Ranieri Cancedda

cDNA cloning, characterization and chromosome mapping of the gene encoding human cartilage associated protein (CRTAP).

We have recently isolated and characterized cDNA clones coding for a novel developmentally regulated avian and mouse embryo protein, CASP for Cartilage Associated Protein. Here we describe the isolation and characterization of the gene coding for the human CASP. The comparison of the putative human and mouse protein sequences with the chick sequence revealed an overall high identity (89% and 51%, respectively). Homology search with known DNA and protein sequences showed that CASPs are related to two mammalian nuclear proteins. Here we demonstrate definitively that CASPs are distinct from these nuclear proteins. However, sequence comparison analyses suggest that all of these proteins belong to a new family. In all human tissues examined two CASP mRNA species were detected, whereas a single mRNA and three mRNAs were found in chick and mouse, respectively. The human CASP gene (CRTAP) was assigned to chromosome 3p22 by fluorescence in situ hybridization.

Acute phase lipocalin Ex-FABP is involved in heart development and cell survival.

Ex‐FABP is an extracellular fatty acid binding protein, expressed during chicken embryo development in cartilage, muscle fibers, and blood granulocytes. Transfection of chondrocytes and myoblasts with anti‐sense Ex‐FABP cDNA results in inhibition of cell proliferation and apoptosis induction. Ex‐FABP expression is dramatically enhanced by inflammatory stimuli and in pathological conditions. In this paper, by in situ whole mount and immunohistochemistry analysis we show that, at early developmental stage, Ex‐FABP is diffuse in all tissues of chick embryos. Particularly high level of transcript and protein are expressed in the heart. During acute phase response (APR) induced by endotoxin LPS injection, a marked increase of Ex‐FABP mRNA was observed in embryos, highest Ex‐FABP expression being in heart and liver. To investigate in vivo the biological role of Ex‐FABP, we have directly microinjected chicken embryos with antibody against Ex‐FABP. Almost 70% of chicken embryos died and the target tissue was the heart. We detected in heart of the treated embryos a significant increase of apoptotic cells and high level of fatty acids. We propose that the accumulation of fatty acid, specific ligand of Ex‐FABP, in the cell microenvironment is responsible of heart cell death, and we suggest that Ex‐FABP may act as a survival protein by playing a role as scavenger for fatty acids.

In vitro translation of chicken type X collagen in the presence of pancreas microsomes

Total RNA from epiphysis of 17-day-old chick embryo tibiae was used to direct protein synthesis in a wheat germ cell free system. The type X collagen chain, identified on the basis of its electrophoretic migration and of peptides obtained by S. aureus V8 protease digestion, was the major translation product. The newly synthesized chain included a signal sequence that was removed when dog pancreas membranes were added at the time of the protein synthesis.

Expression of the extracellular fatty acid-binding protein during muscle fiber formationin Vivo andin Vitro

Extracellular fatty acid-binding protein (Ex-FABP) is a 21-kDa protein developmentally regulated in chick embryo, first observed in vitro in chondrocytes at a late stage of differentiation as a protein secreted into the culture medium (1). Immunolocalization performed in cartilage tissues with polyclonal antibodies obtained against the purified protein showed that the protein is present in vivo in chicken embryo tibia in hypertrophic chondrocytes of the growth plate at the border between hypertrophic cartilage and newly forrned bone, in the cartilage surrounding marrow cavities and blood vessels, and in the epiphysial prearticular cartilage. The complete amino acid sequence of the protein, obtained by combining protein and nucleotide sequence data, assigned the protein to the superfamity of lipocalins or lipophitic molecules carrier proteins (2). The recombinant protein was obtained in the baculovirus system, and studies were performed to identify a possible ligand. By lipidex binding assay with radioactive lipophilic compounds, a specific fatty acid binding to the protein was shown and a preferential binding of long-chain unsaturated fatty acids was observed (3). Calculated dissociation constant was 2 x 10-7M for unsaturated fatty acids and 5 x 10 .7 M for stearic acid. Shortchain fatty acids did not bind to the protein. Other hydrophobic molecules as retinoic acid, retinol, progesterone, prostaglandins, and long-chain alcohols and aldehydes did not bind to the protein. It is widely accepted that free fatty acids in blood are transported by albumin. Ex-FABP is present in chicken serum and represents the first extracellular protein able to selectively bind and transport fatty acids in extracellular fluid and serum. Ex-FABP could represent inblood the high-affinity, low-capacity, specific binding protein that transports fatty acids to target organs while albumin could represent a tow-affinity, high-capacity storage protein for fatty acids. Ex-FABP is also produced by peripheric granutocytes. We performed studies aimed to elucidate a possible role of the protein during chicken embryo development, and we observed that Ex-FABP is expressed in the forming myotubes both in vitro and in vivo. The presence of the protein and of the mRNA was observed in newly formed myotubes at early stages of chick embryo development by im-