PubMed · 8136461
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R Wilson, C R Leitch, J Roberts, C Jenkins, J H McKillop, J J Walker. 1993. Interleukin 8.. https://doi.org/10.3109/08916939309115757
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Our previous studies showed that monkey amniotic epithelial cells (MAEC) synthesize and release catecholamines and possess D1 and D2 dopamine (DA) receptors (Elwan, M.A., Ishii, T., Ono, F. and Sakuragawa, N., Evidence for the presence of dopamine D1 receptor mRNA and binding sites in monkey amniotic epithelial cells. Neurosci. Lett., 262 (1999) 9-12; Elwan, M.A., Ishii, T. and Sakuragawa, N., Detection of dopamine D2 receptor mRNA and binding sites in monkey amniotic epithelial cells. J. Neurosci. Res., 56 (1999) 316-322; Elwan, M.A., Thangavel, R., Ono, F. and Sakuragawa, N., Synthesis and release of catecholamines by cultured monkey amniotic epithelial cells. J. Neurosci. Res., 53 (1998) 107-113). In the present study we tested the presence of DA transporter (DAT) in MAEC using radioligand binding experiments. Saturation studies showed that [3H]mazindol binds to a high affinity site with K(D) and Bmax values of 7.85 +/- 1.25 nM and 123.22 +/- 18.34 fmol/mg protein, respectively. Competition studies indicated that selective DAT inhibitors are potent displacers of [3H]mazindol binding, compared to inhibitors of other types of transporters. The rank order of potency of the competing drugs is consistent with the pharmacology of DAT. These results provide, for the first time, clear evidence that MAEC natively possess DAT binding sites and suggest that MAEC may provide a potential primate cell model to study DA release and uptake processes and to explore new drugs active at this site.
Guanidinoacetate methyltransferase deficiency is a newly recognized inborn error of creatine biosynthesis. Manifestation of neurologic symptoms occurs in infancy and is partly reversible upon oral substitution of creatine. In the first two index patients, enzymatic diagnosis was established in a liver biopsy, and the underlying molecular defect in the GAMT gene has been identified. In order to provide non-invasive biochemical diagnosis, we have developed an enzyme assay based on the formation of radiolabeled creatine from 14C guanidinoacetate and S-adenosylmethionine in concentrated and dialyzed extracts from cultivated skin fibroblasts, Epstein-Barr virus transformed lymphoblasts, and cultivated amniotic cells. Cells were investigated from controls, from 1 index patient with proven GAMT deficiency and from 3 additional patients with clinical and biochemical signs of GAMT deficiency. Separation of 14C guanidinoacetate from 14C creatine in the reaction mixture was accomplished by HPLC on Hypersil ODS column and radioactivity was determined in fractions according to respective UV signals. GAMT activities in control fibroblasts (n = 7), lymphoblasts (n = 8) and in amniotic cells (n = 2) were 0.38-0.56, 0.61-0.84 and 0.38-0.56 nmol/h/mg protein. Apparent Km values were 9.5-14.8 microM for guanidinoacetate and 68-78 microM for S-adenosylmethionine. In the index patient and in the three additional patients at risk, GAMT activity was < 0.1 nmol/h/mg protein. The assay described here allows non-invasive diagnosis of GAMT deficiency in patients at risk.
A tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and its metabolite, 4-[(methylnitrosamino)-1-(3-pyridyl)but-1-yl]beta-O-D-glucosiduronic+ ++ acid (NNAL-Gluc), have been found in the urine of newborns whose mothers smoked during pregnancy. We set out to determine whether this carcinogen is present in the fetus in early pregnancy. Cell-free amniotic fluid (AF) was obtained through routine amniocentesis for prenatal genetic studies from groups of smokers and non-smokers. NNAL and NNAL-Gluc were quantified by previously published methods. A history of smoking was confirmed by assays for cotinine plus N-beta-D-glucosiduronosyl-(S)-(-) cotinine inner salt (cotinine-Gluc) in AF. NNAL was detected in the AF of 11/21 (52.4%) of smokers and in 2/30 (6.7%) of non-smokers, a statistically significant difference (p=0.0006). There was not convincing evidence of NNAL-Gluc in the AF. This study documents for the first time that the tobacco-specific carcinogen NNAL is present in the fetus in early pregnancy. Further rigorous epidemiological studies are needed to determine whether the offspring of smoking mothers have an increased lifetime risk of cancer.