Lysosomal enzyme activities in cultured lymphoid cell lines

Several lysosomal enzyme activities in cultured lymphoid cell lines were studied during 3 phases of cell culture; logarithmic growth phase, stationary phase and decline phase.Enzyme induction during cell growth was found in N-acetyl-hexosaminidase, β-galactosidase and α-l-fucosidase, but no induction in a-d-mannosidase, α-glucosidase and β-glucuronidase. The latter two enzymes were unchanged during all cell culture phases.A drop in α-l-fucosidase and α-d-mannosidase activity was found during the stationary and decline phases of cell culture.     

Angiotensin I-converting enzyme in amniotic fluid

Angiotensin I-converting enzyme (ACE) is present in human amniotic fluid. We characterized the enzyme by both its antigenic and enzymatic properties. Using a specific direct radioimmunoassay, ACE was quantified and characterized in each of the 19 samples tested. Mean level was 136 +/- 83 ng/ml. Amniotic ACE completely crossreacted, like that in plasma and kidney, with antibodies raised against the lung enzyme. ACE activity in amniotic fluid averaged 8.7 +/- 5.6 microU/ml using Hip-His-Leu as substrate and was significantly correlated with ACE antigen levels. ACE was not associated with the cells or the free intracellular organelles in amniotic fluid, and the enzyme was present in soluble form. Angiotensinase activity and high levels of kininase activity were found in amniotic fluid. Inhibition studies with captopril and anti-human ACE antibodies suggest that angiotensinases and kininases other than ACE were also present. Because renin, mostly in inactive form, and angiotensinogen were also found in these amniotic fluids, it appears that a complete, although not fully activated, renin angiotensin system is present in amniotic fluid and fetal membranes during pregnancy.     

Quantitative enzyme antigen immunoassay of acetylcholinesterase in amniotic fluid

An enzyme immunoassay for acetylcholinesterase in amniotic fluid is described. Rabbit antiserum (IgG fraction) against human erythrocyte membrane acetylcholinesterase is first attached to microtitre plates. Samples containing acetylcholinesterase are added and the enzyme activity is then measured, with acetylthiocholine as substrate and Ellman's reagent as coupling salt. Results are comparable with those by qualitative determination of the enzyme after polyacrylamide gel electrophoresis, except for blood-contaminated amniotic fluids. One person can perform 200 enzyme analyses per day. Intra- and interassay coefficients of variation are less than 5% at concentrations from 15 to 450 arb. units/L. The mean catalytic concentration in 400 amniotic fluid samples was 21 arb. units/L (range 5 to 70 arb. units/L) with 1000 arb. units/L for a human serum pool as standard. All 39 cases of neural tube defects and two-thirds of cases with omphalocele and gastroschisis had abnormally high values, exceeding 75 arb. units/L.     

Lysosomal enzyme activities in fresh and frozen chorionic villi and in cultured trophoblasts

Fifteen lysosomal enzyme activities were compared in 14 presumed normal chorionic villus specimens that were each divided, processed and analyzed as fresh tissue, tissue frozen for 1 week, and cultures established from minced whole villi. Most of the activities determined in the chorionic villus tissue were not affected significantly by freezing. However, activities for most enzymes were significantly different from those determined in the cultured cells. Our experience with first trimester prenatal evaluations for several lysosomal disorders showed that the limited amount of tissue obtained is not always sufficient for thorough analysis and thus, cultured trophoblasts derived from the tissue specimen should also be examined. The results of this study stress the importance of using appropriate tissue-type and cell-type controls to establish the normal range in the respective analyses.     

羊水细胞培养技术的临床应用

目的探索羊水细胞染色体培养改良方法,提高培养、收获成功率,满足临床诊断需要。方法对493例羊水标本进行细胞培养和染色体收获,并对实验细节进行优化,建立标准操作程序。结果羊水培养成功率99.8%（492/493）,发现21-三体6例、18-三体2例、其他异常2例。结论采取严格质量控制;保留换液后的上清,解决因收获失败无法诊断的问题;采取肝素抗凝等,消除母血细胞干扰,可使羊水细胞培养成功。     

A new assay for choline-containing phospholipids in amniotic fluid by an enzyme sensor

A new rapid and direct method for the determination of choline-containing phospholipids in the amniotic fluid is proposed. The determination is performed by an amperometric-enzymatic method. The correlation with an enzymatic-spectrophotometric method, already published, is also considered.     

Microenzymatic assays for lysosomal enzymes in primary amniotic fluid cell cultures

A study of three lysosomal enzymes (hexosaminidase, beta-galactosidase and alpha-galactosidase) in normal primary amniotic fluid cell cultures using a microenzymatic assay is presented. No difference in enzyme activity was found between primary and amniotic cell cultures in passage number one. A progressive change in the proportions of hexosaminidase A and hexosaminidase B with time was demonstrated in culture. The feasibility of this procedure for the early prenatal diagnosis of disorders due to lysosomal enzyme deficiency is discussed.     

Organic acids in amniotic fluid

The organic acids of human amniotic fluid were studied by gas chromatography and mass spectrometry. About 30 acids were identified. A "normal" metabolic profile of acids was established which can provide the basis for the identification of abnormal patterns.     

Lysosomal enzyme activity in human aqueous humor.

Activity of the lysosomal enzymes N-acetyl-beta-D-hexosaminidase, alpha-D-mannosidase, beta-D-glucuronidase, and beta-D-galactosidase was detected in aqueous humor from eyes undergoing intraocular surgery. There was no correlation between lysosomal enzyme activity and age. Lysosomal enzyme activity in human released by ocular tissues surrounding the anterior chamber including the cornea, trabecular meshwork, ciliary body, iris, and lens. Their release into aqueous humor may have a role in regulating aqueous outflow in normal and glaucomatous eyes.     

羊水游离RNA中神经发育关键基因分析

目的分析孕中期羊水游离RNA(AfcfRNA)转录组,筛选神经发育共表达关键基因。方法利用基因共表达网络分析,建立AfcfRNA转录组的基因共表达网络模块。筛选各共表达模块中的神经特异性基因,建立神经系统特异性共表达模块。利用基因间相互作用筛选神经组织特异性共表达模块中的关键基因。结果通过加权基因共表达网络分析共建立27个以颜色命名的共表达模块,在Human Protein Atlas数据库中筛选到832个神经组织特异性基因。在蓝色、棕色、蓝绿色以及黄色模块中富集到前脑发育、神经突触组装和功能、神经递质释放过程、轴突发生以及学习和记忆过程相关的GO术语。通过基因间相互作用以及基因在孕中期的平均表达量分析,共发现蓝色模块(SLC18A3、TACR3、SYT2)、棕色模块(SSTR5、STX1A、SNAP25、GHSR、SSTR4、GABBR2)、蓝绿色模块(DRD2、SLC32A1、GNG3、OPN4、PENK)以及黄色模块(RAB3A、HCRT、GRM5)中的17个关键基因。结论该研究获得了神经系统发育密切相关的并且具有共表达关系的关键基因,可作为潜在的产前诊断中监测神经系统发育的标志物。     

Acid esterase activity in cultured skin fibroblasts and amniotic fluid cells using 4-methylumbelliferyl palmitate.

The properties and levels of acid esterase in cultured skin fibroblasts and amniotic fluid cells were investigated using 4-methylumbelliferyl palmitate as substrate. Determinations of acid esterase activity could be made using as little as 1 microgram cell protein. Cardiolipin increased the activity 2--3 fold at the pH optimum 4.0. The apparent KM for both cell types studied was 196 micrometer without and 96 micrometer with cardiolipin. Acid esterase activity was inhibited by cyanide and thiomersal, but not by iodoacetate and N-ethylmaleimide. However activation by cardiolipin was prevented by iodoacetate, N-ethylmaleimide and also sodium chloride. Skin fibroblasts and primary amniotic fluid cells had similar levels with or without cardiolipin. A cyclic activity was found with subculture but no consistent pattern with passage. The acid esterase deficiency in Wolman's and cholesterol ester storage diseases was demonstrated with this substrate.