http://repub.eur.nl/res/aut/606/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/13785/ 2005-07-01T00:00:00Z CONTEXT: Although glucocorticoid hormone, thyroid hormone, and retinoic acid play important roles in fetal development, the expression of their receptors in human lung is still unknown. OBJECTIVE: The aim of this study was to investigate the ontogeny of glucocorticoid receptor (GR)alpha, thyroid hormone receptors (TRs), retinoic acid receptors (RARs), and retinoid X receptors (RXRs) mRNA expression in human lungs. DESIGN: Lungs from human fetuses and neonates (13.5-41 wk gestation; n = 20) as well as adults (n = 5) were analyzed by real-time PCR to monitor the ontogeny of mRNA expression for each receptor. In addition, immunohistochemistry was performed to show the cellular distribution of the different receptors. RESULTS: The expression of GRalpha, TRs, RARs, and RXRs was already detected in the earliest developmental stages analyzed. There was no significant difference in mRNA expression between developmental groups for any of the genes studied. However, for fetal and neonatal samples, there were positive correlations between gestational age and mRNA expression for RARalpha (r = 0.665; P = 0.001), RXRalpha (r = 0.444; P = 0.050), and RXRgamma (r = 0.464; P = 0.039). Immunohistochemical studies showed the presence of GRalpha, TRs, RARs, and RXRs in the nuclei of both epithelial and mesenchymal cells, albeit more pronounced in epithelium of larger airways. CONCLUSIONS: The detection of GRalpha, TRs, RARs, and RXRs expression in human lung as early as 13.5 wk gestation implies an early potential for therapeutic or toxic effects by exogenous analogs or by excess of endogenous ligands. http://repub.eur.nl/res/pub/12904/ 2000-01-01T00:00:00Z The CCAAT boxes of the beta-like globin genes interact with three proteins: NF-Y, GATA-1 and NFE-6. We demonstrate that NFE-6 contains C/EBPgamma, and address its role in globin gene regulation by erythroid overexpression of C/EBPgamma, and a dominant-negative form C/EBPgammaDeltaB, in mice. Elevated levels of C/EBPgamma, but not C/EBPgammaDeltaB, increase expression of the (fetal) gamma-globin relative to the (adult) beta-globin gene. Interestingly, fetal liver erythropoiesis is ablated when the C/EBPgamma and C/EBPgammaDeltaB levels are further increased in homozygous transgenics. We suggest that targeted expression of dominant-negative leucine zipper proteins is a generally applicable approach to ablate specific tissues in mice. http://repub.eur.nl/res/pub/17620/ 1998-11-25T00:00:00Z The lysosome represents a well characterized, membrane-contained intracellular digestive system. Iu this important organelle a battery of lysosomal hydro lases and accessory proteins work in concert on the step-wise conversion of macromolecular substrates into small biological building blocks, which are either reutHized by the cell 01' discarded. A failure of any of these enzymes to properly exert their hydrolytic activity results in the progressive accumulation of partially degraded· metabolltes that are retained ('stored') in the lysosome. The genetic disorders caused by a malfunction of the lysosomal system are collectively known as lysosomal storage disorders, aud are normally associated with a single enzyme deficiency. One known exception is the disease galactosialldosis which is due to partial or complete loss of activity of two glycosidases, acid p-D-galactosidase and N-acetyl-a-neuraminidase, because of a primary defect in the carboxypeptidase protective protein/cathepsin A (PPCA). The latter enzyme associates with both glycosidases soon after syntbesis, and is essential for their proper intracellular routing, lysosomal stability and activity. Aside from the protective function, PPCA has cathepsin Aldeamidase activity on a selected number ofneuropeptides. The aim of the experimental work presented in tbis thesis was to gain insigbts into the transcription regulation of the PPCA gene and the expression of PPCA mRNA and protein In mouse tissues. These studies have coutributed to the understanding of the phenotypic abnormalities in the murine model of galactosialldosis, which reflected to a large extent the distribution pattern of the protein in normal tissues. Given the fact that the observed pathology in galactosialldosis is iu part caused by tbe secondary neuraminidase deficiency, the isolation and characterization of the murine neuraminidase was instrumental to better understand in whicb way tbe two enzymes depend on each otber and cooperate ill vivo. http://repub.eur.nl/res/pub/2563/ 1998-01-01T00:00:00Z The locus control region of the beta-globin cluster contains five DNase I hypersensitive sites (5'HS1-5) required for locus activation. 5'HS3 contains six G-rich motifs that are essential for its activity. Members of a protein family, characterized by three zinc fingers highly homologous to those found in transcription factor Sp1, interact with these motifs. Because point mutagenesis cannot distinguish between family members, it is not known which protein activates 5'HS3. We show that the function of such closely related proteins can be distinguished in vivo by matching point mutations in 5'HS3 with amino acid changes in the zinc fingers of Sp1 and EKLF. Testing their activity in transgenic mice shows that EKLF is a direct activator of 5'HS3. http://repub.eur.nl/res/pub/2514/ 1995-01-01T00:00:00Z The lysosomal storage disorder galactosialidosis results from a primary deficiency of the protective protein/cathepsin A (PPCA), which in turn affects the activities of beta-galactosidase and neuraminidase. Mice homozygous for a null mutation at the PPCA locus present with signs of the disease shortly after birth and develop a phenotype closely resembling human patients with galactosialidosis. Most of their tissues show characteristic vacuolation of specific cells, attributable to lysosomal storage. Excessive excretion of sialyloligosaccharides in urine is diagnostic of the disease. Affected mice progressively deteriorate as a consequence of severe organ dysfunction, especially of the kidney. The deficient phenotype can be corrected by transplanting null mutants with bone marrow from a transgenic line overexpressing human PPCA in erythroid precursor cells. The transgenic bone marrow gives a more efficient and complete correction of the visceral organs than normal bone marrow. Our data demonstrate the usefulness of this animal model, very similar to the human disease, for experimenting therapeutic strategies aimed to deliver the functional protein or gene to affected organs. Furthermore, they suggest the feasibility of gene therapy for galactosialidosis and other disorders, using bone marrow cells engineered to overexpress and secrete the correcting lysosomal protein.