Microfibril-associated glycoprotein-1 (MAGP-1) is specifically located on the beads of the beaded-filament structure for fibrillin-containing microfibrils as visualized by the rotary shadowing technique.

This study used immunoelectron microscopic techniques to define the ultrastructural location of MAGP-1 on the fibrillin-containing microfibrils of the ocular zonule. A specific anti-MAGP-1 monoclonal antibody (MAb), 11B, was produced that did not crossreact with fibrillin-1 or other microfibrillar proteins. MAb 11B was shown by immunofluorescence to localize intensely to zonular tissue. Postembedding immunoelectron microscopy showed that MAGP-1 was associated with microfibrils throughout the zonule, with the exception of a narrow band of microfibrils at the junction with the lens capsule. With preembedding labeling, the anti-MAGP-1 MAb was found to localize in a crossbanding pattern, at intervals of about 50 nm, to microfibrils throughout the zonule and along bundles of microfibrils in surrounding vitreous tissue. Rotary shadowing of isolated microfibrils showed a "beads on a string" morphology with a periodicity of about 50 nm. With immunogold labeling, the anti-MAGP-1 antibody specifically localized on the beads in a symmetrical manner. Occasionally two gold partides were attached to the same bead, suggesting that multiple MAGP-1 molecules were present in the structure. The results indicate that MAGP-1 is intimately and regularly associated with the bead regions of fibrillin-containing microfibrils. The findings are consistent with a major structural role for MAGP-1 in microfibril biology.

Immunoelectron microscopic localization of elastic tissue components in archival tissue samples.

Tissue samples that have been stored for many years, in different media and under a variety of conditions, have been examined by modern techniques of immunoelectron microscopy, using antibodies against elastic tissue components. A range of postembedding restorative procedures has been identified, which will allow reliable immunolocalization of antibodies against the elastic tissue component of such specimens. These methods have been applied successfully to autopsy-derived material, fixed in buffered formaldehyde, to archival material stored frozen at -70 or -20 degrees C, to specimens fixed for electron microscopy and stored for many years in buffer, and even to archival material from formaldehyde-fixed, paraffin-embedded blocks, reprocessed for electron microscopic examination. The successful restorative methods included pre-treatment of the sections with 6 M guanidine hydrochloride, or 1 M Tris/saline, each containing 100 mM dithiothreitol (a reducing agent) followed by alkylation with 220 mM iodoacetamide. The application of these techniques allowed reliable study of elastic tissue antibody distributions in archival tissues that could not be obtained again, as well as comparative studies with tissues processed many years previously.

The structure of the normal human glomerular basement membrane. Ultrastructural localization of type IV collagen and laminin.

Structural models of the glomerular basement membrane (GBM) have been based solely on the localization of antigens in animal kidneys. These models depict a type IV collagen lattice as the structural skeleton along the central portion of the membrane, with the glycoprotein laminin attached predominantly in the laminae rarae where it is thought to be involved with endothelial and visceral epithelial cell attachment. The human GBM is also known to contain type IV collagen and laminin. The present study localizes both of these structural antigens in normal human GBM using the ultrastructural immunogold technique. Type IV collagen is situated in the sub-endothelial third of the GBM and is continuous with the type IV collagen within the mesangial matrix. Laminin is localized throughout the entire thickness of the GBM and mesangial matrix. These results indicate that the structure of human GBM is significantly different from that proposed in animal models. A structure for the normal human GBM is discussed, depicting a thin type IV collagen lattice which is asymmetrically placed in the sub-endothelial third of the membrane, with laminin distributed as an integral component of the membrane in addition to its role in cell attachment.

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.

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.

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.

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).

Dependency levels of elderly people in institutional care in Dunedin.

All elderly people in long term institutional care in the Dunedin area were assessed using a rating scale which assessed self-care, continence, mental function and social integration. Religious and welfare organisations that provide a comprehensive service and the private sector, cared for a higher proportion of medium and high dependency residents in their residential homes, than did religious and welfare organisations which provided residential home care alone. The reasons for this are discussed. Local authority homes in the UK were shown to have a considerably higher proportion of medium and high dependency subjects than residential homes in Dunedin. Of all subjects in residential homes and long term hospitals in Dunedin, 38% were essentially independent in the parameters assessed. There is a real need to explore alternative forms of institutional care and alternative forms of subsidy for those elderly in New Zealand who require care.

Converting a stomach to a uterus: the microscopic structure of the stomach of the gastric brooding frog Rheobatrachus silus.

Rheobatrachus silus is a rare aquatic frog of eastern Australia. The female ingests the eggs after fertilization and broods them in the stomach until fully formed. "Gastric brooding" takes place in the fundus and proximal part of the body of the stomach, which dilates to accommodate the growing young. The surface epithelium becomes attenuated and the cells contain fewer mucus droplets. The acini of the glands are less numerous because of stretching, and they contain oxyntic cells that show evidence of profound suppression or regression. Morphometric studies on the limited number of samples show that the oxyntic cells are attenuated with few surface projections, sparse tubulovesicular reticulum, and a few pepsinogen granules and mitochondria. Eight days after ejection of the young, and 4 days after feeding commences, the lining shows a return of gastric pits and glands. The oxyntic cells show many surface projections and a proliferation of the tubulovesicular reticulum and mitochondria. These findings suggest that the eggs, tadpoles, and juvenile frogs release a substance, or substances, that inhibit acid secretion immediately after the eggs are ingested and that persist throughout brooding.

Ultrastructural changes in isolated rat hepatocytes after incubation with carbon tetrachloride.

Suspensions of rat hepatocytes, isolated by a collagenase-based perfusion technique, were incubated with carbon tetrachloride (CCl4) for up to 60 min. Control incubated cells examined by transmission electron microscopy had an essentially normal substructure. Treatment with CCl4, however, resulted in a loss of microvilli at the cell membrane, disorganization of the RER, the appearance of condensed membranes, vacuoles within mitochondria and cellular protrusions. While some of the alterations are similar to those observed after in vivo CCl4 exposure, others are not. This suggests that the mechanism of in vitro damage may not be identical to that in vivo. Further investigation of the differences, such as the cellular protrusions, may yield increased understanding of the toxicity induced by CCl4 in the whole animal.

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.

Metal complexes as promoters of N-nitrosation reactions: a progress report.

Our research on three classes of transition metal-containing N-nitrosating agents is described. One class, the nitrito complexes, can give rise in organic solvents to quantitative yields of N-nitrosamines on exposure to secondary amines. The second class is capable of activating nitrite as an N-nitrosating agent in aqueous alkaline media; it involves N-coordination of nitrite, followed by removal of oxide ion to form the active metal nitrosyl species. The third class of N-nitrosating agent involves an as yet incompletely characterized metal complex containing a ligand having the formula NO3. The possible significance of compounds in these classes in environmental or in vivo N-nitrosamine formation is discussed.