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D Volpin

Publications and source records attributed to D Volpin.

At least 37 records · Page 2Linked to original sources
PubMed1987

Biosynthesis of chick type VI collagen. I. Intracellular assembly and molecular structure.

A monospecific rabbit antiserum to pepsin-extracted chick gizzard type VI collagen was used to characterize the intact forms of type VI collagen in tissues and cultured cells. Immunoblotting of gizzard extracts revealed polypeptides of Mr ranging from 260,000 to 140,000. Components of about Mr = 260,000, 150,000, and 140,000, each with a different peptide profile, were immunoprecipitated from labeled matrix-free chick embryo cells. Cleavage of the immunoprecipitated polypeptides with pepsin generated pepsin-resistant fragments of about Mr = 70,000, 55,000, and 45,000 that represent the alpha 1(VI), alpha 2(VI), and alpha 3 (VI) fragments. Immunoblotting with affinity-purified antibodies indicated that the Mr = 150,000 is the intact parent polypeptide of the alpha 1(VI) pepsin; the Mr = 140,000 of the alpha 2(VI) pepsin, and the Mr = 260,000 of the alpha 3(VI) pepsin. Association of the three parent chains was studied by pulse-chase experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis under nonreduced conditions. A complex of Mr = 500,000 is already present intracellularly at the end of a 7-min pulse and increases considerably with time while the three unassembled chains show a comparable decrease. After 5-15 min of chase larger forms appeared along with small amounts of aggregated material that did not enter the gel. Analysis of the immunoprecipitate by diagonal electrophoresis indicated that the component of Mr = 500,000 and the larger forms dissociated into the Mr = 260,000, 150,000, and 140,000 polypeptides. Sedimentation profile of a labeled cell extract on a 5-20% sucrose gradient under nondenaturing conditions confirmed the presence of three different peptides in the complex.

Animals↗
PubMed1987

Detection of elastin by immunoelectronmicroscopy. A comparison of different procedures.

Elastin components have been identified in chick aorta by different immunoelectronmicroscopic procedures (peroxidase-antiperoxidase, immunoferritin and immunogold) using affinity purified antibodies to chick tropoelastin. The PAP method used in a preembedding procedure stained the outer portion of amorphous elastin and the microfibrils very intensively. The surface of the cells was also slightly stained. On the contrary immunogold labelling on Epon or Lowicryl embedded sections produced a strong decoration only of amorphous elastin, while microfibrils remained almost completely unlabelled. The result is not due to loss of antigenicity of microfibrils during embedding, since similar data were obtained with immunoferritin in a preembedding procedure. Experiments performed under different stringency conditions showed that the products of the peroxidase reaction diffuse and redistribute in the tissue, indicating that the positive staining of microfibrils and cell surface is an artifact. The value of different immunological reagents and procedures in studying the fine mapping of elastin components is discussed.

Animals↗
PubMed1987

Fine mapping of tropoelastin-derived components in the aorta of developing chick embryo.

Affinity-purified antitropoelastin antibodies have been used to localize tropoelastin-derived components in aortas from chick embryos of different age by immunoelectron microscopy. Staining in the matrix is first noted at day 3 associated with irregular bundles of filaments resembling microfibrils, in the absence of amorphous elastin deposits. Amorphous material, which rapidly accumulates at later stages, is heavily labelled, while surrounding microfibrils are only poorly labelled. By contrast, a more intense staining of microfibrils persists in regions in which amorphous material is not morphologically evident. These observations indicate that the initial accumulation of elastin requires microfibrils, while the two components are not in close association in the subsequent growth of the amorphous core of the fibre. Intracellular staining is evident in the secretory apparatus of the cell and in peripheral large vesicles. Differentiated cells also show regions of close contact with elastic fibres in which immunological staining for elastin is very close to the cell membrane.

Animals↗
PubMed1987

Widespread codistribution of glycoprotein gp 115 and elastin in chick eye and other tissues.

Frozen 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.

Animals↗
PubMed1987

Multiple binding reactivities of an IgG1 mouse monoclonal antibody raised against the extracellular matrix glycoprotein Gp 115.

An IgG1 mouse monoclonal antibody that recognized five different polypeptides from chick and man is described. The antibody was raised against the elastin associated chick glycoprotein gp 115. In addition to the immunizing antigen, antibody 106D6 bound to chick smooth muscle cell myosin and to the alpha, beta and gamma chains of human fibrinogen. Cross reaction was demonstrated by radioimmunobinding, immunofluorescence and immunoblotting experiments. The immunoblotting assays were carried out using identical amounts of the various antigens to exclude the possibility that relevant quantitative differences in their concentration might result in non-specific binding.

Amino Acid Sequence↗
PubMed1986

Relevance of aggregation properties of tropoelastin to the assembly and structure of elastic fibers.

Solutions of tropoelastin incubated under different experimental conditions were examined by electron microscopy after negative staining and after fixation and embedding. Below 37 degrees C only polymorphous structureless elements of variable size could be found. In samples kept for a few minutes at 40 degrees C, flexible, isolated filaments of 5 nm diameter and variable length, together with a few small aggregates of filaments, were seen. No single filaments, but only bundles of filaments were detectable after incubation at 40 degrees C for longer than 5-10 min. Tropoelastin kept at 40 degrees C for longer than 10 hr formed a white precipitate, which, when fixed and embedded as in conventional electron microscopy, consisted of 0.5-2 microns thick, amorphous and branching fibers, identical to those seen in identically processed normal tissues. From these observations a model for the assembly and structure of elastic fibers is proposed.

Animals↗
PubMed1986

Aortic elastin abnormalities in osteogenesis imperfecta type II.

Skin and aortic samples from two patients who died by lethal perinatal Osteogenesis Imperfecta (O.I.) were studied by optical and electron microscopy and compared with similar samples from two normal human fetuses and one newborn child. No significant abnormalities were observed in the dermis of O.I. patients apart from small differences in the diameter of reticular collagen fibrils. On the contrary, in the aortas of both patients collagen fibrils were significantly smaller than in the controls; moreover, elastin lamellae were deeply altered and consisted of roundish aggregates of elastin, massively permeated by cytochemically recognizable glycosaminoglycans. As identical features were described in experimental lathyrism by using inhibitors of the enzyme lysyl oxidase (Pasquali Ronchetti et al., 1984), the conclusion is reached that in the two cases of lethal perinatal O.I. examined, a severe lysyl oxidase deficiency could account for the observed ultrastructural abnormalities of elastin and that, besides defects of collagen type I, additional alterations of cellular metabolism might be responsible for the clinical heterogeneity of the disease.

Aorta↗
PubMed1985

Monoclonal antibodies against chick gp 115, a matrix glycoprotein with broad distribution.

Hybridoma cell lines were generated producing monoclonal antibodies to chick gp 115, a 115,000-dalton glycoprotein widely distributed in the connective tissue. The specificity of the antibodies was determined by indirect radioimmunobinding: the extent of binding was a function of i) antigen and ii) antibody concentration; iii) inhibition of binding of radiolabelled antibody by unlabelled antibody and iv) among many known extracellular collagenous or noncollagenous glycoproteins tested only gp 115 gave a strong positive binding reaction. The antibodies were used for indirect immunofluorescence and a strong staining reaction was detected in all blood vessels, around smooth muscle cells in several organs, and in the connective matrix of other tissues such as the liver, and the lung. Based on the competition of binding of [125I]-labeled purified antibody by unlabeled antibodies, two separate epitopes were identified on gp 115. Further analysis of the localization of the epitope was obtained by CNBr cleavage and partial digestion of gp 115 with Staphylococcus aureus V8 protease and alpha-chymotrypsin digestion. Following CNBr cleavage a major fragment of Mr = 35,000 was recognized by 4 monoclonal antibodies, and fragments of comparable Mr were detected following V8 protease and alpha-chymotrypsin digestion.

Animals↗
PubMed1985

Glycoprotein 115, a glycoprotein isolated from chick blood vessels, is widely distributed in connective tissue.

An extracellular glycoprotein (gp 115) with an apparent Mr = 115,000 isolated from chick aortas (Bressan, G. M., I. Castellani, A. Colombatti, and D. Volpin, 1983, J. Biol. Chem., 258:13262-13267), was used to immunize mice. The antisera were shown to specifically recognize gp 115 by numerous criteria: a major band around Mr = 115,000 plus minor bands of lower Mr were visible by immunoblotting on aorta extracts, and a similar pattern was observed with a monoclonal antibody; no cross-reactivity was detected by radioimmunobinding with other extracellular proteins, namely, fibronectin, laminin, and collagen types I, III, IV, V, and VI. Antigen distribution on frozen tissue sections from newborn chicks was investigated by using affinity-purified antibody. Strong immunoreactivity was always found in blood vessels. In the digestive tract, the fluorescent staining was localized both at the level of muscular layers and in the stromal matrix of the villi. Within skeletal muscle and myocardium, staining was associated with large connective tissue bundles and the matrix around each muscle fiber. Intense fluorescence was observed in the kidney, in smooth muscle cells rich areas of parabronchi, and within the portal space and along liver sinusoids. The antigen was not detected at the epidermal-dermal junction; immunoreactivity in the dermis was present as a diffuse fibrillar pattern. That the antigen detected by immunofluorescence in the various organs was indeed gp 115 was demonstrated by immunoblotting analysis: as in aorta extracts, a major band around Mr = 115,000 was detected in several tissues. Antibody-reacting material was also incorporated into the extracellular matrix produced by embryo smooth muscle cells grown in vitro and was organized as a meshwork of fine fibrils.

Animals↗
PubMed1984

Elastin fiber-associated glycosaminoglycans in beta-aminopropionitrile-induced lathyrism.

Ruthenium red and toluidine blue O precipitates were described associated with lathyritic elastic fibers in aortas of chickens treated with beta-aminopropionitrile fumarate (I. Pasquali-Ronchetti, C. Fornieri, I. Castellani, G. M. Bressan, and D. Volpin (1981). Alterations of the connective tissue components induced by beta-aminopropionitrile. Exp. Mol. Pathol. 35, 42-56). In this report evidence is given that these precipitates reveal the presence of proteoglycans, as they are completely removed by 5 M guanidine-HCl incubation and by specific enzymatic digestions. In particular, proteoglycans associated with the poorly cross-linked lathyritic elastin can be removed by testicular hyaluronidase, chondroitinase ABC, heparitinase, and nitrous acid treatments, whereas they are rather resistant to streptococcal hyaluronidase and chondroitinase AC. On the contrary, proteoglycans of the matrix or associated with collagen fibers are particularly sensitive to these latter enzymatic treatments. The conclusion is reached that glycosaminoglycans associated with beta-aminopropionitrile-induced lathyritic elastin (i) are different from those of the matrix or associated with collagen, and (ii) include mainly dermatan and heparan sulfates.

Aminopropionitrile↗
PubMed1983

Isolation and characterization of a 115,000-dalton matrix-associated glycoprotein from chick aorta.

Chick aortas were extracted sequentially with phosphate-buffered saline, 6 M guanidine HCl, and 6 M guanidine HCl containing dithioerythritol. The proteins present in the guanidine HCl + dithioerythritol extract were separated by DEAE-cellulose chromatography, and the fractions recovered were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Five major glycoprotein components with apparent Mr = 205,000, 195,000, 150,000, 135,000, and 115,000 (gp 115) were identified. gp 115 was further studied since it was the only noncollagenous protein based on amino acid analysis. The protein was purified to homogeneity by preparative electrophoresis. Its amino acid composition was characterized by a high content of glutamic acid and arginine and a relatively high content of leucine, glycine, and alanine. The concentration of gp 115 in the guanidine HCl + dithioerythritol extract was about 15-fold that in the guanidine and saline extracts. Overall, about 80% of the protein was solubilized with guanidine HCl + dithioerythritol, suggesting that most of it formed large aggregates stabilized by disulfide bonds in vivo. Immunofluorescence studies with specific antibodies showed that gp 115 formed an extracellular fibrillar network in the aorta wall. One-dimensional finger printing with Staphylococcus aureus V8 protease and immunological studies indicated that the protein was unrelated to fibronectin and laminin. The data led us to conclude that gp 115 is a novel extracellular component of chick aorta.

Amino Acids↗
PubMed1983

Banded fibers in tropoelastin coacervates at physiological temperatures.

Tropoelastin was purified from aortas of chicks grown on a beta-aminopropionitrile-containing diet. The preparation could be considered pure following the criteria of amino acid composition and gel electrophoresis. When aqueous solutions of tropoelastin (5 mg/ml) were warmed to 40 degrees C (physiological temperature for chicken) for 10 min, and observed by negative-staining electron microscopy, it revealed the presence of two kinds of ordered structures. One consisted of densely packed parallel filaments with a center-to-center distance of about 5 nm, and the other of banded fibers, 100-150 nm in diameter, with a cross periodicity of about 55 nm. In some areas the fibers appeared to be formed by lateral aggregation of 1.5-2-nm-thick microfilaments. The fibers were similar to those previously obtained with the synthetic polypentapeptide of elastin (Val-Pro-Gly-Val-Gly)n and degradation products of elastin at temperatures much higher than the physiological one. The results indicate that the property of tropoelastin to form ordered structures is intrinsic to some of the polypeptide sequences of the molecule and that hydrophobic forces are involved in the formation of the aggregates.

Amino Acids↗
PubMed1981

Pseudoxanthoma elasticum. Biochemical and ultrastructural studies.

Ultrastructural and biochemical studies of the dermis from patients with pseudoxanthoma elasticum are described. Elastin fibers showed mineral deposits that were associated with an elastin material exhibiting altered affinity for the electron microscopical stains and altered ultrastructural organization. In the most affected patient the elastin was shown to be increased and to have an abnormal amino acid composition. In some patients the collagen fibers were laterally fused. The ground substance was increased as evidenced by electron microscopy and confirmed by a slight increase in uronic acids, hexosamines, and neutral sugars.

Amino Acids↗
PubMed1979

X-ray diffraction study of bovine lens capsule collagen.

The wide angle X-ray diffraction pattern of air-dried lens capsule collagen under tension is the same as the tendon collagen diffraction pattern with regard to the main reflections, and indicates that lens capsule collagen has the characteristic three-stranded helical structure with an axial repeat of 0.29 nm as tendon collagen. The low angle X-ray diffraction pattern shows several weak diffraction maxima corresponding to the meridional reflections of capsule collagen which show orders of 63.0 nm periodicity. This is an evidence of quarter staggered molecular assembly typical of tendon collagen even if less ordered. The results are consistent with the existence in lens capsule collagen of clearly defined molecular units, which can be oriented by stress and are packed in a poor-ordered fibrillar assembly.

Animals↗
PubMed1978

Structural organization of collagen fibrils in media aortic wall.

Small-angle X-ray diffraction patterns of bovine, human and porcine media aortic wall show meridional reflections corresponding to a periodicity which suggest a molecular packing typical of tendon collagen. However the meridional intensity distribution of stretched air dried aortic samples appears different from that of air-dried tendon, probably because of the presence of a large amount of type III collagen with the environment, which are specific for aortic tissue. The stretched wet aortic samples show a marked decrease in intensity, revealing an extensive disorder in the axially-projected structure of the fibrils. When a loading system simulating the effect of blood pressure is applied to a ring of aorta, no evidence of orientation of collagen is seen by X-ray diffraction, as would be expected if collagen fibrils had an isotropic distribution inside the aorta media. Scanning electroni microscopy supports the existence of a network of collagen fibrils surrounding elastic lamellae.

Adult↗