Developmental expression of dermatan sulfate proteoglycans in the elastic bovine nuchal ligament.

The nuchal ligament of bovines is a useful system in which to study elastic fibre formation since it contains up to 83% elastin and undergoes a period of rapid elastinogenesis during the last trimester of fetal development and in the first four post-natal months. To identify proteoglycans (PGs) which may be involved in this process we initially investigated changes in the glycosaminoglycan (GAG) profiles during nuchal ligament development. In contrast to the collagenous Achilles tendon, nuchal ligament exhibited: (a) elevated hyaluronan (HA) levels in the peak period of elastin-associated microfibril (fibrillin) synthesis (130-200 days) which precedes elastinogenesis; and (b) markedly increased synthesis of a glucuronate-rich copolymeric form of dermatan sulfate (DS) in the period corresponding to elastin formation (200-270 days). Analysis of DSPGs isolated from 230-day nuchal ligament showed that this copolymer was predominantly associated with a glycoform of biglycan which was specifically elevated at this stage in development. This finding was consistent with Northern blot analysis which showed that steady-state biglycan mRNA levels increased significantly during the elastinogenic period. In contrast, the mRNA levels for decorin, the only other DSPG detected in this tissue, declined rapidly after 140 days of fetal development. In conclusion, the results suggest that HA may play a role in microfibril assembly and that a specific glycoform of biglycan may be associated with the elastinogenic phase of elastic fibre formation.

Attitudes towards community medicine: a comparison of students from traditional and community-oriented medical schools.

OBJECTIVE: To compare the attitudes towards community medicine of first and final year students from two Australian medical schools. METHOD: In 1995, medical students from Newcastle University (a problem-based, community-oriented curriculum) and Adelaide University (a more traditional lecture-based curriculum) were asked to complete the Attitudes to Community Medicine questionnaire. This is a valid and reliable 35 item survey assessing six key domains of community medicine. The two medical schools differ in their methods of selection and curriculum delivery, and also in curriculum content. RESULTS: Response rates averaged 95% for first year and 81% for final year students. Students selected into both medical schools were found to have positive attitudes with respect to most aspects of community medicine. However, those entering Newcastle had more positive attitudes toward community medicine overall than their Adelaide counterparts. They also scored more positively on subscales relating to holistic care and evaluation of health care interventions. Students who were older and female scored more positively on some subscales, but correction for age and gender did not change the conclusions about medical school differences. CONCLUSION: This study suggests that selection criteria, and probably curriculum style and emphasis, have an influence on the attitudes that medical students possess and later develop toward community medicine.

Molecular cloning of a novel human PAPS synthetase which is differentially expressed in metastatic and non-metastatic colon carcinoma cells.

Subtractive hybridisation was used to select for genes which are differentially expressed between a highly metastatic human colon carcinoma cell line, KM12SM, and the isogenetic non-metastatic cell line, KM12C. This led to the isolation of cDNA clones for a novel human adenosine 5'-phosphosulphate kinase/ATP sulphurylase (PAPS synthetase). Northern hybridisation revealed a single 4.2 kb mRNA species which showed an approximately 20-fold higher level of expression in the non-metastatic cell line than in the metastatic cell line. The overlapping cDNA clones together covered 3,774 bp including the entire coding region of 1,842 bp encoding a protein of 614 amino acids (calculated molecular mass of 69,496 Da). The protein contains consensus sequences for APS kinase and ATP sulphurylase, in its amino- and carboxy-terminal regions, respectively, as well as other sequences that are highly conserved amongst ATP sulphurylases and APS kinases. Interestingly, consensus sequences for GTPase activity were also identified, indicating that enzyme activity may be regulated by an intrinsic GTPase mechanism. Overall the new protein is 78% homologous with a previously described human PAPS synthetase (PAPSS1) indicating that we have identified the second member of a gene family which we have provisionally named PAPSS2. The gene locus for PAPSS2 was identified on chromosome 10 at 10q23.1-q23.2. This locus has synteny with the mouse brachymorphic gene recently identified as a PAPS synthetase (SK2). PAPSS2 appears to be the human homologue of this gene and thus PAPSS2 is likely to be important in human skeletogenesis.

Preferential pre-mRNA utilisation of an upstream cryptic 5' splice site created by a single base deletion mutation in exon 37 of the FBN-1 gene.

A heterozygous deletion of a single base (A4704) from exon 37 of the fibrillin-1 gene was defined in a patient with Marfan syndrome and subsequently in his previously undiagnosed father. The deletion created a cryptic 5' splice site in exon 37 which was utilised in preference to the normal 5' splice site during pre-mRNA processing in skin fibroblasts cultured from the proband. The mutant mRNA showed a 48-bp deletion from the 3' end of exon 37 which was predicted to restore the reading frame in the mutant mRNA and result in the deletion of a 16-amino-acid sequence from a central eight-cysteine repeat motif of the fibrillin-1 molecule. Interestingly, the cryptic 5' splice site in exon 37 and the normal 5' splice site had equally strong consensuses for splice-site selection. The preferential utilisation of the cryptic site is discussed in relation to current theories on the mechanisms involved in pre-mRNA splicing. Analysis by reverse-transcription PCR indicated that, in the patients skin fibroblasts, the steady-state level of the mis-spliced mutant mRNA was close to that from the normal allele. In addition, evidence from immunoblotting and pulse-chase biosynthetic labelling indicated that close to normal amounts of fibrillin-1 were being synthesised and secreted by the cells. However, in contrast to control cells cultured from an unaffected individual, little fibrillin-1 was detected, either biosynthetically or by immunofluorescence, in the extracellular matrix produced by the proband's fibroblasts. Thus, the slightly shorter mutant fibrillin-1 molecules appeared to be exerting a powerful dominant-negative effect on the incorporation of normal fibrillin-1 molecules into microfibrils in this culture system. This severe inhibition of microfibril synthesis in cell culture contrasts with the 'classic' phenotype of the proband, suggesting that factors influencing microfibril formation may differ greatly between in vivo and in vitro environments.

Microfibril-associated glycoprotein-2 (MAGP-2) is specifically associated with fibrillin-containing microfibrils but exhibits more restricted patterns of tissue localization and developmental expression than its structural relative MAGP-1.

We developed an affinity-purified anti-MAGP-2 peptide antibody that specifically identified MAGP-2 on Western blots of purified matrix proteins and extracts of nuchal ligament. Immunolocalization studies on tissues from a 210-day-old fetus and a mature bovine showed that MAGP-2 was located in similar regions to MAGP-1 and fibrillin-1 but that the distribution of MAGP-2 was more restricted. In fetal nuchal ligament, skeletal muscle, and spleen the distribution of MAGP-2 was indistinguishable from that of MAGP-1. In contrast to MAGP-1, MAGP-2 was not detected in the medial layer of fetal thoracic aorta and in much of the peritubular matrix of fetal and mature kidney and in the mature ocular zonule. Some differences in the immunolocalization patterns were also evident in fetal lung, cartilage, skin, and heart. Immunoelectron microscopy confirmed that MAGP-2 was specifically associated with fibrillin-containing microfibrils in nuchal ligament, dermis, adventitia of aorta, glomerular mesangium and perimysium. Northern blotting of RNA from tissues of a 210-day-old fetus indicated that steady-state MAGP-2 mRNA levels were highest in nuchal ligament. Significant expression was also detected in lung, heart, skeletal muscle, skin, and Achilles tendon. The tissue pattern of MAGP-2 expression differed significantly from that of MAGP-1. MAGP-2 expression appeared to be higher in nuchal ligament, heart, and skeletal muscle and lower in aorta and kidney. In nuchal ligament, MAGP-2 mRNA expression appeared to peak around 180 days of fetal development, which correlates with the period of onset of elastinogenesis in this tissue. Overall, the immunolocalization and expression patterns of MAGP-2 appeared to be distinct from those of other microfibrillar components. This is consistent with the view that MAGP-2 plays a unique role in the biology of the microfibrils, perhaps by mediating their interaction with cell surfaces at specific stages of development and differentiation. (J Histochem Cytochem 46:871-885, 1998)

An exploratory student learning model of clinical diagnosis.

This paper investigates how students vary in their conception and engagement of the diagnostic process. The research question is whether it is possible to isolate appropriate sources of variation that are sufficiently psychometrically and conceptually robust for modelling purposes. Eleven such sources of variation are reported, together with an associated common-factor empirical model that is readily interpretable in conceptual terms.

Immunohistochemical and ultrastructural localization of MP78/70 (betaig-h3) in extracellular matrix of developing and mature bovine tissues.

MP78/70 is a matrix protein, with 78-kD and 70-kD isoforms, which was initially identified in bovine tissue extracts designed to solubilize elastin-associated microfibrils. Peptide analysis has shown that MP78/70 is closely related to the human protein, betaig-h3. In the present study an antibody raised to a synthetic betaig-h3 peptide was shown specifically to identify MP78/70 in purified form and in bovine tissue extracts. This is consistent with MP78/70 and betaig-h3 being the bovine and human forms, respectively, of the same protein. The antibody was further affinity-purified on MP78/70 bound to Sepharose and used to localize the protein in a range of bovine tissues. Immunofluorescence showed that MP78/70 was localized to collagen fibers in tissues such as developing nuchal ligament, aorta and lung, and mature cornea; to reticular fibers in fetal spleen; and to capsule and tubule basement membranes in developing kidney. No general localization to elastic fibers was observed. The staining pattern in most tissues more closely resembled that of Type VI collagen, which occurs as collagen fiber-associated microfibrils, than that of fibrillin-1, a component of elastin-associated microfibrils. However, MP78/70 appeared to be less widely distributed than Type VI collagen. Immunoelectron microscopy showed that MP78/70 was predominantly found in loose association with collagen fibers in most tissues examined and was also located on the surface of the capsule basement membrane in developing kidney. Double labeling experiments indicated that MP78/70 is co-distributed with Type VI collagen microfibrils located in these regions. In some elastic tissues significant immunolabel was detected in regions of interface between collagen fibers and fibrillin-containing microfibrils of adjacent elastic fibers, and at the outer margins of the latter structures. Overall, the evidence points to MP78/70 having a bridging function, perhaps in association with Type VI collagen microfibrils, linking or stabilizing the interaction between interstitial collagen fibrils and other matrix structures, including some basement membranes and elastin-associated microfibrils.

Graded check lists to assist undergraduate students in self-directed learning and assessment in general and systematic anatomical pathology.

To guide undergraduate medical, dental and health science students undertaking self-directed learning and preparing for examinations in general and systematic pathology, comprehensive graded check lists of selected learning topics have been prepared. Each topic is allocated a grade of 0 (knowledge of the topic not required), 1 (awareness of existence of condition needed, but detailed information not required), 2 (moderately important) or 3 (very important, substantial knowledge required). As students advance through the undergraduate medical course they are provided with replacement lists in which the gradings of many topics have been increased to match requirements for increased knowledge. Over the five years that these check lists have been used, they have received a high level of approval, students finding them increasingly useful as self-directed learning progressively replaces didactic teaching methods. The introduction of the check lists has markedly reduced student inquiries regarding the levels of knowledge required for examinations, and has proved useful to teachers involved in the setting and marking of student assessments. As a result of student pressure, other departments have introduced graded topic lists.

Further characterization of proteins associated with elastic fiber microfibrils including the molecular cloning of MAGP-2 (MP25)

Together with the 31-kDa microfibril-associated glycoprotein (MAGP), four polypeptides designated MP340 (340 kDa), MP78 (78 kDa), MP70 (70 kDa), and MP25 (25 kDa) have previously been identified in tissue extracts designed specifically to solubilize the microfibrillar component of elastic fibers. In the present study, both MP78 and MP70 were shown to be forms of a protein which is closely related to the human protein beta ig-h3, and MP340 was confirmed to be the bovine form of fibrillin-1. Peptide sequences from MP25 proved to be unique, and affinity-purified anti-MP25 antibodies were shown, by immunofluorescence and immunoelectron microscopy, to localize specifically to the elastin-associated microfibrils. This confirmed that MP25 was a distinct component of these structures. Expression screening of nuchal ligament cDNA libraries yielded a cDNA, cM10A (770 base pairs) which encodes amino acid sequences matching those of the MP25 peptides. Further library screening with cM10A identified cDNAs which encode the complete primary structures of bovine and human MP25. Bovine and human MP25 were found to be around 80% homologous and contain 170 and 173 amino acids, respectively. Data base searches revealed that MP25 had significant similarity of structure only with MAGP, indicating that the two proteins form a new family of microfibrillar proteins. In acknowledgment, MP25 has been formally renamed MAGP-2, and MAGP is referred to as MAGP-1. The close similarity between the two proteins (57%) is confined to a central region of 60 amino acids where there is precise alignment of 7 cysteine residues. Elsewhere the MAGP-2 molecule is rich in serine and threonine residues and contains an RGD motif. MAGP-2 lacks the proline-, glutamine-, and tyrosine-rich sequences and a hydrophobic carboxyl terminus, characteristic of MAGP-1. These structural differences suggest that MAGP-2 has some functions which are distinct from those of MAGP-1. The locus of the human MAGP-2 gene was identified on chromosome 12 in the region of 12p12.3-12p13.1.

Microfibril-associated glycoprotein binds to the carboxyl-terminal domain of tropoelastin and is a substrate for transglutaminase.

Microfibril-associated glycoprotein (MAGP) is an integral component of microfibrillar structures that play a critical role in the organization of elastic fibers in the extracellular matrix. To study possible molecular interactions between MAGP and other elastic fiber components, we have generated native MAGP using a baculovirus expression system and tested its ability to associate with tropoelastin and fibrillin. MAGP produced by SF9 cells underwent processing similar to the mammalian protein, including correct cleavage of the signal peptide and sulfation of tyrosine residues. When tested in solid-phase binding assays, native MAGP specifically bound to tropoelastin but not fibrillin-1. Binding to tropoelastin was divalent cation-independent and was completely blocked by reduction and alkylation of either protein. Antibody inhibition studies indicated that the carboxyl terminus of tropoelastin mediated its interaction with MAGP. In addition to binding to elastin, MAGP was also a substrate for transglutaminase, which might explain its propensity to form high molecular weight aggregates that cannot be dissociated with reduction or denaturation. Together, the results of this study provide new insights into the functional relationship between microfibrillar proteins and have important implications for understanding elastic fiber assembly.

Plasma elastin-derived peptide levels in normal adults, children, and emphysematous subjects. Physiologic and computed tomographic scan correlates.

Pulmonary emphysema is likely to be the result of elastic tissue digestion by unrestrained elastase activity in the lung. Elastin breakdown by elastases results in the release of soluble elastin fragments (EDP), which may be measured in plasma by an ELISA. Plasma EDP levels measured using an ELISA were determined in the following groups: disease-free children (n = 24), 0.162 +/- 0.082 ng/ml; disease-free adult nonsmokers (n = 114), 1.74 +/- 0.8 ng/ml; smokers (n = 68), 2.76 +/- 4.59 ng/ml; reformed smokers (n = 43), 1.91 +/- 1.14 ng/ml. Adults with established pulmonary emphysema (n = 50), as defined by bullous formation on the chest radiograph, had levels of 50.83 +/- 24.8 ng/ml, significantly higher than the disease-free groups at p < 0.01. Pulmonary emphysema can be reflected by pulmonary function tests, especially those that measure the pulmonary elastic properties, and by computed tomographic (CT) scan percent emphysema score. We therefore examined the relationship of plasma EDP to these other indicators of pulmonary emphysema in a separate group of 26 subjects using elastic recoil measurements (K), and a further group of 30 subjects with CT scan percent emphysema score. A significant correlation of p < 0.001 was shown for plasma EDP and K and a significant correlation of p < 0.01 was shown for plasma EDP and CT scan percent emphysema score, these correlations suggesting that plasma EDP levels are indicators of the loss of pulmonary distensibility and of mild to moderate pulmonary emphysema. These findings suggest that pulmonary emphysema is characterized by active elastin breakdown.

Effects of enalapril and hydralazine treatment and withdrawal upon cardiovascular hypertrophy in stroke-prone spontaneously hypertensive rats.

OBJECTIVE: To test the hypothesis that effects of angiotensin converting enzyme (ACE) inhibitors upon resistance vessel structure are responsible for their ability to cause long-term reduction in blood pressure. DESIGN: Stroke-prone spontaneously hypertensive (SHRSP) and Wistar-Kyoto (WKY) rats were treated with enalapril or hydralazine from 4 to 15 weeks of age. Effects upon tail-cuff blood pressure, left ventricular hypertrophy and structural indices of the superior mesenteric artery (SMA) and its resistance vessels were assessed at 11 weeks of treatment and up to 11 weeks post-treatment. METHODS: Left ventricular hypertrophy was assessed by left ventricular weight:body weight ratios. Evidence of vascular structural change was obtained from tissue weight:body weight ratios, levels of RNA, DNA and expression of alpha-actin and elastin messenger (m)RNA. RESULTS: The effects of enalapril and hydralazine upon left ventricular hypertrophy in SHRSP were consistent with their respective effects upon blood pressure. Both drugs prevented the development of medial hypertrophy in SMA and resistance vessels. This was accompanied by substantial reductions in RNA:DNA ratios. Alpha-actin mRNA levels were not affected by either drug but elastin mRNA levels were reduced by both drugs. During the first 12 days post-treatment there was evidence of structural change in SMA accompanying the increases in blood pressure but importantly not in the resistance vessels. CONCLUSION: The effects of enalapril upon left ventricular hypertrophy and mesenteric arterial hypertrophy are totally consistent with responses to blood pressure and the persistence of structural changes post-treatment does not underlie the ability of the ACE inhibitors to persistently suppress hypertension.

Elastofibroma: disturbed elastic fibrillogenesis by periosteal-derived cells? An immunoelectron microscopic and in situ hybridization study.

Monospecific antibodies to elastic tissue components have been used for immunoelectron microscopy of two examples of elastofibroma. The elastic-staining fibers typically seen in these lesions exhibited a variety of morphologies with differing ratios of the amorphous and microfibrillar components usually seen in elastic fibers. The amorphous elastic material in these fibers had variable affinity for ionic stains and exhibited several substructural morphologies. Despite this, each form reacted specifically with anti-elastin antibodies. Most of the elastic fibers were associated with relatively large numbers of 12-nm diameter microfibrils that were typical of those associated with normal elastic fibers, and were specifically reactive with monospecific antibodies to microfibril-associated glycoprotein. In situ hybridization studies with a cRNA probe for human elastin confirmed that active elastin biosynthesis was occurring patchily within the lesions. The appearances and staining characteristics of the elastic tissue elements, the morphology of the cells, and the structure of the collagen fibers in these lesions were shown to have many features in common with those of normal periosteum. It is proposed that elastofibromas arise from the periosteum as a result of chronic irritation and that the different elastic fiber morphologies represent disturbances of elastic fibrillogenesis by periosteal-derived cells.

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.

Complementary DNA cloning establishes microfibril-associated glycoprotein (MAGP) to be a discrete component of the elastin-associated microfibrils.

Affinity-purified antibodies to microfibril-associated glycoprotein (MAGP) were used to screen a random-primed, bovine nuchal ligament cDNA library in lambda gt11. A 303-base pair clone, cM5, was isolated which encoded an amino acid sequence homologous with that determined directly from a Lys-C peptide of MAGP. A 936-base pair cDNA clone, cM32, was identified in an oligo(dT)-primed cDNA library using plaque hybridization with clone cM5. Clone cM32 encoded amino acid sequences corresponding to sequences obtained from three Lys-C peptides of MAGP, indicating that the clone was an authentic cDNA for the glycoprotein. The cDNA coded for the entire MAGP polypeptide (21 kDa) of 183 amino acids including a putative signal peptide of 17-19 amino acids. This was confirmed by in vitro translation of synthetic mRNAs transcribed from cM32. The amino acid composition of the encoded protein was virtually identical to that previously published for MAGP. DNA sequence analysis of cM32 indicated that MAGP contains two structurally dissimilar regions, an amino-terminal domain containing high levels of glutamine, proline, and acidic amino acids and a carboxyl-terminal domain containing all 13 of the cysteine residues and most of the basic amino acids. Northern blot hybridization of poly(A+) RNA from fetal nuchal ligament with clone cM32 identified a single mRNA species for MAGP of approximately 1.1 kilobases. The evidence indicates that MAGP is a distinct component of 12-nm microfibrils and that it is not derived from a larger microfibrillar glycopolypeptide.

Elastin gene expression in elastotic human breast cancers and epithelial cell lines.

Elastosis is a prominent feature of the desmoplastic reaction in many invasive breast cancers. It is widely held that the elastic tissue is produced by fibroblastic cells of the breast stroma, but several studies have suggested that it derives from breast cancer epithelium. In studies directed to examining the mechanisms regulating desmoplasia in breast cancers, cell lines of human breast cancer derivation have been shown to synthesize immunoreactive tropoelastin in cell culture. Stromal fibroblasts, grown out from breast cancers, produced as much elastin as did nuchal ligament fibroblasts at similar passages. The human breast cancer cell lines, grown under similar conditions, produced elastin in culture at rates equivalent to 1.6-15% of those of the control fibroblastic cells. These included two estrogen receptor positive and one estrogen receptor negative cell types. Northern blot analysis of total RNA showed the presence, under high stringency conditions, of a 3.5-kilobase elastin mRNA band in both the fibroblastic cells and the cancer cell lines. In situ hybridization, with an elastin complementary RNA probe (prepared from a short segment of the translated region of human elastin mRNA), has been carried out on a selection of 21 invasive ductal breast cancers and 9 normal breast samples. It has been found that, while fibroblastic cells of the stroma and of the periductal region are responsible for elastin synthesis in most breast cancers, the malignant epithelium is a source of the elastin in the desmoplastic tissue of a significant proportion of such neoplasms. Vascular endothelium also expresses the elastin gene in some breast cancers. The elastotic elastin may have different cellular origins in different portions of a single ductal breast cancer. The results indicate that elastosis in breast cancers is very likely to be a complex process with multifactorial regulatory mechanisms. Subclassifying cancers according to the cellular source of the desmoplastic elastin, on the basis of in situ hybridization of elastin mRNA, may provide insights into the prognostic significance of elastosis in breast cancers.

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.

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.