Porphyrin synthesis and mitochondrial respiration in acute intermittent porphyria: studies using cultured human fibroblasts.

The formation of variation of delta-aminolevulinic acid (ALA) and of porphyrins, as well as respiratory metabolism, have been studied in skin fibroblasts from six normal control subjects and seven patients with acute intermittent porphyria. The mean activity of ALA synthetase was the same in both groups, whereas the mean activity of uroporphyrinogen I synthetase (as measured by the conversion of porphobilinogen [PBG] to porphyrins) was significantly decreased in fibroblasts from porphyric subjects, the mean value being 52 per cent that of control subjects (p less than or equal to 0.001). The findings of decreased uroporphyrinogen synthesis without an increase in ALA synthetase in mitochondria-containing cells from subjects with acute intermittent porphyria are compatible with the concept that defective PBG ultilization is the fundamental defect in heme biosynthesis in this disease and the possibility that ALA synthetase is "irreversibly" repressed in nonhepatic tissues. Respiration of the cells was studied polarographically. The two types of cells showed similar overall rates of respiration and in general responded to substrates and inhibitors as expected. Of the inhibitors tested (rotenone, amytal, antimycin, and cyanide), only rotenone showed a differential effect: respiration of fibroblasts from porphyric patients was not as sensitive to the inhibitor as was that of the control subjects. These results are interpreted as suggesting a possible defect in mitochondrial NADH oxidation in acute intermittent porphyria.     

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.     

Heme synthetase deficiency in human protoporphyria. Demonstration of the defect in liver and cultured skin fibroblasts.

The final step in heme biosynthesis is chelation of porphyrin with Fe++ catalyzed by the mitochondrial enzyme heme synthetase. We have employed a sensitive radiochemical assay for this enzyme, using 59Fe and deuteroporphyrin or protoporphyrin as substrates. In this method iron is maintained in the ferrous state, oxygen is excluded from the incubation system, and labeled heme product is extracted into ethyl acetate. This assay has been used to measure the activity of heme synthetase in homogenates of liver, obtained by needle biopsy, and in sonicates of human skin fibroblasts, cultured in vitro. In addition, activity of the first enzyme of the heme synthetic pathway, delta-aminolevulinic acid synthetase, has been measured in fibroblast lysates. Lysates of fibroblasts from eight patients with protoporphyria had activities of delta-aminolevulinic acid synthetase which did not differ significantly from those of eight normal fibroblast lines, whereas activity of heme synthetase, with either deuteroporphyrin or protoporphyrin as substrate, was markedly decreased in sonicates of skin fibroblasts from these patients, the mean being 8% of control with deuteroporphyrin and 14% with protoporphyrin as substrate. In homogenates of liver from five patients with protoporphyria, activity of heme synthetase was also significantly less than that found in six patients without prophyria, the mean being 13% of control with protoporphyrin as substrate. These results provide evidence that decreased activity of heme synthetase is the basic defect in the heme synthetic pathway in protoporphyria. This deficiency is probably responsible for protoporphyrin accumulation and hence the biochemical and clinical features observed in protoporphyria.     

Internalization of exogenous gangliosides in cultured skin fibroblasts for the diagnosis of mucolipidosis IV

The internalization of exogenous mixed brain gangliosides in ML IV cultured skin fibroblasts indicated an impairment of ganglioside catabolism in these cells. Incubation of ML IV, normal and various other lysosomal storage disorders cell lines for five days with exogenous tritium labelled GM3, GD1a or GT1 gangliosides allowed accurate quantitation of the retained gangliosides. This in vitro approach provides a reliable method for the diagnosis of ML IV.     

Two alpha-glucosidases in cultured amniotic fluid cells and their differentiation in the prenatal diagnosis of Pompe's disease.

A sensitive fluorometric assay utilizing 4-methylumbelliferyl-alpha-D-glucopyranoside has been developed for the determination of alpha-glucosidase. The enhanced sensitivity was achieved by increasing the solubility of the substrate with a water miscible organic solvent. With this system, cultured amniotic fluid cells were found to have two major forms of alpha-glucosidase with somewhat overlapping acidic pH optima; one with pH optimum at 4.5 is deficient in Pompe's disease (type II glycogenosis), while one with pH optimum at 6.0 is not affected in this disease. Specificity for the pH 4 form of alpha-glucosidase was achieved by exploiting the greater thermal lability of the pH 6 enzyme. The pH 6 form of the enzyme was also detectable in freshly prepared extracts of cultured fibroblasts. The procedure is direct and simple and has been applied to the prenatal diagnosis in two pregnancies at risk for Pompe's disease.     

Assay of galactose-1-phosphate uridyl transferase in cultured amniotic cells for prenatal diagnosis of galactosaemia.

We report studies designed to establish optimal conditions for the assay of amniotic cell galactose 1-phosphate uridyl transferase (Gal-PUT) for early prenatal diagnosis of galactosaemia. Methods based on linkage of the reaction to cause of non-specific reactions occurring even in the absence of Gal-1-P. In the final method, sonicates of confluent cultures are incubated with (14-C) Gal-1-P is degraded by treatment with alkaline phosphatase. Gal-PUT specific activities of both control and galactosaemic amniotic cells are higher in non-confluent that confluent cultures.     

Sulphamidase activity in leucocytes, cultured skin fibroblasts and amniotic cells: diagnosis of the Sanfilippo A syndrome with the use of radiolabelled disaccharide substrate

1. Sulphamidase activity was assayed by incubation of the radiolabelled disaccharide O-(alpha-2-sulphamino-2-deoxy-D-glucopyranosyl)- (1 leads to 3)-L-[6-3H]idonic acid with homogenates of leucocytes and cultured skin fibroblasts and concentrates of urine derived from normal individuals, patients affected with sulphamidase deficiency disorder [mucopolysaccharidosis type IIIA (MPS IIIA): the Sanfilippo A syndrome], parents of such patients and patients affected with other mucopolysaccharidoses and lysosomal enzyme deficiencies. 2. The assay clearly distinguished affected homozygotes from normal controls, heterozygotes and other mucopolysaccharidoses types. 3. Sulphamidase displayed remarkable thermal stability; reaction rates were constant for at least 24 h at 60 degrees C for leucocyte and 20 h at 37 degrees C for cultured fibroblast preparations. Apparent Km values for fibroblast sulphamidase were 71 mumol/l at 37 degrees C and 100 mumol/l at 50 degrees C; the corresponding Vmax, values were 21 and 72 pmol min-1 mg-1 of protein respectively. An incubation temperature of 60 degrees C was used for the routine assay of sulphamidase activity in leucocytes, urine and amniotic supernatant preparations. The specific activities of fibroblast and amniotic cell sulphamidase, assessed at incubation temperatures of 37 degrees C, were more than 10-fold the leucocyte enzyme activity at 60 degrees C. 4. We recommend the use of radiolabelled disaccharide substrate for the assay of sulphamidase in leucocytes, skin fibroblasts and urine, for the routine enzymic detection of the sulphamidase deficiency disorder of the Sanfilippo A syndrome.     

Diagnosis of Pompe's disease in cultured skin fibroblasts and primary amniotic fluid cells using 4-methylumbelliferyl-alpha-D-glucopyranoside as substrate.

The possible interference of neutral alpha-D-glucosidase in the diagnosis of Pompe's disease using 4-methylumbelliferyl-alpha-D-glucopyranoside as substrate for the assay of acid alpha-D-glucosidase was investigated. The pH profile of alpha-D-glucosidase in control skin fibroblasts and amniotic fluid cells showed two peaks of activity. The shape of the pH profile depended upon whether or not the extract was added to the buffer before the substrate. If extract was added to the buffer before the substrate, a greater separation was obtained between the two peaks of activity. The neutral alpha-D-glucosidase activity could be totally removed by preliminary precipitation at pH 5.0. Following acid region whilst Pompe's cells had no activity enabling a clear distinction to be made between carriers and the disease state.     

Regulation of high density lipoprotein receptor activity in cultured human skin fibroblasts and human arterial smooth muscle cells.

Cultured human skin fibroblasts and human arterial smooth muscle cells possess high-affinity binding sites specific for high density lipoproteins (HDL). Results from the present study demonstrate that binding of HDL to these sites is up-regulated in response to cholesterol loading of cells. When fibroblasts or smooth muscle cells were preincubated with nonlipoprotein cholesterol, cellular binding of 125I-HDL3 was enhanced severalfold. This enhancement was sustained in the presence of cholesterol but was readily reversed when cells were exposed to cholesterol-free medium. The stimulatory effect of cholesterol treatment was prevented by cycloheximide, suggesting the involvement of protein synthesis. Kinetic analysis of HDL3 binding showed that prior exposure to cholesterol led to an induction of high-affinity binding sites on the cell surface. In the up-regulated state, the apparent dissociation constant (Kd) of these sites was approximately 2 micrograms protein/ml. Competition studies indicated that the HDL binding sites recognized either HDL3 or HDL2 but interacted weakly with low density lipoprotein (LDL). Exposure of cells to lipoprotein cholesterol in the form of LDL also enhanced HDL binding by a process related to delivery of sterol into cells via the LDL receptor pathway. Enhancement of HDL binding to fibroblasts by either nonlipoprotein cholesterol or LDL was associated with an increased cell cholesterol content, a suppressed rate of cholesterol synthesis, decreased LDL receptor activity, and an enhanced rate of cholesterol ester formation. A comparison of HDL3 binding with the effects of HDL3 on cholesterol transport from cells revealed similar saturation profiles, implying a link between the two processes. Thus, cultured human fibroblasts and human arterial smooth muscle cells appear to possess specific receptors for HDL that may function to facilitate cholesterol removal from cells.     

Adenoviral-mediated expression of porphobilinogen deaminase in liver restores the metabolic defect in a mouse model of acute intermittent porphyria.

The aim of this study was to investigate the potential of gene therapy in the treatment of acute intermittent porphyria (AIP), a disorder caused by a partial deficiency of porphobilinogen deaminase (PBGD), the third enzyme in heme synthesis. The condition is biochemically characterized by accumulation of the porphyrin precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG). Here we present the first experiments in vivo using adenoviral vectors to replace the deficient enzyme in the liver of an AIP mouse model. The use of adenoviral vector carrying the cDNA of luciferase in wild-type mice confirmed that transgene expression after intravenous administration was found mainly in liver. When PBGD-deficient mice were administered with adenoviral vector carrying the cDNA of mouse PBGD, the hepatic PBGD activity increased in a dose- and time-dependent manner. The highest activity was found 7 days after injection and remained high after 29 days. The expressed enzyme was shown to correct the metabolic defect in the PBGD-deficient mice as no accumulation of ALA or PBG occurred in plasma, liver, or kidney after induction of heme synthesis by phenobarbital. The study demonstrates that hepatic PBGD expression prevents the accumulation of porphyrin precursors, suggesting a future potential for gene therapy in AIP.     

New mutations of the hydroxymethylbilane synthase gene in German patients with acute intermittent porphyria

Acute intermittent porphyria (AIP) is a low-penetrant, autosomal dominant disorder caused by decreased activity of hydroxymethylbilane synthase (HMBS; MIM 176 000), the third enzyme in the heme biosynthetic pathway. We report the first molecular analysis of HMBS gene mutations in classical AIP patients of German origin. The HMBS gene of 5 German AIP patients was analysed by DGGE-screening and direct sequencing of amplified genomic DNA. Five different mutations including four novel mutations were found. Three of them are single base substitutions that affected exon 3 (R16C), exon 10 (V202L), and intron 13 (T to A, IVS13+2) The two remaining mutations are frameshifts which produce a stop codon (del GA in exon 6 and insA in exon 14). These mutations are likely to be responsible for the decrease in HMBS activity found in both erythrocytes and non-erythroid cell lines (lymphocytes). Our results demonstrate the allelic heterogeneity of HMBS mutations in AIP patients of German origin.     

Antibody method for measurement of dihydrotestosterone receptors in cultured human skin fibroblasts.

We have adapted the method of Casta?eda and Liao to the assay of DHT receptors in cultured fibroblasts arising from human skin. An antitestosterone antibody (100% crossreactivity for DHT) was coupled to CNBr-activated Sepharose. Confluent monolayers of fibroblasts were incubated with 3H-DHT (2 nM) at 37 degrees C for 30 min. Fibroblasts were then collected, sonicated, and centrifuged at 1200 x g for 15 min. The receptor assay was carried out on the supernatant; the antibody-sepharose was used to remove both unbound and nonspecifically bound DHT. Experience showed that the antibody did not entirely remove the nonspecifically bound and free DHT. A blank (sample heated at 60 degrees C for 3 min) was therefore subtracted to obtain an accurate value of specifically bound DHT. In spite of this, the antibody method, when compared to the gel filtration method, was more rapid and more convenient. Its reproducibility was similar to that of the gel filtration method, and its sensitivity was somewhat greater in patients with low levels of DHT-receptor complex. Improved sensitivity could be particularly useful when dealing with partial AIS.     

The change in the pH 4 and pH 6 forms of alpha-glucosidase in cultured amniotic fluid cells and its implication in prenatal diagnosis of Pompe's disease.

Activities of two major forms of alpha-glucosidase in cultured amniotic fluid cells have been measured by the 4-methylumbelliferyl-alpha-D-glucoside assay after 3, 6 and 9 weeks of culturing. Activity of the pH 4 forms of alpha-glucosidase, which is deficient in Pompe's disease, was low in early culture but increased rapidly as the culture time was increased. The cells harvested at 3 weeks had a low absolute activity of the pH 4 form as well as low ratio of the pH 4 to pH 6 enzyme. The pH 6 form is not affected in Pompe's disease. The results suggest cautions when attempting early diagnosis by use of microtechniques and re-emphasizes the need for differentiation of these two forms of alpha-glucosidases in prenatal diagnosis of Pompe's disease.     

Low-density lipoprotein receptor activity in cultured human skin fibroblasts. Mechanism of insulin-induced stimulation.

Low-density lipoproteins (LDL) receptor activity, as reflected by LDL degradation, was stimulated by the addition of insulin to cultures of human skin fibroblasts. These changes occurred independently of the glucose concentration of the incubation medium and occurred whether or not LDL receptor activity was suppressed. A comparison of the saturation kinetics of LDL receptor activity in the presence and absence of insulin indicated that insulin produced a 35% increase in Vmax with no difference in "apparent Km". These results suggest that insulin enhances LDL receptor activity by increasing the number of LDL receptors rather than by influencing binding affinity. In confirmation, LDL degradation by receptor negative cells was not enhanced by insulin. Sterol synthesis from [14C]acetate was also stimulated by insulin, but egress of cholesterol and cellular cholesterol content were unaffected by the hormone. The effect of insulin on LDL receptors was not dependent on its known ability to enhance cellular DNA synthesis and proliferation, because insulin stimulated LDL receptor activity in cells kept quiescent by maintenance in plasma-derived serum that was devoid of platelet derived growth factor. Nevertheless, the effect of insulin in enhancing LDL receptor number, coupled with stimulation of endogenous cholesterol synthesis, provides a mechanism whereby the cell could theoretically increase its supply of cholesterol during times of additional need.     

Comparative enzymology of eleven acid hydrolases in cultured amniotic fluid cells, skin fibroblasts and embryonic lung fibroblasts, and the respective changes with the increasing age of the cell cultures

Assay conditions were studied for eleven lysosomal enzymes (beta-D-galactosidase, alpha-D-mannosidase, beta-hexosaminidase, beta-D-glucuronidase, alpha-D-galactosidase, alpha-D-glucosidase, arylsulfatase, beta-D-glucosidase, alpha-L-fucosidase, alpha-D-neuraminidase and alpha-L-iduronidase) in cultured amniotic fluid cells (CAFC), cultured skin fibroblasts (CSF) and cultured embryonic lung fibroblasts (CELF), and the properties of the enzymes were compared among these cultured cells. In addition, changes in these enzymes from the three cell types were investigated between 4-6 earlier passages and 24-26 later passages. With the exception of alpha-D-glucosidase, alpha-D-neuraminidase and alpha-L-fucosidase, all enzymes assayed for the 4-6 earlier passages and the 24-26 later passages had the same Km values and the same pH optima, and were also unchanged with the increasing age of cell cultures, with regard to their points. The specific activities of beta-D-glucuronidase, arylsulfatase, alpha-D-glucosidase and beta-D-glucosidase for the 4-6 earlier passages increased significantly with development, though no change was observed with development in the specific activities of other enzymes. Variations were observed between the levels of these enzymes in the three cell types with the increasing age of cell cultures, such as increases in some, decreases in others and no change in still others.     

体外培养人羊膜上皮细胞的表型及分化能力

背景：人羊膜上皮细胞具有多系分化能力,是再生医学中重要的细胞来源。目前的研究多集中于对其分化能力的考察,而体外培养过程中羊膜上皮细胞的生物学特征如何变化尚不清楚。 目的：分析体外培养对人羊膜上皮细胞生长、表型及向心肌样细胞分化的能力等生物学特性的影响,探讨原代人羊膜上皮细胞干性标志物SSEA-4的表达水平与人羊膜上皮细胞生物学特性变化之间的关联性。 方法：使用统一分离方法获得原代羊膜上皮细胞并进行体外培养。利用CCK-8、流式细胞仪及real-time PCR等手段检测不同培养阶段人羊膜上皮细胞的增殖、表型以及向心肌样细胞分化的能力。 结果与结论：不同胎儿样本来源的原代人羊膜上皮细胞的SSEA-4表达在26.7%-97%,存在很大的个体差异。并且,随着传代次数的增加,人羊膜上皮细胞的SSEA-4表达水平显著降低,其下降程度与原代SSEA-4的表达水平无关。另外,培养后人羊膜上皮细胞的心肌分化潜能也存在很大个体差异,且其差异与原代人羊膜上皮细胞的SSEA-4表达水平的高低无关。结果提示,不同胎儿样本来源的原代人羊膜上皮细胞的SSEA-4表达水平受到个体差异的影响,需要建立更准确的临床样本筛选指标来稳定获得原代高表达SSEA-4的胎儿样本,以实现对人羊膜上皮细胞的质量监控。另外,体外培养过程中SSEA-4的表达水平受到培养条件的影响,需要继续优化培养条件以维持其高表达。此外,人羊膜上皮细胞向心肌样细胞分化的能力受到样本个体差异以及培养条件的影响,在今后还需要进一步研究。     

羊水细胞低密度脂蛋白受体基因突变分析在家族性高胆固醇血症产前诊断中应用

目的探讨羊水细胞低密度脂蛋白受体（low density lipoprotein receptor,LDLR）基因突变分析在家族性高胆固醇血症（familial hypercholesterolemia,FH）产前诊断中的应用价值。方法 3例曾生育FH重症患儿并再次妊娠的妇女及其核心家系成员,提取其外周血基因组DNA,筛查LDLR基因突变;于妊娠16～20周在超声引导下行羊膜腔穿刺术抽取羊水,提取胎儿脱落细胞DNA,分别对家系存在的LDLR基因突变进行检测,判断胎儿是否为重症FH。结果 3个家系均符合FH诊断,并分别在LDLR基因检测到2个互不相同的杂合突变位点;胎儿LDLR基因核苷酸序列分析证实,1号家系胎儿仅携带该家系1个突变位点判断为杂合（轻症）,2号家系胎儿携带该家系2个突变位点判断为复合杂合（重症）,3号家系胎儿未检到该家系的突变位点推测为正常个体。结论 FH孕妇羊水脱落细胞LDLR基因分析安全有效,可尽早发现FH纯合子患儿。