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E G Cleary

Publications and source records attributed to E G Cleary.

At least 37 records · Page 2Linked to original sources
PubMed1989

The protein components of the 12-nanometer microfibrils of elastic and nonelastic tissues.

A procedure has been developed which is much more specific for the solubilization of the elastin-associated microfibrils from fetal bovine nuchal ligament using treatment with reductive saline in place of reductive guanidine hydrochloride buffer. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reductive saline extracts were shown to contain only five major protein bands with Mrs of 340,000, 78,000, 70,000, 31,000, and 25,000. The 31-kDa species was identified immunologically as the previously described macromolecule named microfibril-associated glycoprotein (MAGP) (Gibson, M. A., Hughes, J. L., Fanning, J. C., and Cleary, E. G. (1986) J. Biol. Chem. 261, 11429-11436). The proteins were purified by gel permeation, ion exchange, and affinity chromatography. Amino acid analyses showed that each protein had a profile which was distinct from that of MAGP although each was also high in acidic amino acids and cystine. The 340- and 78-kDa species were each demonstrated by immunoelectron microscopy with affinity-purified antibodies to be derived from the elastin-associated microfibris, and these were provisionally named microfibrillar protein 340 (MP340) and microfibrillar protein 78 (MP78), respectively. Each of the above antibodies gave a tissue distribution identical to that of anti-MAGP antibodies, and thus MP340 and MP78 also were identified with the 12-nm microfibrils of nonelastic tissues. MP340 was shown to absorb out completely the microfibrillar immunoreactivity of anti-(reductive guanidine hydrochloride extract) antibodies, indicating that MP340 was (a) the major microfibrillar constituent in these extracts and (b) the second unidentified microfibrillar antigen described previously. The relationship of the 70- and 25-kDa proteins to microfibrils is yet to be established. Immunoblot and immunoabsorption studies showed that MAGP and MP78 were immunologically related to MP340 but not to each other. Cyanogen bromide peptide mapping indicated that MAGP was structurally related to MP340. It is postulated that MAGP and MP78 are constituents of MP340 which in turn is the subunit of which the 12-nm microfibrils are composed.

Amino Acids↗
PubMed1989

The tissue distribution of microfibrils reacting with a monospecific antibody to MAGP, the major glycoprotein antigen of elastin-associated microfibrils.

Elastic tissue, when viewed in the electron microscope, consists of an amorphous component that is immunoreactive with anti-tropoelastin (TE) antibodies and microfibrils, that react with monospecific antibodies against a 31 kDa microfibrillar glycoprotein constituent, called MAGP. A detailed study of the tissue distribution of microfibrils and of the two elastic tissue antibodies has been carried out, using single and double-labeled immunogold techniques in high resolution electron microscopy. Microfibrils similar in appearance to those associated with elastic tissue and immunoreactive with the anti-MAGP antibody, have been demonstrated in many tissues in the absence of amorphous elastic tissue. In the majority of these tissues, specific anti-TE antibody localization was demonstrated in the immediate vicinity of the microfibrils, or alternatively, the microfibrils were shown to be in direct continuity with microfibrils of similar morphology, which were associated with material immunoreactive with anti-TE antibody. The diameter of these microfibrils varied between 8 nm and 16 nm. They were unbranched structures of indefinite length, with a tubular profile on cross section and periodic staining in longitudinal section. In some tissues, notably in the ciliary zonule and in the mesangial region of the renal glomerulus, microfibrils of similar morphology were demonstrated which were immunoreactive with anti-MAGP antibody, but which were unrelated to amorphous elastic tissue and with which anti-TE antibody localization could not be demonstrated. The evidence available supports the conclusion that all these microfibrils are members of a single class of structures, which are widely distributed in the tissues and which are secreted by a range of cell types. Attention is directed to the close relationship between these microfibrils and the basement membrane of the glomerulus, of uterine smooth muscle, of the basal cells of the epidermis and of the reticulum cells of the spleen.

Animals↗
PubMed1988

Development of immunoreagents to ciliary zonules that react with protein components of elastic fiber microfibrils and with elastin-producing cells.

We describe the generation of a monoclonal antibody library to ocular zonule components and the characterization of three monoclonal antibodies: 1) one specific for microfibrillar associated glycoprotein (MAGP), a component of both ocular zonules and microfibrils of elastin fibers, 2) an antibody to an as yet unidentified 70,000 dalton antigen that is present in abundance in the extracellular matrix (ECM) of elastin-producing cells, and 3) an antibody reacting with the 67000 dalton subunit of the elastin receptor. The presence of antigenic determinants common to the ocular zonule and elastic fiber microfibrils suggests that zonules, which can be obtained in relatively pure form, can provide a valuable resource for characterizing proteins common to both microfibrillar structures.

Animals↗
PubMed1988

Post-embedding methods for immunolocalization of elastin and related components in tissues.

Elastic tissue is composed of amorphous-appearing elastin and 12-nm diameter microfibrils, one component of which has recently been isolated and characterized as the 31 KD microfibril-associated glycoprotein MAGP. Monospecific antibodies to each of these components have been developed in this laboratory. The parameters that determine optimal localization of colloidal gold probes for post-embedding immunolabeling of elastic tissue components have been systematically studied in a variety of normal and developing tissues in mammals and birds. Protein A-gold probes stabilized with dextran have been shown to provide complexes that remain stable after more than 2 years. Conditions have been defined that permit precise localization within the extracellular matrix of antibodies to MAGP and to elastin, singly and together. Best results were obtained with acrylic resins (Lowicryl K4M or LR White). Fixation in glutaraldehyde or other aldehydic fixatives, with or without osmium, did not affect the immunostaining of elastic tissue with affinity-purified antibodies to tropoelastin, or to anti-[alpha-elastin] or anti-[alkali-insoluble elastin]. Immunostaining with the anti-MAGP antibody was less robust and was possible in tissues which had been fixed only lightly before embedding in Lowicryl K4M or LR White. This staining was enhanced by metaperiodate oxidation of the sections as well as by reduction of the tissues with sodium borohydride en bloc, followed by hyaluronidase digestion of the sections. The effects on immunostaining of a range of enzyme digestions have also been examined. Conditions have thus been defined that make possible detailed study of the relationship between elastic tissue, elastin-associated microfibrils, and other microfibrillar structures in normal and abnormal tissues during development and aging.

Animals↗
PubMed1987

The immunohistochemical localisation of microfibril-associated glycoprotein (MAGP) in elastic and non-elastic tissues.

We have previously identified the major antigen of elastin-associated microfibrils as a 31kD glycoprotein which we named microfibril-associated glycoprotein or MAGP. Affinity-purified antibodies to MAGP were shown to localise specifically to elastin-associated microfibrils in sections of bovine foetal nuchal ligament. In the present paper we compare the localisation of anti-MAGP antibodies and anti-tropoelastin antibodies in a range of bovine elastic and non-elastic tissues. The results show that anti-MAGP antibodies invariably localised to immuno-reactive elastic fibres, wherever they occurred. Extensive additional localisation was observed in a number of tissues. This extra distribution of anti-MAGP antibodies was found to correspond to those structures exhibiting the oxytalan histochemical staining reaction in tissues such as skin, periodontal ligament and ocular zonule. Since these oxytalan fibres have been shown to consist of 12 nm microfibrils which are morphologically similar to those of elastic fibres (and unpublished data from this laboratory confirm this conclusion), the results suggest that MAGP is a component of 12 nm microfibrils in both elastic and non-elastic tissues. Anti-tropoelastin antibodies did not localise to these oxytalan fibres, suggesting that tropoelastin is not a component of 12 nm microfibrils. MAGP was also detected in extracellular matrix regions of tissues such as skeletal muscle, Achilles tendon and spleen, suggesting that 12 nm microfibrils, containing one or more macromolecular constituents in common, make up an important structural system within the extracellular matrix in a wide range of elastic and non-elastic tissues.

Animals↗
PubMed1986

The major antigen of elastin-associated microfibrils is a 31-kDa glycoprotein.

The major antigen derived from elastic fiber microfibrils was identified as a Mr = 31,000 glycoprotein, using immunoblotting and immunohistochemical techniques with antisera raised to "reductive guanidine extracts" of fetal bovine nuchal ligament, and to subfractions of these. A second, elastic fiber-derived, but unidentified, antigen of large molecular size (Mr greater than 200,000) was present in these extracts. Antisera raised to the purified 31-kDa glycoprotein were shown, by immunoelectron microscopy, to localize specifically to the elastin-associated microfibrils. Thus, the macro-molecule was called "microfibril-associated glycoprotein" or MAGP. MAGP is an acidic glycoprotein with a distinctive amino acid composition, being exceptionally rich in glutamic acid, rich in cystine, and low in glycine. MAGP was extractable from tissue homogenates using NaCl, urea, or guanidine hydrochloride solutions, only if a strong reducing agent was present. Thus, disulfide bonding is important for the strong association of MAGP with elastic fibers. Immunoblotting with anti-MAGP antiserum identified two additional reactive species, of Mr = 60,000 and Mr approximately 300,000, in tissue extracts. As only the 31-kDa species was detected in fibroblast culture medium, these additional species were probably aggregates, rather than precursors. MAGP did not react with antilysyl oxidase antiserum on immunoblots or by enzyme-linked immunosorbent assay. MAGP is the first macromolecule to have been established to be a constituent of elastin-associated microfibrils in both developing and mature elastic tissues.

Amino Acids↗
PubMed1986

Influence of prednisolone and methotrexate on cellular and biochemical aspects of acute and chronic inflammation studied in subcutaneously implanted sponges in rats.

The effects of prednisolone and methotrexate on the histology and the content of collagen and proteoglycans of the inflammatory tissue in polyurethane sponges impregnated with heat killed M. tuberculosis and implanted subcutaneously in hooded Wistar rats for up to 63 days were studied. Drugs were administered during the 6-day period before sponge removal 7, 21, 35, 49 and 63 days after implantation. Our findings indicate that prednisolone and methotrexate have profound effects on the cellular events of acute and chronic inflammation, and influence the synthesis or degradation of connective tissue macromolecules at certain stages of the inflammatory process.

Acute Disease↗
PubMed1985

CL glycoprotein is the tissue form of type VI collagen.

CL glycoprotein (CLGP), the 140,000-dalton collagenous glycoprotein, has been isolated from fetal bovine aorta and nuchal ligament, in milligram amounts in its reduced and alkylated form, using a multistage procedure. This material exhibited a characteristic amino acid composition with a consistent ratio of hydroxylysine to hydroxyproline (approximately 1:1). Digestion of CLGP with bacterial collagenase yielded three discrete noncollagenous fragments. Monospecific anti-CLGP antiserum exhibited strong cross-reactivity with the pepsin-resistant polypeptides of type VI collagen. CLGP was also prepared in the unreduced disulfide-bonded form and in a partially reduced form, using brief treatment with cysteine. On treatment with pepsin these preparations yielded resistant peptides corresponding in size to the longer and shorter forms, respectively, of type VI collagen. A slightly larger, soluble form of CLGP (Mr = 150,000) was detected in the media from cultures of aortic smooth muscle cells and nuchal ligament fibroblasts. The evidence indicates that CLGP is the native form in which type VI collagen is present in the tissues and that it consists of three structurally distinct polypeptide chains, each about 140 kDa in mass, which are disulfide bonded into a triple-helical molecule. The CLGP molecules appear to be present in the tissues as dimers and larger aggregates, stabilized by intermolecular disulfide bonding. The distribution of type VI collagen will thus be as described in our earlier immunofluorescence studies with anti-CLGP antiserum (Gibson, M.A., and Cleary, E.G. (1983) Collagen Relat. Res. 3, 469-488).

Amino Acids↗
PubMed1985

Identification of glycoproteins associated with elastin-associated microfibrils.

The microfibrils associated with elastic tissue have been shown to be predominantly proteinaceous. On the basis of their affinity for cationic stains, including ruthenium red, they have been assumed to be glycoprotein, but more evidence to support this claim has not been adduced. Despite repeated investigation of glycoprotein materials obtained by extraction of elastic tissues with reagents that appear to remove microfibrils, the chemical composition of elastin-associated microfibrils remains obscure. An electron microscopic study of the microfibrils in two elastin-rich tissues (bovine nuchal ligament and aorta) during their development was pursued using more specific histochemical methods. The periodic acid-alkaline bismuth stain (analogous to the periodic acid-Schiff stain for glycoproteins in light microscopy) has been adapted for this study. Specific aldehyde groups (confirmed by blocking with m-aminophenol or sodium borohydride) were identified after periodate oxidation as fine granules of bismuth stain. These were shown to localize specifically along the elastin-associated microfibrils in a finely punctate form. Staining of the amorphous elastic component did not occur except for a fine rim adjacent to the microfibrils. Lectin binding with concanavalin A (with ferritin markers) confirmed that there are glucose- or mannose-containing proteins associated with the microfibrillar component of elastic tissue. This was true of these microfibrils in all layers of the aortic wall and throughout the ligament. It was also true of mature adult tissues in which there was a lesser proportion of microfibrils. It is concluded that elastin-associated microfibrils really are associated with glycoprotein(s).

Animals↗
PubMed1984

Microfibrillar protein from elastic tissue: a critical evaluation.

Many workers have claimed to have isolated proteins which have been derived from the microfibrillar components of elastic tissue. Virtually all of these preparations have been derived from extracts made with strong solutions of guanidinium chloride (GuHCl) under reducing conditions following Ross and Bornstein (1969). The products have ranged from heterogeneous mixtures of proteins to discrete glycoproteins. In no case has identity between an individual protein and the elastin-associated microfibrils been confirmed by immunoelectron microscopy. We have undertaken a detailed re-examination of the extractability of elastin-associated microfibrils and of the composition of the extracts from foetal bovine nuchal ligament. Finely homogenized samples were subjected to a series of extractions (including cyclical treatments with GuHCl and purified bacterial collagenase) in the presence of inhibitors of protease activity. Under these conditions it has been shown that--(i) microfibrils were removed progressively by GuHCl, throughout the extraction schedule, without the need for reduction; (ii) all remaining microfibrils were removed by reductive GuHCl extraction; (iii) the product from this reductive extraction consisted of a heterogeneous mixture of proteins including several glycoproteins; (iv) a major antigenic constituent of the mixture of proteins localized to elastin-associated microfibrils, as shown by immunoelectron microscopy. It is concluded that, while reductive GuHCl extracts do contain components with antigenic activity that is localized on elastin-associated microfibrils, they have many non-microfibrillar components. We stress that claims that a macromolecule is microfibrillar must be substantiated by immunoelectron microscopy.

Animals↗
PubMed1983

Distribution of CL glycoprotein in tissues: an immunohistochemical study.

CL glycoprotein is a collagen-like glycoprotein which we have recently isolated from rapidly growing fetal bovine, elastin-rich tissues. This protein has a molecular weight of approximately 140,000 daltons, contains hydroxyproline and hydroxylysine and is digested by highly purified collagenase to yield three large polypeptides. A specific antibody has been developed against this protein and has been used for immunofluorescence microscopy to study the distribution of CL glycoprotein in a range of tissues. It has been shown that the antibody localized in the intercellular matrix of nuchal ligament and aorta, of the non-elastic Achilles tendon and in complex tissues such as kidney, lung, skin and spleen. The antibody also localized to the surface of aortic smooth muscle cells-presumably to the basement membrane, but did not bind to other basement membranes, including the vascular subendothelial basement membrane. The pattern of distribution was similar in adult bovine tissues. As this antibody showed no avidity for elastic tissue elements, it is most unlikely that CL glycoprotein is a constituent of elastin-associated microfibrils. When the pattern of the CL glycoprotein distribution within the tissues was studied, it was found that, apart from its concentration around vascular smooth muscle cells, CL glycoprotein exhibited considerable overlap in distribution with the interstitial collagens. On the basis of these observations and having regard to its biochemical characteristics, it is proposed that CL glycoprotein has a structural role inter-linking interstitial components to one another and to vascular smooth muscle cells.

Animals↗
PubMed1981

Possible roles of microfibrils in elastogenesis.

On examination in the electron microscope elastic tissue is seen to consist of an amorphous component surrounded by microfibrillar components. The exact relationship between these components is unknown, although during development the microfibrils appear before the amorphous material. In this report we summarize our recent observations on the microfibrillar material. At high magnification the microfibrils are seen to have a poorly staining central core around the periphery of which are arranged more densely staining filaments which appear to wind around the microfibrils in a spiral fashion. Careful measurements of microfibrillar diameters from the aorta of four large species show that there are significant differences in the mean diameter and population distributions with species. The mean diameter of the microfibrils changes with age during fetal and postnatal development. The results of immunoelectronmicroscopic localization of an antibody to a microfibrillar component are reported and the possible roles of microfibrils in elastic tissue formation are examined.

Adolescent↗