Applications of a new fluorimetric enzyme assay for the diagnosis of aspartylglucosaminuria

L-Aspartic acid-β-7-amido-4-methylcoumarin is a sensitive and specific fluorogenic substrate for lysosomal glycoasparaginase (aspartylgluco-saminidase). Fibroblasts and leukocytes from 8 patients with aspartylglucosaminuria, showed 1-7% of the mean normal glycoasparaginase activity. Heterozygotes showed intermediate activities. Glycoasparaginase activity in chorionic villi, cultured trophoblasts, cultured amniotic fluid cells and amniotic fluid was readily detectable, indicating that prenatal analysis of aspartylglucosaminuria should be possible with this assay. β-Aspartyl-4-methylumbelliferone was synthesized but this potential substrate can not be used to assay glycoasparaginase since it hydrolyses spontaneously

Regulation of chloride transport in cultured normal and cystic fibrobis keratinocytes

Abstract Cultured normal (N) and cystic fibrosis (CF) keratinocytes were evaluated for their Cl−-transport properties by patch-clamp-, Ussing chamber- and isotopic efflux-measurements. Special attention was paid to a 32 pS outwardly rectifying Cl− channel which has been reported to be activated upon activation of cAMP-dependent pathways in N, but not in CF cells. This depolarization-induced Cl− channel was found with a similar incidence in N and CF apical keratininocyte membranes. However, activation of this channel in excised patches by protein kinase (PK)-A or PK-C was not successfull in either N or CF keratinocytes. Forskolin was not able to activate Cl− channels in N and CF cell-attached patches. The Ca2+-ionophore A23187 activated in cell-attached patches a linear 17 pS Cl− channel in both N and CF cells. This channel inactivated upon excision. No relationship between the cell-attached 17 pS and the excised 32 pS channel could be demonstrated. Returning to the measurement of Cl− transport at the macroscopic level, we found that a drastic rise in intracellular cAMP induced by forskolin did in N as well as CF cells not result in a change in the short-circuit current (Isc) or the fractional efflux rates of 36Cl− and 125I−. In contrast, addition of A23187 resulted in an increase of the Isc and in the isotopic anion efflux rates in N and CF cells. We conclude that Cl−-transport in cultured human keratinocytes can be activated by Ca2+, but not by cAMP-dependent pathways

Asymptomatic and late-onset ornithine transcarbamylase (OTC) deficiency in males of a five-generation family, caused by an A208T mutation

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Asymptomatic and late-onset ornithine transcarbamylase deficiency caused by a A208T mutation : Clinical, biochemical and DNA analyses in a four-generation family

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Asymptomatic and late-onset ornithine transcarbamylase (OTC) deficiency in males of a five-generation family, caused by an A208T mutation.

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Characterization and localization of the FMR-1 gene product associated with fragile X syndrome

THE fragile X syndrome is the most frequent form of inherited mental retardation after Down's syndrome, having an incidence of one in 1,250 males1,2. The fragile X syndrome results from amplification of the CGG repeat found in the FMR-1 gene3–6. This CGG repeat shows length variation in normal individuals and is increased significantly in both carriers and patients3–6; it is located 250 base pairs distal to a CpG island6 which is hypermethylated in fragile X patients4–7. The methylation probably results in downregulation of FMR-1 gene expression8. No information can be deduced about the function of the FMR-1 protein from its predicted sequence. Here we investigate the nature and function of the protein encoded by the FMR-1 gene using polyclonal antibodies raised against the predicted amino-acid sequences. Four different protein products, possibly resulting from alternative splicing, have been identified by immunoblotting in lymphoblastoid cell lines of healthy individuals. All these proteins were missing in cell lines from patients not expressing FMR-1 messenger RNA. The intracellular localization of the FMR-1 gene products was investigated by transient expression in COS-1 cells and found to be cytoplasmic. Localization was also predominantly cytoplasmic in the epithelium of the oesophagus, but in some cells was obviously nuclear