1,721,029 research outputs found
Synthesis of elastin in aortas from chick embryos. Conversion of newly secreted elastin to cross-linked elastin without apparent proteolysis of the molecule.
No full textThe biosynthesis of elastin was examined in matrix-free cells isolated by enzymic digestion of aortas from 17-day old chick embryos. After the cells were incubated with [14C]proline and then were rapidly boiled in buffer containing high concentrations of protease inhibitors and sodiumdodecyl sulfate, about one-quarter of the intracellular 14C-labeled protein was recovered as an elastin component with an apparent molecular weight of about 72 000. Examination of the medium from the cell suspension indicated that the largest elastin component secreted by the cells also had an apparent molecular weight of about 72 000. Pulse-chase experiments with intact aortas demonstrated that about two-thirds of the 72 000-dalton component disappeared in 2 h, apparently because it was converted to cross-linked fibers. When cross-linking was inhibited with penicillamine, the 72 000-dalton component persisted in the tissue 5 h. When cross-linking was inhibited with beta-aminopropionitrile, the elastin component of 72 000 daltons persisted for about 2 h, but thereafter it was gradually degraded to small peptides which were recovered in the incubation medium. The results suggest that elastin is secreted by cells in chick aorta as a polypeptide of about 72 000 daltons and that the secreted protein is incorporated into elastin fibers without cleavage to a protein of considerably smaller size
Is newly-secreted elastin cleaved to a smaller molecule before being incorporated into crosslinked elastin fibers?
No full textThe biosynthesis of elastin was examined in matrix-free cells isolated by enzymatic digestion of aortas from 17 day old chick embryos. When the cells were incubated with (14C) proline and then were rapidly boiled in buffer containing high concentrations of protease inhibitors and sodium dodecylsulfate, about one-quarter of the intracellular (14C)-protein was recovered as an elastin component with apparent molecular weight of about 72,000. Examination of the medium from the cell suspension indicated that the largest elastin component secreted by the cells also had an apparent molecular weight of about 72,000
Spatial and temporal changes of type VI collagen expression during mouse development.
No full textThe expression of type VI collagen has been studied in mouse tissues. By Northern blotting, the mRNA for the alpha 1 (VI) chain was detectable in whole embryos at 10.5 days postcoitum and steeply increased afterward. The messenger levels were high at birth, but decreased rapidly in the following days, reaching low levels in adult animals. In 2-month-old mice, lung, skin, adrenal gland, heart, skeletal muscle and tail and fat were among the most active producers of alpha 1 (VI) mRNA. In situ hybridization first identified mRNA for alpha 1 (VI) collagen in mesenchymal cells of 10.5-day embryos in various locations, including serosae, branchial arches, large blood vessels and the cephalic mesenchyme. Staining increased at later stages of development and most connective tissues were positive at 16.5 days and later. Strongly staining tissues were joints, intervertebral disks, perichondrium, periostium, dermis, skeletal muscle and heart valves, whereas cartilage and bone were very poorly labelled. Epithelia and the central nervous system were completely negative. In several organs, notably lung, salivary glands and the digestive tract, staining was concentrated underneath epithelia. This staining pattern was different from that for collagen type I, which was evenly distributed in the subepithelial mesenchyme. The pattern of distribution of the protein, revealed by immunocytochemistry, was coincident with that of the alpha 1 (VI) mRNA. In addition, the results confirmed that type VI collagen is preferentially deposited in the pericellular environment. This was particularly evident in skeletal muscle. The data show that type VI collagen is mainly produced by mesenchymal cells and suggest a role for the protein in delineating the boundary of distinct domains in connective tissue
Changes of cellular expression of mRNA for tropoelastin in the intraembryonic arterial vessels of developing chick revealed by in situ hybridization.
No full textThe pattern of expression of tropoelastin mRNA in the arterial tree of developing chick has been studied by in situ hybridization. Significant hybridization was noted in 5.5-day embryos in the region of the truncus arteriosus where aorta and pulmonary artery had newly separated. The activation of expression then propagated centrifugally and longitudinal gradients of mRNA decreasing from the heart to the periphery were established. For almost two-thirds of the embryonic period, the hybridization signal was rather uniform over the entire wall of the arterial vessels. Later, however, its distribution varied depending on the type of artery (elastic or muscular) and on the developmental stage. A radial gradient of tropoelastin mRNA expression decreasing in the in-out direction was formed in elastic arteries. This was first seen in the pulmonary artery (15-day chick embryos) and became detectable in the vessels of the general circulation only much later (2 weeks after hatching). The appearance of the radial gradient was followed by a general reduction of mRNA synthesis. In muscular arteries radial gradients were also established, but had, however, an opposite polarity; in small arteries a ring of hybridization was evident at the media-adventitia border. The results indicate that the expression of the tropoelastin gene in cells of the arterial wall is finely regulated, depending on the coordinates in the arterial tree, the type of artery and the organ supplied
Kinetics of the incorporation of tropoelastin into elastic fibers in embryonic chick aorta.
No full textMatrix-free cells isolated by enzymic digestion of chick embryo aortas were labeled with [14C]proline for 20 to 60 min and the kinetics of the secretion of tropoelastin were followed by chasing the label and assaying [14C]tropoelastin in the cells and in the medium. The results indicated that secretion of tropoelastin followed the kinetics of a single first order process with a half time of 60 min. In parallel experiments tissue explants of chick embryo aortas were labeled with [14C]proline and the kinetics for the incorporation of tropoelastin into elastic fibers were followed by chasing the label and assaying the soluble [14C]tropoelastin and insoluble [14C]elastin in tissues. It was found that the incorporation of tropoelastin into elastic fibers also followed a single first order process with a half time of 85 min, similar to the secretion of tropoelastin from cells. In further studies, antibodies directed against tropoelastin were utilized to isolate soluble [14C]elastin components in the tissues after 0 to 4 hr chase of the 14C label. The results demonstrated that all soluble elastin components were recovered as monomeric tropoelastin and no soluble oligomeric elastin could be detected. These results are consistent with the proposition that elastic fiber growth occurs by addition of individual tropoelastin molecules to existing fibers and that oligomers of elastin were not intermediates in the process
Repeating structure of chick tropoelastin revealed by complementary DNA cloning.
No full textA cDNA library was constructed from chick aorta poly(adenylic acid)-containing RNA in the expression vector pEX1. Several clones were identified by screening the library with a polyclonal antiserum raised against chick tropoelastin and confirmed by DNA sequencing. Analysis of the deduced amino acid sequence, corresponding to the mature tropoelastin and most of the signal peptide, revealed that the molecule is composed of at least 8, and possibly 13, repeating units. The common features of each unit include an N-terminal region composed largely of alanines and lysines and ending with an aromatic amino acid, followed by a GAG span and then a C-terminal region consisting mostly of valines, prolines, and glycines often present in several copies of the sequence (VPGV). This structure is discussed in terms of the functional properties of the molecule
The elastin associated glycoprotein Gp 115: synthesis and secretion by cells in colture
No full textSynthesis of gp115 by aorta smooth muscle cells and tendon fibroblasts isolated from chick embryos was investigated. gp115 was specifically immunoprecipitated by both polyclonal and monoclonal antibodies from cell lysates and culture medium of matrix free cells metabolically labeled with [3H]leucine and [35S]methionine. The component of gp115 isolated from the cell lysate had an apparent Mr in reduced sodium dodecyl sulfate polyacrylamide gels lower (105,000) than the protein isolated from the culture medium (Mr = 115,000). In immunoblot experiments, the latter corresponded in apparent Mr to the form isolated from chick tissues. gp115 was glycosylated in vitro; it was labeled with [3H]fucose, and when cells were cultured and labeled in the presence of tunicamycin, a lower Mr form with an apparent Mr = 90,000 was immunoprecipitated in both the cell lysate and the culture medium. In pulse-chase experiments, the intracellular and the extracellular forms were clearly suggestive of a direct precursor-product relationship in the absence of intermediate forms. The kinetics of secretion appeared very slow compared with that of other proteins of the extracellular matrix investigated in the same system; about 50-70% of gp115 in the form of the Mr = 105,000 species was still cell-associated after 4 h, whereas the half-time for secretion of fibronectin, type VI collagen, and tropoelastin was about 60 min, 3 h, and 60 min, respectively. Newly synthesized and processed cell-associated gp115 migrated in both reduced and non-reduced gels as a monomer. On the contrary, the secreted protein was present in the culture medium as large aggregates that did not enter the gel in the absence of reducing agents
Widespread codistribution of glycoprotein gp 115 and elastin in chick eye and other tissues.
No full textFrozen sections of chick tissues were exposed to affinity-purified monoclonal antibodies raised against chick gp 115 and to affinity-purified antibodies raised against chick tropoelastin to study the distribution pattern of the corresponding antigens by the avidin-biotin immunoperoxidase technique. Laminin and fibronectin antibodies were used for comparison. Gp 115 and tropoelastin antibodies localized to the same structure in several of the tissues examined. The endothelial membrane of the cornea and Bruch's membrane in the choroid were positive, while the corneal epithelial membrane was negative. Both antibodies displayed a peculiar punctate reactivity in the corneal stroma and a very fine fibrillar pattern in the conjunctiva and at the corneal-scleral junction. Liver, heart and large vessels, striated muscle and skin showed a similar pattern both for tropoelastin and gp 115 antibodies. Few differences were seen in the distribution of the reactivity: the pericellular matrix of intestinal smooth muscle cells was stained by gp 115 but not by tropoelastin antibodies. However, the reactivity of gp 115 and tropoelastin antibodies was similarly distributed in the lung smooth muscle cell clusters. The peritubular matrix in the kidney did also not react with tropoelastin antibodies as did the brain intraparenchymal vessels; whereas gp 115 antibody reactivity was present in both sites. We interpret these lack of apparent codistribution in some tissues as a variation in the relative availability of the target antigen for the reaction with the antibody and not as a consequence of a qualitative difference in the distribution of gp 115 and tropoelastin. By the use of anti gp 115 monoclonal antibodies that do not cross-react, and presumably recognize different epitopes, it was shown that some but not all antibodies, react with brain intraparenchymal blood vessels; whereas the pattern of distribution in other tissues was the same. This suggests that in vessels with an undetectable level of elastin, certain epitopes of gp 115 molecule might not be recognized as a result of being masked by other components or by a different conformation of the molecule
Efficient construction of cDNA libraries in plasmid expression vectors using an adaptor strategy.
No full textWe describe a method for the construction of large DNA fragment libraries in plasmid vectors, in which complementary, single-stranded extensions are ligated onto both vector and insert DNA using un-phosphorylated adaptor oligonucleotides. Special consideration has been taken of the requirements of expression screening as follows: cDNA synthesis using random oligonucleotide primers is described which maximises the probability of obtaining open reading frame fragments from large mRNA molecules, the adaptors use codons found in high abundance E. coli proteins to minimise problems of premature termination when using strong promoters, and the sequence encoded by the adaptors, when cloned into the bacterial expression vector pEX1, promotes a surface location for the foreign antigenic determinant where it is accessible to antibodies used for screening
- …
