Upon direct injection of a mixture of 3,4‐cyclohexenoesculetin β‐D‐galactopyranoside and ferric ammonium citrate, intense contrast was observed immediately in MCF7‐lacZ tumors, but not in wild‐type tumors. 3,4‐Cyclohexenoesculetin β‐D‐galactopyranoside activation in combination with ferric ions introduces a novel approach for assaying enzyme activity by MRI in vivo. Magn Reson Med, 2010. © 2010 Wiley‐Liss, Inc.     

Morquio-B syndrome (MPS-IV B) associated with β-galactosidase deficiency in two siblings

In the present article we describe two cases with Morquio-B syndrome characterized by β-galactosidase deficiency in a Muslim family. They were found to have skeletal dysplasia, short stature and short trunk dwarfism with undetectable level of β-galactosidase in leucpcytes. Probands¹ sister who had no clinical signs of mucopolysaccharidosis was investigated and found to have normal levels of the enzyme. Mother was found to have a deficient activity of β-galactosidase and father was not available for the study. Since mother was pregnant, prenatal study from chorionic cells was carried out to investigate βgalactosidase activity in the chorionic villus. An intermediate level of β-galactosidase activity was found in the chorionic villus cells suggesting a carrier status. The diversity and rarity of the study makes it worth presenting. An erratum to this article is available at .     

Development of a free beta-galactosidase in vitro test for the assessment of heavy metal toxicity

The effect of heavy metals and organic compounds on the activity of the enzyme beta-galactosidase in a standardized bioassay has been evaluated, considering future applications in environmental monitoring. The tests were done using a commercial extract of a hydrolase from the eukaryote yeast Kluyveromyces lactis and o-nitrophenyl-beta-D-galactopyranoside (ONPG) as substrate. The enzyme was exposed to Cr(VI), Cd(II), Cu(II), Ni(II), Pb(II), Hg(II), phenol, sodium dodecyl sulfate, methanol and pentachlorophenol for 5, 15, 30, and 60 min. According to the results, a 15 min exposure time was considered optimum for the performance of the assay. Results of tests with metals showed IC50 values ranging between 9.25mg/L for Cd(II) and 0.015mg/L for Hg(II), with an order of sensitivity of: Cd(II) < Ni(II) < Cr(VI) = Pb(II) < Cu(II) < Hg(II). Sensitivity to organic compounds ranged from 200 to 4,000 mg/L, showing a higher specificity to heavy metals. The present in vitro free enzyme test showed a similar behavior to other tests based on beta-galactosidase such as the MetPlate. Furthermore, when compared to data from the literature on acute toxicity assays currently used in environmental assessment, test results show good agreement regarding the sensitivity to metals. After standardization, the proposed test could be used as a rapid and low-cost assay when evaluating biological effects of heavy metals in monitoring programs.     

B→O血型改造制备通用型血过程中残留工具酶的检测

目的：检测B→O血型改造制备通用型血过程中的残留工具酶-α-半乳糖苷酶。方法：血清学反应确定α-半乳糖苷酶残酶量安全值，用SDS—PAGE银染法和酶活性检测法测定通用型血中微量残留工具酶量。结果：确定了α-半乳糖苷酶残酶量安全值为10ng／ml，建立了检测微量残留α-半乳糖苷酶的方法。确定经过6-7次洗涤后。残留酶量可达到安全水平。结论：本研究为B→O血型改造制备的通用型血进一步的临床应用奠定了基础。     

一个Fabry病家系的GLA基因突变分析

目的 对一个临床诊断的非典型Fabry病患者进行α半乳糖苷酶基因(GLA)进行突变分析。方法 抽取患者家系中4名成员的外周血基因组DNA，PCR分段扩增位于Xq22的GLA基因的7个外显子，产物纯化后直接进行DNA测序检测突变。结果 测序显示，男性患者GLA基因的第6外显子存在CGA301CAA(Arg301Gln)突变，该患者为带有突变基因的半合子，母亲为携带突变基因的杂合子，哥哥及父亲为CGA301野生型的半合子。结论 对临床诊断的Fabry病患者及其亲属，进行GLA基因突变检测可以进行基因诊断，并有助于早期筛选出家系中的其他患者。     

Markers of senescence?

The specific identification of cellular senescence in clinical material has important implications for determining the role of senescence in age-related pathologies and in neoplasia in certain tumours. One suggested marker of senescence is the histochemical identification of a specific beta-galactosidase enzyme operative at pH 6.0. However, recent data indicate that this enzyme may not be specific for senescence in all tissues and probably represents the expression of endogenous lysosomal acid beta-galactosidase, which is expressed by a variety of differentiated cell types.     

以抗原半乳糖苷酶融合蛋白为酶结合物检测丙型肝炎抗体

目的　为了降低检测丙型肝炎（ 丙肝） 病毒（ＨＣＶ） 抗体的假阳性,建立基于双抗原夹心法的酶联免疫吸附测定（ＥＬＩＳＡ） 系统。方法　利用基因工程手段,在大肠杆菌中表达ＨＣＶ 嵌合抗原（ 含核心、Ｃ３３ｃ ,ＮＳ４ 及ＮＳ５ 抗原） 与大肠杆菌β半乳糖苷酶的融合蛋白,以该融合蛋白为酶结合物进行ＥＬＴＳＡ。结果　构建了重组质粒,并在大肠杆菌中表达了融合蛋白,融合蛋白具有ＨＣＶ 抗原的抗原性,并且保持了的酶活性。以重组融合蛋白为酶结合物,用于检测抗ＨＣＶ 抗体时,阳性检出率和阴性特异性均接近使用辣根过氧化物酶系统的水平。灵敏度接近以辣根过氧化物酶为标记酶的ＥＬＩＳＡ 系统。结论　重组ＨＣＶ 抗原β半乳糖苷酶融合蛋白可以用作酶结合物,应用于ＨＣＶ 感染的诊断。