Biomedical subjects

J Moroianu

Publications and source records attributed to J Moroianu.

22 records · Page 2Linked to original sources

PubMed1994

Nuclear translocation of angiogenin in proliferating endothelial cells is essential to its angiogenic activity.

The intracellular pathway of human angiogenin in calf pulmonary artery endothelial (CPAE) cells has been studied by immunofluorescence microscopy. Proliferating CPAE cells specifically endocytose native angiogenin and translocate it to the nucleus, where it accumulates in the nucleoli. Nuclear translocation of angiogenin does not occur in nonproliferative, confluent CPAE cells. These cells were previously found to express an angiogenin-binding protein (AngBP) that was identified as smooth muscle alpha-actin. Exogenous actin, an anti-actin antibody, heparin, and heparinase treatment all inhibit the internalization of angiogenin, suggesting the involvement of cell surface AngBP/actin and heparan sulfate proteoglycans in this process. It has been established that two regions of angiogenin are essential for its angiogenic activity, one is its endothelial cell binding site and the other its catalytic site capable of cleaving RNA. CPAE cells do not internalize four enzymatically active angiogenin derivatives whose cell binding site is modified, but they do internalize two enzymatically inactive mutants whose cell binding site is intact. Thus, the putative cell binding site of angiogenin is necessary for both endocytosis and nuclear translocation, but the catalytic site is not. Three other angiogenic molecules are also translocated to the nucleus of growing CPAE cells. Overall, the results suggest that nuclear translocation of angiogenin and other angiogenic molecules is a critical step in the process of angiogenesis.

Actins↗

PubMed1993

Actin is a surface component of calf pulmonary artery endothelial cells in culture.

An angiogenin binding protein isolated previously from endothelial cells has been shown to be a member of the actin family. Calf pulmonary artery endothelial (CPAE) cells were investigated for the presence of surface actin by immunoblotting of isolated surface proteins and by immunofluorescence. CPAE cell surface proteins were isolated by selective apical biotinylation and recovery of biotinylated proteins by avidin affinity chromatography. Immunoblotting with a specific smooth muscle alpha-actin antibody detected the presence of this type of actin among the isolated cell surface proteins. Immunofluorescence confirmed that smooth muscle alpha-actin is localized at the surface of nonpermeabilized CPAE cells. Exposure of CPAE cells to angiogenin prior to cell surface immunostaining diminished the signal. When CPAE and rat aortic smooth muscle cells were made permeable before staining, stress fibers could be recognized by the antibody in smooth muscle cells but not CPAE cells. The results indicate that a smooth muscle type of alpha-actin is localized specifically on the surface of cultured CPAE cells where it might interact with angiogenin and other actin binding proteins present in the extracellular environment.

Actins↗

PubMed1990

Albumin binding proteins are highly expressed in actively proliferating fetal and adult tissues.

The expression of albumin binding proteins (ABP, 31 and 18 kDa peptides) in various organs as a function of their ontogenic development was investigated in fetuses (20 days old), neonates (1 day old) and adult rabbits. At each of these stages, tissue extracts of brain, lung, thymus, heart, skeletal muscle and liver as well as whole embryos (11 days old) were examined by ligand blotting and quantitative immunoblot assays. Blots were either incubated with [125I]albumin followed by autoradiography and radioassay or exposed to a radioiodinated antibody raised against affinity-isolated 31 kDa peptide. Anti-31 kDa IgG cross-reacted with both 31 and 18 kDa peptides. Both methods used revealed that ABP are well expressed in embryos and in all fetal organs investigated. By comparison, in neonates, the ABP expression was diminished (by approximately 2-fold) in brain, heart and skeletal muscle. These changes were even more pronounced in the adult rabbit brain, heart, skeletal muscle and liver; no significant modification was detected in the lung. Prompted by these results, which inferred a high level of ABP in actively proliferating/differentiating tissues, we checked for the presence of ABP in other adult cells and tissues. In bone marrow cells, thymocytes and splenocytes, the 31 and 18 kDa peptides represented the major sodium dodecyl sulfate-urea extracted proteins, whereas in mature circulating white blood cells they were moderately expressed. The results indicate that ABP 1) are present early in embryogenesis, 2) are particularly well expressed in organs (fetal or adult) and cells characterized by active proliferation and differentiation, and 3) are not tissue specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumins↗

PubMed1989

Affinity isolation of albumin-binding proteins using nitrocellulose-bound albumin.

Albumin immobilized on a nitrocellulose membrane was used as an affinity matrix to purify albumin-binding proteins (ABP) from extracts of lung, heart, thymus, and isolated microvascular endothelial cells. Albumin was immobilized onto nitrocellulose either (i) directly (physically adsorbed), (ii) cross-linked by treatment with 0.25% glutaraldehyde, or (iii) covalently coupled to the matrix using NaIO4 and Na-borohydride. The affinity support was incubated with a membrane-enriched fraction (obtained from tissue homogenates) in the presence of protease inhibitors; specific binding of ABP occurred within 30 min of incubation. The adsorbed proteins were eluted with 0.5% sodium dodecyl sulfate (SDS) and analyzed by SDS-polyacrylamide gel electrophoresis and ligand blotting. Analysis of electrophoretic mobility of eluted proteins showed that they consisted exclusively of the two sets of polypeptides of 31 000 Da and 18 000 Da previously identified as ABP (N. Ghinea et al., J. Cell Biol. 107, 231-239 (1988]. As demonstrated by ligand blotting, the ABP purified on nitrocellulose-bound albumin maintain the ability to interact specifically with albumin. Preliminary experiments showed that the method employed may be of a broader use for the isolation of receptor proteins from tissue extracts by incubating the latter with the cognate ligand immobilized on nitrocellulose membranes.

Albumins↗