Beta-N-acetyl-D-glucosaminidase activity in embryonic chick skin and lung tissue and cultured fibroblasts

During development the content of mesenchymal glycosaminoglycans (GAG) undergoes prominent changes, currently considered to act as regulatory signals in the epithelial-mesenchymal interactions. The factors involved in controlling GAG composition are as yet completely unknown. Lysosomal enzymes play a key role in GAG turnover. A possible mechanism for regulating GAG content could therefore be linked to developmental modulation of lysosomal glycosidases activity. We have examined the activity of the beta-N-acetyl-D-glucosaminidase (EC 3.2.1.30; a lysosomal hydrolase cleaving glycosidic linkage of the non-reducing terminal beta-N-acetyl-D-glucosamine residues) in chick embryo skin and lung (rudiments whose GAG composition has previously been studied) at various embryonic stages. Determinations were carried out on whole organs as well as on primary cultures of fibroblasts obtained from the two rudiments. beta-N-acetyl-D-glucosaminidase activity varied greatly during development, and it was significantly different in embryonic skin and lung tissues at various incubation days. In cultured fibroblasts, the enzymatic activity varied at different incubation days correlating with the in vivo data. Developmental changes of beta-N-acetyl-D-glucosaminidase paralleled mesenchymal GAG pattern both in vivo and in vitro. Our results, therefore, support the possibility that lysosomal enzymes could be involved in the regulation of mesenchymal GAG content during development.     

Extracellular glycosaminoglycans (GAG) released by chick embryonic fibroblasts

Summary Administration of Concanavalin A (Con A) to cultured skin fibroblasts derived from chick embryos at two developmental stages produce variations in the relative concentration of individual glycosaminoglycan (GAG) secreted by the cells. This effect is different: at 7 days (increase of hyaluronic acid and dermatan sulphate and decrease of chondroitin sulphate) and at 14 days (dermatan sulphate is not detectable).All the cells bind the Con A specifically, but a different pattern of agglutination is present in fibroblasts of the two embryonic ages. Since Con A is well known to bind carbohydrate-containing surface proteins, the result suggests that the release of GAG by chick embryonic fibroblasts can be modulated by cell surface receptors.     

Interleukin secretion, proteoglycan and procollagen alpha(1)(I) gene expression in Crouzon fibroblasts treated with basic fibroblast growth factor

The present study provides the first evidence that fibroblasts obtained from patients affected by Crouzon syndrome, a rare craniosynostosis, despite mutations in the high-affinity bFGF receptor retain their capacity to respond to bFGF. The growth factor reduces IL-1 secretion, downregulates biglycan and procollagen alpha(1)(I), and increases betaglycan expression. Since betaglycan is a co-receptor for bFGF signalling, an alternative signal transduction pathway is suggested in Crouzon fibroblasts, to explain the documented changes in ECM macromolecule production.     

Extracellular matrix and growth factors in the pathogenesis of some craniofacial malformations

The normal development of cranial primordia and orofacial structures involves fundamental processes in which growth, morphogenesis, and cell differentiation take place and interactions between extracellular matrix (ECM) components, growth factors and embryonic tissues are involved. Biochemical and molecular aspects of craniofacial development, such as the biological regulation of normal or premature cranial suture fusion, has just begun to be understood, thanks mainly to studies performed in the last decade. Several mutations has been identified in both syndromic and non-syndromic craniosynostosis patients throwing new light onto the etiology, classification and developmental pathology of these diseases. In the more common craniosynostosis syndromes and other skeletal growth disorders, the mutations were identified in the genes encoding fibroblast growth factor receptor types 1-3 (FGFR1, 2 and 3) where they are dominantly acting and affect specific and important protein binding domain. The unregulated FGF signaling during intramembranous ossification is associated to the Apert and Crouzon syndrome. The non syndromic cleft of the lip and/or palate (CLP) has a more complex genetic background if compared to craniosynostosis syndrome because of the number of involved genes and type of inheritance. Moreover, the influence of environmental factor makes difficult to clarify the primary causes of this malformation. ECM represents cell environment and results mainly composed by collagens, fibronectin, proteoglycans (PG) and hyaluronate (HA). Cooperative effects of ECM and growth factors regulate regional matrix production during the morphogenetic events, connective tissue remodelling and pathological states. In the present review we summarize the studies we performed in the last years to better clarify the role of ECM and growth factors in the etiology and pathogenesis of craniosynostosis and CLP diseases.     

Incrimination of Anopheles (Anopheles) intermedius Peryassú, An. (Nyssorhynchus) nuneztovari Gabaldón, An. (Nys.) oswaldoi Peryassú as natural vectors of Plasmodium falciparum in French Guiana

Anopheles darlingi Root is the major vector of human malaria in the Neotropics and has been considered to be the sole malaria vector in French Guiana. The presence of other potential vectors suggests that malaria may be transmitted by other species under certain conditions. From 2006-2011, all anopheline specimens collected from 11 localities were assayed to determine if the Plasmodium circumsporozoite protein was present. In addition to An. darlingi, we found Anopheles oswaldoi, Anopheles intermedius and Anopheles nuneztovari specimens that were infected with Plasmodium sp. Further investigations on the behaviour and ecology of An. oswaldoi, An. intermedius and An. nuneztovari are necessary to determine their role in malaria transmission in French Guiana.     

Linkage disequilibrium between GABRB3 gene and nonsyndromic familial cleft lip with or without cleft palate

The malformation of nonsyndromic cleft lip with or without cleft palate (CL/P) is a common congenital disease that affects approximately 1/1000 newborns in Caucasian populations. Genetic studies indicate that CL/P has the characteristics of a complex genetic trait. Linkage analysis and mouse-model knockout studies have suggested several candidate genes mapping in different chromosome regions for CL/P malformation. On these grounds, we have investigated, by linkage disequilibrium (LD) and parametric and nonparametric linkage analyses, five different candidate genes, including those for the beta3 subunit of the gamma-aminobutyric acid receptor (GABRB3), glutamic acid decarboxylase 1 (GAD1), retinoic acid receptor alpha (RARA), transforming growth factor beta3 (TGFB3), and msh ( Drosophila) homeobox homolog 1 (MSX1). Interestingly, a significant LD between GABRB3 and CL/P was obtained ( P-value=0.008 in the allele-wise analysis for multiallelic markers), suggesting that the GABRB3 gene is involved in this congenital disease. This new finding in humans is in agreement with previously reported data obtained with the murine model. Indeed, mouse studies indicate a role for gamma-aminobutyric acid (GABA) and its receptor in normal palate development. Exclusion of the GAD1 gene, which encodes the GABA-producing enzyme, in CL/P pathogenesis was obtained in our study. Moreover, we were unable to confirm the involvement of the MSX1 gene in nonsyndromic CL/P. Modest evidence of LD between marker alleles and CL/P was found at the RARA and TGFB3 loci suggesting a minor role for these genes in our family set of nonsyndromic CL/P.     

Regulation of protein synthesis by estradiol-17beta in cultured hepatocytes

Estradiol-17beta added to cultured chick embryo hepatocytes induced the appearance in the medium of a phosphoprotein, identified as phosvitin on the basis of: (i) its behaviour on ionic exchange columns; (ii) its SDS-acrylamide gel electrophoretic mobility; (iii) its amino acid composition. The hormone treatment was also followed by a decreased synthesis of other proteins secreted by the hepatocytes.