Bacterial Collagenase Digestion Research Articles

In-depth correlation analysis demonstrates that 4-hydroxyproline at the Yaa position of Gly-Xaa-Yaa repeats dominantly stabilizes collagen triple helix

There is a general consensus that collagen stability is largely maintained by Pro and its major hydroxylated form, 4-hydroxyproline (4Hyp). However, positional difference in their stabilizing effect at the Xaa or Yaa position of collagenous Gly-Xaa-Yaa sequences has remained inconclusive. Here, we position-specifically evaluated the correlation of imino acid contents to denaturation temperature (Td) of collagen among various vertebrate and invertebrate species, using a recently developed LC–MS methodology. 4Hyp at the Yaa position showed the highest positive correlation with Td, followed by Pro at the Xaa position, which was even further increased by excluding invertebrates. We confirmed that Gly-Pro-4Hyp liberated after bacterial collagenase digestion was highly positively correlated with Td. Furthermore, other tripeptides with Yaa position 4Hyp also had comparable positive correlation, excepting negative correlation of Gly-Gly-4Hyp, while tripeptides with Xaa position Pro did not. These data provide evidence that 4Hyp dominantly contributes to thermal stability of collagen depending on its sequence position, especially in vertebrates.

Investigation of fibrillin microfibrils in the canine cruciate ligament in dogs with different predispositions to ligament rupture

Cranial cruciate ligament disease (CCLD) is the most common cause of pelvic limb lameness in dogs but its precise aetiopathogenesis is uncertain. Fibrillin microfibrils (FM) are complex macro-molecular assemblies found in many tissues including ligaments, where they are thought to play an important mechanical role. We hypothesised that FM ultrastructural variation correlates with the differing predisposition of canine breeds to CCLD.Non-diseased cranial and caudal cruciate ligaments (CCLs and CaCLs) were obtained from Greyhound (GH) and Staffordshire Bull Terrier (SBT) cadavers. Fibrillin microfibrils were extracted from the ligaments by bacterial collagenase digestion, purified by size-exclusion chromatography and subsequently visualized by atomic force microscopy (AFM). With AFM, FMs have a characteristic beads-on-a-string appearance. For each FM, periodicity (bead-bead distance) and length (number of beads/FM) was measured.Fibrillin microfibril length was found to be similar for GH and SBT, with non-significant inter-breed and inter-ligament differences. Fibrillin microfibril periodicity varied when comparing GH and SBT for CCL (GH 60.2 ± 1.4 nm; SBT 56.2 ± 0.8 nm) and CaCL (GH 55.5 ± 1.6 nm; SBT 61.2 ± 1.2 nm). A significant difference was found in the periodicity distribution when comparing CCL for both breeds (P < 0.00001), further, intra-breed differences in CCL vs CaCL were statistically significant within both breeds (P < 0.00001). The breed at low risk of CCLD exhibited a periodicity profile which may be suggestive of a repair and remodelling within the CCL.

Effect of tea root-derived proanthocyanidin fractions on protection of dentin collagen

ObjectivesProanthocyanidins (PAs) have been widely used as effective agents for dentin collagen cross-linking to enhance the biomechanics and biostability of dentin in vitro. However, the effects and protective mechanisms of various tea root-derived PA components on dentin remain undefined. This study evaluated the effects of these tea root-derived PA components on dentin biomechanics and biostability.MethodsIn this study, ethyl acetate and n-butyl alcohol were used to extract PAs with different degrees of polymerization from tea roots; the effects of these PA extracts on dentin were evaluated.ResultsDentin was treated with glutaraldehyde, ethyl acetate, n-butyl alcohol, or water. PAs with a high degree of polymerization, extracted using n-butyl alcohol, were able to more effectively improve dentin collagen cross-linking, increase resistance to bacterial collagenase digestion, and enhance dentin elasticity, relative to treatment with glutaraldehyde or PAs with a low degree of polymerization (extracted using ethyl acetate). Additionally, treatment with aqueous extract of tea roots was detrimental to dentin stability and function.ConclusionsPAs with a high degree of polymerization were effective for dentin protection and restoration in vitro, suggesting clinical treatment potential for tea root-derived PAs.

Addition of Grape Seed Extract Renders Phosphoric Acid a Collagen-stabilizing Etchant

Previous studies found that grape seed extract (GSE), which is rich in proanthocyanidins, could protect demineralized dentin collagen from collagenolytic activities following clinically relevant treatment. Because of proanthocyanidin’s adverse interference to resin polymerization, it was believed that GSE should be applied and then rinsed off in a separate step, which in effect increases the complexity of the bonding procedure. The present study aimed to investigate the feasibility of combining GSE treatment with phosphoric acid etching to address the issue. It is also the first attempt to formulate collagen-cross-linking dental etchants. Based on Fourier-transformed infrared spectroscopy and digestion assay, it was established that in the presence of 20% to 5% phosphoric acid, 30 sec of GSE treatment rendered demineralized dentin collagen inert to bacterial collagenase digestion. Based on this positive result, the simultaneous dentin etching and collagen protecting of GSE-containing phosphoric acid was evaluated on the premise of a 30-second etching time. According to micro-Raman spectroscopy, the formulation containing 20% phosphoric acid was found to lead to overetching. Based on scanning and transmission electronic microscopy, this same formulation exhibited unsynchronized phosphoric acid and GSE penetration. Therefore, addition of GSE did render phosphoric acid a collagen-stabilizing etchant, but the preferable phosphoric acid concentration should be <20%.

In vitro evaluation of plant-derived agents to preserve dentin collagen

ObjectiveBiomodification of dentin by a natural crosslinker has been recommended to improve a mechanical property of demineralized dentin. This study investigated the effect of various plant-derived agents (hesperidin, proanthocyanidin, epigallocatechin gallate and genipin) on the stability of dentin collagen matrix to resist collagenase degradation. MethodsThe dentin specimens were treated with glutaraldehyde (0.5% and 5.0%) and each plant-derived test solution (0.5%). They were subjected to ultimate tensile strength (UTS) and swelling ratio measurements. Demineralized human dentin powder was incubated with 0.02%, 0.1% and 0.5% of each test agent and followed by bacterial collagenase digestion. The extent of collagen degradation was investigated using hydroxyproline assay. ResultsThe UTS and swelling ratio measurements revealed that the mechanical property of dentin was improved by the use of these natural agents. The greatest reduction in collagen degradation was shown following the use of hesperidin, proanthocyanidin, and epigallocatechin gallate at 0.5%. SignificanceThe use of hesperidin, proanthocyanidin, and epigallocatechin gallate could improve the mechanical properties of collagen and resist enzymatic degradation, leading to functional repair of pathological dentin lesion.

Conformational diversity of the Goodpasture antigen, the noncollagenous-1 domain of the α3 chain of collagen IV

The noncollagenous-1 domain of the alpha3 chain of collagen IV networks of basement membranes is the target of an antibody-mediated inflammatory response in Goodpasture autoimmune disease. This domain when excised from basement membranes by bacterial collagenase digestion exists in two molecular forms, M(H) and M(L), that differ in cleavage site and mobility in SDS-PAGE. In the present study, M(H) and M(L) were shown to also differ with respect to epitope exposure, susceptibility to endoprotease digestion, and redox states of specific cystene residues, as determined by MS. Moreover, M(H) and M(L) assemble to form different quaternary structures, critically influencing pathogenic epitope(s) exposure and autoantibody binding. Collectively, our findings reveal that M(H) and M(L) are conformational isomers stabilized by a distinct disulfide bond connectivity, and coexist in basement membranes. The hitherto unrecognized conformational diversification of the Goodpasture autoantigen may be of relevance in pathogenesis.

Effect of UV on the susceptibility of acid-soluble Skh-1 hairless mouse collagen to collagenase.

Photoaging of the skin is a result of chronic exposure to environmental ultraviolet radiation (UV). The milieu provided by the extracellular matrix, which significantly influences the behaviour of resident fibroblasts, depends critically on the supermolecular collagen structure. We ask whether direct photochemical treatment of type I collagen with solar wavelengths capable of reaching the dermis can modify the substrate's susceptibility to collagenase in a model in vitro system. Acid- extracted Skh-1 hairless mouse collagen samples were irradiated with 0-140 J/cm2 of radiation from bank of filtered FS lamp (UVB/UVA = 0.33, fluence rate = 0.81 mW/cm2). Subsequent to UV irradiation, collagen samples were coupled with fluorescein isothiocyanate (FITC) and assayed for susceptibility to bacterial collagenase by monitoring the appearance of supernatant FITC fluorescence (a measure of lysed collagen) over time of incubation. As a 'reference', unirradiated commercial FITC-labelled citrate-soluble collagen (Elastin Products, Owensville, MO 65066, USA) was similarly analysed. Unirradiated mouse collagen had a lower rate of cleavage than did the calfskin sample. Irradiation of unlabelled mouse collagen for 0-48 h (0-140 J/cm2 total UV) rendered the sample more soluble, with concomitant chain degradation, cross-linking and loss of intrinsic collagen fluorescence. At irradiation time's >/= 4 h (>/=11.7 J/cm2), the irradiated collagen was significantly more susceptible to bacterial collagenase digestion. It appears that the rate of cleavage depends on the superstructure of the collagen, since the kinetics of collagen cleavage differ for two collagen samples having essentially the same primary structure. Cleavage kinetics may depend on the 'maturity' (solubility) of the collagen. The observation that UV-damaged mouse collagen is a better substrate for collagenase than the intact sample may be illustrative of a mechanism whereby damaged collagen targets itself for selective attack by collagenase.

Changes in distribution of the long form of type XII collagen during chicken corneal development.

The expression and distribution of the long form of Type XII collagen were investigated histochemically during chicken corneal development using a monoclonal antibody (P3D11) raised against the N-terminal domain of chicken Type XII collagen. Specificity of the antibody was confirmed by immunoprecipitation before and after bacterial collagenase digestion. Immunofluorescent microscopic studies showed that during chicken cornea formation, the long form of Type XII collagen is initially detected on Day 3 embryo (stage 19) in the sub-epithelial matrix of the corneal periphery and in the matrix around the optic cup. On Day 5 embryo (stage 27) the long form was expressed in the primary stroma. Thereafter, as the secondary stroma was formed, the long form localized in the sub-epithelial and sub-endothelial matrices and in the anterior region of the limbus (corneoscleral junction) before the formation of Descemet's and Bowman's membranes. After hatching, the immunoreactivity decreased predominantly in the sub-epithelial and sub-endothelial matrices but remained at the anterior region of the limbus. Immunoelectron microscopic examination demonstrated that the long form localizes in the Descemet's and Bowman's membranes and along the collagen fibrils in the stroma with a periodic repeat. Based on the distribution of the long form of Type XII collagen in the sub-epithelial and sub-endothelial matrices and limbus, it was suggested that the long form of Type XII collagen is involved in formation of the Descemet's and Bowman's membranes and in stabilization of the limbus.

Synthesis and processing of bone sialoproteins during de novo bone formation in vitro

Bone sialoprotein (BSP) and osteopontin (OPN) are sulphated and phosphorylated sialoglycoproteins that regulate the formation of hydroxyapatite crystals during de novo bone formation. To gain insights into the relationship between the synthesis and posttranslational modification of BSP and OPN and the mineralization of bone, pulse-chase studies were conducted on cultures of newly forming bone nodules produced by fetal rat calvarial cells in vitro. Cultures were pulse labelled with 35SO4, or with either 32PO4 or [gamma-32P]ATP to study intracellular and extracellular phosphorylation, respectively, and chased in isotope-free medium for various times up to 24 h. The presence of radiolabelled BSP and OPN was determined in the cells, in culture medium, and in various tissue compartments obtained by dissociative extraction with 4 M GuHCl (G1), 0.5 M EDTA (E), and again with 4 M GuHCl (G2) and a bacterial collagenase digestion of the demineralized collagenous tissue residue. With each isotope employed, radiolabelled BSP and OPN were detected in the E extract within the 1-h chase period and increased in amount with time. Similarly, 35SO4- and 32PO4-labelled BSP increased in the G2 extract, but OPN was not detected. In the G1 extract the 35SO4-labelled BSP decreased with chase time, whereas the 32PO4-labelled BSP increased. No differences were evident in the profiles of BSP labelled with 32PO4 or [gamma-32P]ATP. In the absence of beta-glycerophosphate, which is required for optimal mineralization of the bone nodules, 35SO4-labelled BSP was increased in the medium and G1 extract and decreased in the E extract and G2 extract after 3 h. In addition to differences in the tissue compartmentalization of BSP and OPN, these studies indicate that 35SO4 is lost from BSP during mineralization and that isoforms of BSP exist with a selective affinity for the organic and mineral phases. Moreover, the additional phosphorylation of BSP and OPN catalyzed by ectokinase activity does not appear to alter the distribution of these sialoproteins.

Purification of Fibrillin-Containing Microfibrils and Collagen VI Microfibrils by Density Gradient Centrifugation

A method is described for the purification of collagen VI microfibrils and fibrillin-containing microfibrils, respectively. HighMrmicrofibril-rich preparations isolated from nuchal ligament by bacterial collagenase digestion and size fractionation were purified by CsCl density gradient centrifugation. Localization of collagen VI and fibrillin within the gradient was achieved by SDS–PAGE/Western blotting. Large collagen VI microfibrillar aggregates were present at the top of the gradient. Hyaluronidase pretreatment dissociated these aggregates and enabled purification of collagen VI microfibrils at a density of 1.33 g/ml. Fibrillin-containing microfibrils separated at 1.37 g/ml and copurified with MAGP1, but not LTBP1, LTBP2, or fibronectin. Confirmation of the intact status of the purified microfibrils was obtained by rotary shadowing. The ability to separate and purify these complex macromolecules provides a powerful means of addressing their molecular composition, organization, and structure:function relationships.

Biosynthesis and processing of type XVI collagen in human fibroblasts and smooth muscle cells.

The alpha 1(XVI) collagen chain, recently identified by cDNA cloning, exhibits structural similarity to a subgroup of collagens that associate with collagen fibrils. Recombinant alpha 1(XVI) collagen chains produced in embryonic kidney cells are able to form stable homotrimers, which are rapidly converted into smaller polypeptides after secretion into the culture medium. In this study, we investigated the biosynthesis of native type XVI collagen by immunoprecipitation of metabolically labeled human cells. Dermal fibroblasts and arterial smooth muscle cells were precipitated with three antibodies raised against distinct regions in the N- and C-terminal part of the human alpha 1(XVI) collagen chain. A disulfide-bonded polypeptide of 220 kDa was obtained from the culture medium, cells and extracellular matrix with all three antibodies. This polypeptide is sensitive to bacterial collagenase digestion and partially resistant to pepsin digestion, suggesting that it is the endogenous alpha 1(XVI) collagen chain. Pulse/chase experiments showed that the newly synthesized alpha 1(XVI) chains are secreted into the medium and deposited in the extracellular matrix in a time-dependent manner. Unlike the recombinant chain, the native type XVI collagen does not undergo extensive proteolytic processing upon secretion. Both cell types deposit a substantial amount of the newly synthesized alpha 1(XVI) chain into the extracellular matrix, in which the 220-kDa polypeptide is the only product immunoprecipitated. There is little evidence for the presence of another constituent chain. The data are consistent with a nomotrimeric chain composition for type XVI collagen. No apparent difference exists in the rate of synthesis and secretion between fibroblasts and smooth muscle cells. Indirect immunofluorescence microscopy showed an extracellular distribution of type XVI collagen, which is located close to cells but not associated with fibrillar structures.

Type VI collagen is present in human and bovine vitreous.

Heterotypic (mixed) collagen fibrils that contain collagen types II, IX and V/XI have previously been identified in vitreous gel. The purpose of this study was to determine whether vitreous gel also contains type VI collagen microfibrils, as they are widely distributed in other tissues where they may play an important structural role. Bovine and human vitreous gel was analysed by rotary shadowing electron microscopy after bacterial collagenase digestion and gel filtration chromatography. Bovine vitreous was extracted in 4 M guanidine hydrochloride, and following isopycnic centrifugation fractions were analysed by western blotting. Type VI collagen microfibrils were identified by electron microscopy in human and bovine vitreous. Type VI collagen was identified by western blot analysis. Type VI collagen is present in vitreous and may participate in the structural assembly of the vitreous gel.

Biosynthesis and processing of type XVI collagen in human fibroblasts and smooth muscle cells

The α1(XVI) collagen chain, recently identified by cDNA cloning, exhibits structural similarity to a subgroup of collagens that associate with collagen fibrils. Recombinant α1(XVI) collagen chains produced in embryonic kidney cells are able to form stable homotrimers, which are rapidly converted into smaller polypeptides after secretion into the culture medium. In this study, we investigated the biosynthesis of native type XVI collagen by immunoprecipitation of metabolically labeled human cells. Dermal fibroblasts and arterial smooth muscle cells were precipitated with three antibodies raised against distinct regions in the N- and C-terminal part of the human α1(XVI) collagen chain. A disulfide-bonded polypeptide of 220 kDa was obtained from the culture medium, cells and extracellular matrix with all three antibodies. This polypeptide is sensitive to bacterial collagenase digestion and partially resistant to pepsin digestion, suggesting that it is the endogenous αl(XVI) collagen chain. Pulse/chase experiments showed that the newly synthesized αlXVI) chains are secreted into the medium and deposited in the extracellular matrix in a time-dependent manner. Unlike the recombinant chain, the native type XVI collagen does not undergo extensive proteolytic processing upon secretion. Both cell types deposit a substantial amount of the newly synthesized αl(XVI) chain into the extracellular matrix, in which the 220-kDa polypeptide is the only product immunoprecipitated. There is little evidence for the presence of another constituent chain. The data are consistent with a homotrimeric chain composition for type XVI collagen. No apparent difference exists in the rate of synthesis and secretion between fibroblasts and smooth muscle cells. Indirect immunofluorescence microscopy showed an extracellular distribution of type XVI collagen, which is located close to cells but not associated with fibrillar structures.

The 160k alpha 1(IV) chain, a short form of a type IV collagen polypeptide, of bovine lens capsule retains the NC1 domain.

We recently reported that the bovine lens capsule contained a shorter alpha 1(IV) chain (160k) as a major polypeptide in addition to the 180k alpha 1(IV) chain [J. Biochem. (1995) 117, 1298-1304]. Two experiments were performed to examine whether or not the 160k polypeptide retained the carboxyl-terminal NC1 domain. On immunoblotting analysis with a monoclonal antibody (H11) raised against the NC1 domain of the human alpha 1(IV) chain [positions 1643-1650; near the carboxyl-terminal end of the human alpha 1(IV) chain], the 180k and 160k polypeptides showed identical immunoreactivity, suggesting that the two chains had the same human alpha 1(IV) collagen NC1 domain sequence. Another monoclonal antibody (H21) specific for the NC1 domain of human alpha 2(IV) did not react with these polypeptides, but with the bands corresponding to 175k and 155k. The 160k polypeptide was selectively solubilized from bovine lens capsules, leaving the other major polypeptides, 180k and 175k, insoluble. The 160k polypeptide was separated by preparative electrophoresis. Bacterial collagenase digestion of the separated 160k polypeptide produced collagenase-resistant segments of about 29k and 30k in size based on globular standards. These sizes corresponded well with those of the NC1 domains of type IV collagen alpha chains (25-30k). The results indicated that the 160k polypeptide retained the carboxyl-terminal NC1 domain of the alpha 1(IV) chain. In turn, the 20k polypeptide of the amino-terminal region or the 7S domain of 180k alpha 1(IV) would have been excised to yield 160k alpha 1(IV), assuming that the 160k alpha 1(IV) chain is a processed form of the 180k alpha 1(IV) one and not an alternatively spliced chain of the alpha 1(IV) gene.

Schwann Cells Secrete a Novel Collagen-like Adhesive Protein That Binds N-Syndecan

A heparin-binding glycoprotein was purified from conditioned medium of cultured rat Schwann cells. The protein, p200, which has an apparent molecular mass of approximately 200 kDa, was identified by its ability to bind the cell surface heparan sulfate proteoglycan N-syndecan (syndecan-3) in a membrane overlay assay. Soluble heparin but not chondroitin sulfate inhibited the binding, suggesting the involvement of heparan sulfate chains of proteoglycan in the interaction. Purified p200 promoted the attachment and spreading of Schwann cells. Adhesion to p200 was blocked by heparin, suggesting that heparan sulfate proteoglycans are cell surface receptors for p200. The tissue distribution of p200 was determined by immunoblot analysis with anti-p200 antibodies. Among neonatal rat tissues examined p200 was detected only in sciatic nerve and, at lower levels, in skeletal muscle. p200 expression in sciatic nerve was detectable only during the first 2-3 weeks of postnatal development and was not detected in adult rats. Immunofluorescent staining of rat sciatic nerve showed that p200 was localized in the extracellular matrix surrounding individual Schwann cells-axon units. Two tryptic peptides from p200 were purified and sequenced. These contained multiple GXX collagen-like repeats. Bacterial collagenase digestion of p200 produced a product with an apparent molecular mass of approximately 90 kDa. These data suggest that Schwann cells secrete an apparently novel collagen-like adhesive protein that interacts with cells through cell surface heparan sulfate proteoglycans.

Evaluation of different chemical methods for cros-linking collagen gel, films and sponges

Collagen-based films and sponges are widely used as biomaterials. The rate of their biodegradation can be reduced by treating them with different cross-linking agents. The efficiency of different reticulation procedures on thermal stability (measured by differential scanning calorimetry) and susceptibility to bacterial collagenase digestion of the final material (films or sponges) is compared. The chemical agents used on collagen gels or directly on collagen sponges and films were glutaraldehyde (GTA), hexamethylene diisocyanate (HMDC), cyanamide, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and the two acyl azide methods (with hydrazine or diphenylphosphorylazide (DPPA)) developed in the authors' laboratory. Under these experimental conditions, collagen stabilization by the different agents increased in the following order: cyanamide <EDC<HMDC<hydrazine<DPPA<GTA. Sponges with the highest thermal stability were obtained by treating collagen gel with 0.5% GTA for 1 h at pH 5.5 (initial T d=75.8°C; after 3 months, T d=75.6°C). Direct treatment of collagen films with DPPA gave similar thermal stability (T d=72.6°C) and collagenase resistance when compared with treatment with 0.6% GTA for 96 h (T d=74.6°C). It is demonstrated that collagen sponges and films can be prepared with a wide range of thermal stability (49–75.8°C) and collagenase digestion resistance (10–100%). Recent biocompatibility studies show, however, that DPPA and EDC are the best choices for preparing cross-linked collagen sponges and films.

Stimulation of cell proliferation and autoregulation of elastin expression by elastin peptide VPGVG in cultured chick vascular smooth muscle cells

Synthetic elastin peptides, VPGVG or its polymer (VPGVG) n , enhanced the proliferation of smooth muscle cells 1.5-fold during 48 h treatment at the concetrations over 10 −6 M or 1.0 μg/ml, respectively. Monomeric and polymeric VPGVG sequences reduced elastin synthesis and its mRNA level to one-third and one-half of control respectively under the conditions in which the proliferation of cells were enhanced, but did not change collagen synthesis as measured by bacterial collagenase digestion. The elastin-specific autoregulation by elastin fragments may reflect the feedback regulation of elastin expression which may play an essential role in elastin metabolism under the normal and diseased conditions.

Biochemical study on the tissue compartmentalization of phosphophoryn, osteopontin and bone sialoprotein (BSP) in rat incisor dentin

The tissue compartmentalization of phosphophoryn, osteopontin (OPN) and bone sialoprotein (BSP) in dentin was biochemically analyzed. The radicular portion of rat incisor was excised and sequentially extracted; i.e., proteins non-covalently bound to matrix such as soft tissues were solubilized with 4 M guanidinium chloride (G1), mineral-binding proteins were then extracted with 0.5 M EDTA (E), proteins non-covalently bound to the collagen matrix were extracted with 4 M guanidinium chloride (G2) after E, and finally the proteins covalently bound to the collagen matrix were extracted by bacterial collagenase digestion (C). Stains-All and Rhodamine B staining, which selectively stain calcium-binding proteins, revealed a 50∼80 kDa broad band in the E extract on 10% polyacrylamide gels. This intense band disappeared after precipitation with CaCl2, indicating that the protein is phosphophoryn. After removing phosphophoryn from the E extract, 67 kDa intense and 50 kDa weak bands appeared on the gels stained with Stains-All. The 67 kDa protein was observed similarly in the G2 and C extracts. Western blots using monoclonal antibodies revealed the presence of 67 kDa BSP and 50 kDa OPN. When the tissue compartmentalization was compared, these three proteins were not detected in the G1 extract, phosphophoryn and osteopontin were localized only in the E extract, and BSP was observed not only in the E extract but also in the G2 and C extracts. These findings indicate that phosphophoryn, OPN and BSP are rapidly incorporated into the mineralized fraction and that BSP also binds to collagen-related fraction, suggesting the association with intial mineralization in the dentin.

Normal elastin content of aorta in bovine Marfan syndrome

Samples from the ascending aortae from two calves affected by bovine Marfan syndrome were subjected to biochemical analyses of the connective tissue and were compared to age-matched controls. Elastin was extracted from the aortic samples with 5 M guanidine-HCl, bacterial collagenase digestion, and dithiothreitol reduction. Amino acid analysis revealed that desmosine and isodesmosine levels were the same in Marfan calves as in control animals. Gravimetric measurements of elastin, amino acid composition, soluble protein, and uronic acid values also showed no significant difference between Marfan and control tissue. In contrast to elastin, collagen in aortae of Marfan calves was significantly higher than the mean of several controls. These findings, along with other observations of this animal model, support the conclusion that the microscopic and biochemical lesions of aortic elastin in bovine Marfan syndrome likely result from defective microfibrillar metabolism. Absence of cystic medial necrosis in bovine Marfan aortae may explain normal elastin content in the animal model.

Type VI collagen microfibrils: evidence for a structural association with hyaluronan.

Type VI collagen, a widespread structural component of connective tissues, has been isolated in abundance from fetal bovine skin by a procedure involving bacterial collagenase digestion under nonreducing, nondenaturing conditions and gel filtration chromatography. Rotary shadowing electron microscopic analysis revealed that the collagen VI was predominantly in the form of extensive intact microfibrillar arrays. These microfibrils were seen in association with hyaluronan, which was identified by its ability to bind the G1 fragment of cartilage proteoglycan. Treatment with highly purified hyaluronidase largely disrupted the collagen VI microfibrils into component tetramers, double tetramers, and short microfibrillar sections. Subsequent incubation of disrupted collagen VI in the presence of hyaluronan facilitated a partial repolymerization of the microfibrils. In vitro binding studies have also demonstrated that type VI collagen binds hyaluronan with a relatively high affinity. These studies demonstrate that a specific structural relationship exists between type VI collagen and hyaluronan. This association is likely to be of primary importance in the growth and remodeling processes of connective tissues.