Search

Several recent studies have demonstrated the presence of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in healthy males with infertility caused by congenital absence of the vas deferens (CBAVD), previously recognized as an idiopathic genetic condition distinct from CF. In order to document further the genetic commonality of these two disorders, we undertook a double screening of the entire coding and flanking sequences of the CFTR gene, by using single-strand conformational polymorphism analysis and denaturing gradient gel electrophoresis in 12 unrelated infertile men with abnormalities of the vas deferens and/or epididymis. This strategy allowed us to identify 11 DNA sequence alterations considered as CF-causing mutations and several variations. Despite this double analysis, only two patients out of eight with CBAVD could be demonstrated as compound heterozygotes for CF mutations.

In order to characterize the non-delta F508 mutations that account for 36% of cystic fibrosis (CF) chromosomes in Southern France in a sample of 137 patients, we have systematically screened the entire coding region and adjacent sequences of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by the single strand conformation polymorphism (SSCP) technique followed by direct sequencing of the mutant DNAs. We identified 13 novel mutations (9 reported in this paper) and 4 novel rare nucleotide sequence variations. Forty different mutations including delta F508, located in 15 exons, account for only 91.2% of mutants in a population originating from Southern France, in contrast with a recent report on the Celtic population of Brittany demonstrating that 90% of mutations can be detected with only three mutations. We present a very large spectrum of different CF mutations identified in a small geographical area.

Annexins represent a widespread family of Ca(++)-dependent phospholipid binding proteins. Although their precise functions are still unknown, they probably play an important role in cell regulation because they are major substrates for various growth factor receptor kinases. We characterized annexins in human skin using three different antisera raised against annexin II, annexin V, and a synthetic peptide that resembles the consensus sequence of all annexins. In normal human skin, using SDS-PAGE, two-dimensional gel electrophoresis and immunoblot analysis, we identified two major 34-kDa proteins and one 36-kDa protein, with respective isoelectric points of 6.5, 5.2, and 7.2-7.9. According to these criteria they were identified as annexins, I, V, and II, respectively. Minor 45-51 kDa and 68-kDa proteins with 6.1-6.7 and 6.8-7.1 isoelectric points were also present, and likely corresponded to annexins VII and VI, respectively. We investigated the ability of these proteins to bind phospholipids in the presence of calcium using liposomes formed from a mixture of phosphatidylserine and phosphatidylcholine. The cellular distribution of annexins in normal human skin was determined by immunofluorescence with antiannexin II and anti-annexin V antibodies. Labeling with both antibodies was observed predominantly at the cell membrane with some cytoplasmic staining also being apparent. Specificity was confirmed by the absence of staining using pre-immune sera or after the absorption of the antibodies with their corresponding antigens. These proteins were also characterized in vitro in a reconstituted human skin model. All were present in this system except annexin VI and VII, which were lost after phospholipid purification. Further experiments should now be carried out using this system to clarify the role and regulation of these proteins within the epidermis.

Human skin is a unique organ, which can be reconstituted in vitro and represents an interesting system for studying cell proliferation and differentiation. A simple technique for producing reconstituted skin with optimal epidermal differentiation is described and characterized. A 4-mm punch biopsy of normal human skin is deposited on the epidermal side of mortified de-epidermized human dermis maintained at the air-liquid interface with a metallic support. The culture medium contains insulin, epidermal growth factor (EGF), cholera toxin, hydrocortisone, penicillin/streptomycin and fungizone. A well-differentiated epidermis develops within 15 days. Morphological and ultrastructural studies show a neoepidermis resembling normal skin. Differentiation markers such as involucrin, filaggrin, and various cytokeratins detected with pancytokeratin antibody are present and confirm this resemblance. The keratin profile is comparable to that observed in other skin culture models. A basement-membrane-like structure is reconstituted with hemidesmosomes and anchoring-filament formation. Bullous pemphigoid (BP) antigen is observed at the dermo-epidermal junction after 21 days of culture. Moreover, both dermal substrates and punch biopsies can be kept frozen for long-term storage, with little or no loss of epidermal growth kinetics and morphology. This skin culture technique is rapid, simple, economical and reproducible. Characterization has here shown high-quality epidermal differentiation. Scientists interested in epidermal in vitro studies should take interest in all these advantages.