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The Ehlers–Danlos syndromes (EDS) are a group of rare inherited connective tissue disorders. Vascular EDS (vEDS) is caused by pathogenic variants in COL3A1, most frequently glycine substitutions. We describe the phenotype of the largest series of vEDS patients with glutamic acid to lysine substitutions (Glu>Lys) in COL3A1, which were all previously considered to be variants of unknown significance. Clinical and molecular data for seven families with three different Glu>Lys substitutions in COL3A1 were analyzed. These Glu>Lys variants were reclassified from variants of unknown significance to either pathogenic or likely pathogenic in accordance with American College of Medical Genetics and Genomics guidelines. All individuals with these atypical variants exhibited skin hyperextensibility as seen in individuals with classical EDS and classical-like EDS and evidence of tissue fragility as seen in individuals with vEDS. The clinical data demonstrate the overlap between the different EDS subtypes and underline the importance of next-generation sequencing gene panel analysis. The three different Glu>Lys variants point toward a new variant type in COL3A1 causative of vEDS, which has consistent clinical features. This is important knowledge for COL3A1 variant interpretation. Further follow-up data are required to establish the severity of tissue fragility complications compared with patients with other recognized molecular causes of vEDS.

Hip pathology is common amongst athletes and the general population. The mechanics of cycling have the potential to exacerbate symptomatic hip pathology and progress articular pathology in patients with morphologic risk factors such as femoroacetabular impingement. A professional fit of the bicycle to the individual which aims to optimize hip joint function can allow patients with hip pathology to exercise in comfort when alternative high impact exercise such as running may not be possible. Conversely improper fit of the bicycle can lead to hip symptoms in otherwise healthy individuals who present with risk factors for hip pain. Accordingly a bike fit can form part of the overall management strategy in a cyclist with hip symptoms. The purpose of this clinical commentary is to discuss hip pathomechanics with respect to cycling, bicycle fitting methodology and the options available to a physical therapist to optimize hip mechanics during the pedaling action.

M13 bacteriophage is a key instrument in phage display applications, as well as a possible antibacterial therapeutic agent due to its highly restrictive bacterial pathogenesis, and other applications. The traditional phage purification process is usually achieved by gradient ultracentrifugation or a combination of precipitation, centrifugation and microfiltration. These approaches easily lead to long process times, high operational costs, phage aggregation and consequent product loss (approximately 60%). This work is thus focused on an alternative potential large-scale process to achieve high yield and purity while minimizing the operational costs. Electrostatic-based separation processes are also common biomolecules purification techniques. Although anion exchange chromatography has been used before to purify several viral particles, this technique has been poorly reported for the purification of M13 phage. In a recent work, our group has demonstrated the use of a predominant anion exchange process, where a polymeric ionic liquid (PIL) was used as an alternative separation matrix for M13 bacteriophage. In this work, a variety of system parameters was studied, including chemical structure of the cation and the anion, the crosslinker nature and its concentration, either in batch adsorption/elution or chromatographic operation mode. The PIL-based chromatographic operation mode revealed to be a suitable separation process for M13 from directly filtered E. coli supernatant, reaching over 70% M13 recovery and 4.6 purification factor in a single step. To our knowledge, this is the first time that PILs have been reported as separation agents for bioproducts from complex mixtures.

M13 is a filamentous, non-lytic bacteriophage that infects Escherichia coli via the F pilus. Currently, phage M13 is widely used in phage display technology and bio-nanotechnology, and is considered a possible antibacterial therapeutic agent, among other applications. Conventional phage purification involves 5-7 operational steps, with high operational costs and significant product loss (approximately 60%). In this work, we propose a scalable purification process for M13 bacteriophage using a novel stationary phase based on a polymeric ionic liquid (PIL) with a positively charged backbone structure. Poly (1-vinyl-3-ethyl imidazolium bis(trifluoromethylsulfonyl) imide) - poly(VEIM-TFSI) predominantly acted as an anion exchanger under binding-elution mode. This revealed to be a rapid and simple method for the recovery of phage M13 with an overall separation yield of over 70% after a single downstream step. To the best of our knowledge, PILs have never been used as separation matrices for biological products and the results obtained, together with the large number of cations and anions available to prepare PILs, illustrate well the large potential of the proposed methodology.

Familiarity with how MRI sequences can distinguish different tissues when coupled with an understanding of pathology aids in narrowing differentials or making a specific diagnosis. Utilizing specific MRI pulse sequences allows for identification of key tissue substances such as fat, paramagnetic substances, protein, fibrous tissue, or free or bound water. The identification of these tissue substances allows the radiologist to form narrow or specific diagnoses efficiently. A tissue-based approach to understanding MRI sequences allows the radiologist to both systematically and effectively interpret MRIs despite the large number of pulse sequences particularly in basic MRI body protocols.

Vascular Ehlers–Danlos syndrome (vEDS) is a heritable disorder of connective tissue caused by pathological variants in the COL3A1 gene, which encodes the α1 chain of type III collagen. Type III collagen is a major component of skin, arterial walls, and the gastrointestinal tract. Collagen III protein deficiency manifests as an increased risk of rupture, perforation, and dissection of these structures. The most disruptive gene variants affect the collagen helix via glycine substitutions or splice donor site mutations. The C-propeptide region of COL3A1 includes exons 49–52 and has a crucial role in initiating the C-terminal assembly of procollagen monomers in the early stages of collagen biosynthesis. Nineteen COL3A1 variants have previously been reported in these exons, of which four were associated with a severe vEDS phenotype. We identified two novel C-propeptide missense variants; p.Pro1440Leu, p.Arg1432Leu, and a non-stop mutation, c.4400A > T, p. (*1467Leuext*45). These variants produce variable phenotypes ranging from obvious acrogeria to classical or hypermobile EDS. A previously reported variant p.Lys1313Arg is of unknown clinical significance but likely benign, based on this study. Assigning disease pathogenicity remains complex, clinical phenotyping and crystal structure evidence being crucial. We briefly compare reported phenotypes for patients with missense variants in the C-propeptide domain for other human collagen disorders including COL1A1 and COL1A2 (osteogenesis imperfecta). © 2015 Wiley Periodicals, Inc.

The C propeptides of fibrillar procollagens have crucial roles in tissue growth and repair by controlling both the intracellular assembly of procollagen molecules and the extracellular assembly of collagen fibrils. Mutations in C propeptides are associated with several, often lethal, genetic disorders affecting bone, cartilage, blood vessels and skin. Here we report the crystal structure of a C-propeptide domain from human procollagen III. It reveals an exquisite structural mechanism of chain recognition during intracellular trimerization of the procollagen molecule. It also gives insights into why some types of collagen consist of three identical polypeptide chains, whereas others do not. Finally, the data show striking correlations between the sites of numerous disease-related mutations in different C-propeptide domains and the degree of phenotype severity. The results have broad implications for understanding genetic disorders of connective tissues and designing new therapeutic strategies.

A disintegrin and metalloproteinase with thrombospondin type I motif (ADAMTS)2, 3, and 14 are collectively named procollagen N-proteinases (pNPs) because of their specific ability to cleave the aminopropeptide of fibrillar procollagens. Several reports also indicate that they could be involved in other biological processes, such as blood coagulation, development, and male fertility, but the potential substrates associated with these activities remain unknown. Using the recently described N-terminal amine isotopic labeling of substrate approach, we analyzed the secretomes of human fibroblasts and identified 8, 17, and 22 candidate substrates for ADAMTS2, 3, and 14, respectively. Among these newly identified substrates, many are components of the extracellular matrix and/or proteins related to cell signaling such as latent TGF-β binding protein 1, TGF-β RIII, and dickkopf-related protein 3. Candidate substrates for the 3 ADAMTS have been biochemically validated in different contexts, and the implication of ADAMTS2 in the control of TGF-β activity has been further demonstrated in human fibroblasts. Finally, the cleavage site specificity was assessed showing a clear and unique preference for nonpolar or slightly hydrophobic amino acids. This work shows that the activities of the pNPs extend far beyond the classically reported processing of the aminopropeptide of fibrillar collagens and that they should now be considered as multilevel regulators of matrix deposition and remodeling.-Bekhouche, M., Leduc, C., Dupont, L., Janssen, L., Delolme, F., Vadon-Le Goff, S., Smargiasso, N., Baiwir, D., Mazzucchelli, G., Zanella-Cleon, I., Dubail, J., De Pauw, E., Nusgens, B., Hulmes, D. J. S., Moali, C., Colige, A. Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF-β signaling as primary targets.

BMP-1/tolloid-like proteinases (BTPs) are major enzymes involved in extracellular matrix assembly and activation of bioactive molecules, both growth factors and anti-angiogenic molecules. Although the control of BTP activity by several enhancing molecules is well established, the possibility that regulation also occurs through endogenous inhibitors is still debated. Secreted frizzled-related proteins (sFRPs) have been studied as possible candidates, with highly contradictory results, after the demonstration that sizzled, a sFRP found in Xenopus and zebrafish, was a potent inhibitor of Xenopus and zebrafish tolloid-like proteases. In this study, we demonstrate that mammalian sFRP-1, -2, and -4 do not modify human BMP-1 activity on several of its known substrates including procollagen I, procollagen III, pN-collagen V, and prolysyl oxidase. In contrast, Xenopus sizzled appears as a tight binding inhibitor of human BMP-1, with a K(i) of 1.5 ± 0.5 nM, and is shown to strongly inhibit other human tolloid isoforms mTLD and mTLL-1. Because sizzled is the most potent inhibitor of human tolloid-like proteinases known to date, we have studied its mechanism of action in detail and shown that the frizzled domain of sizzled is both necessary and sufficient for inhibitory activity and that it acts directly on the catalytic domain of BMP-1. Residues in sizzled required for inhibition include Asp-92, which is shared by sFRP-1 and -2, and also Phe-94, Ser-43, and Glu-44, which are specific to sizzled, thereby providing a rational basis for the absence of inhibitory activity of human sFRPs.

Background: Procollagen C-proteinase enhancer-1 (PCPE-1) is an extracellular glycoprotein that increases activity of certain zinc metalloproteinases involved in tissue development and repair. Results: PCPE-1 binds uniquely to the C-propeptide region of the procollagen molecule. Conclusion: PCPE-1 enhances proteolysis by binding solely to the procollagen C-propeptides. Significance: These data may lead to future applications in the development of antifibrotic therapies., Bone morphogenetic protein-1 (BMP-1) and the tolloid-like metalloproteinases control several aspects of embryonic development and tissue repair. Unlike other proteinases whose activities are regulated mainly by endogenous inhibitors, regulation of BMP-1/tolloid-like proteinases relies mostly on proteins that stimulate activity. Among these, procollagen C-proteinase enhancers (PCPEs) markedly increase BMP-1/tolloid-like proteinase activity on fibrillar procollagens, in a substrate-specific manner. Here, we performed a detailed quantitative study of the binding of PCPE-1 and of its minimal active fragment (CUB1-CUB2) to three regions of the procollagen III molecule: the triple helix, the C-telopeptide, and the C-propeptide. Contrary to results described elsewhere, we found the PCPE-1-binding sites to be located exclusively in the C-propeptide region. In addition, binding and enhancing activities were found to be independent of the glycosylation state of the C-propeptide. These data exclude previously proposed mechanisms for the action of PCPEs and also suggest new mechanisms to explain how these proteins can stimulate BMP-1/tolloid-like proteinases by up to 20-fold.

The netrin-like (NTR) domain is a feature of several extracellular proteins, most notably the N-terminal domain of tissue inhibitors of metalloproteinases (TIMPs), where it functions as a strong inhibitor of matrix metalloproteinases and some other members of the metzincin superfamily. The presence of a C-terminal NTR domain in procollagen C-proteinase enhancers (PCPEs), proteins that stimulate the activity of astacin-like tolloid proteinases, raises the possibility that this might also have inhibitory activity. Here we show that both long and short forms of the PCPE-1 NTR domain, the latter beginning at the N-terminal cysteine known to be critical for TIMP activity, show no inhibition, at micromolar concentrations, of several members of the metzincin superfamily, including matrix metalloproteinase-2, bone morphogenetic protein-1 (a tolloid proteinase), and different ADAMTS (a disintegrin and a metalloproteinase with thrombospondin motifs) proteinases from the adamalysin family. In contrast, we report that the NTR domain within PCPE-1 leads to superstimulation of bone morphogenetic protein-1 activity in the presence of heparin and heparan sulfate. These observations point to a new mechanism whereby binding to cell surface-associated or extracellular heparin-like sulfated glycosaminoglycans might provide a means to accelerate procollagen processing in specific cellular and extracellular microenvironments.

Tissue remodelling results from the concerted action of numerous extracellular and cell surface proteases. These act to synchronize the synthesis and degradation of the extracellular matrix with the control of cytokine activity and cell signalling in order to create appropriate environments for cell proliferation, migration and differentiation. Wound healing is a complex example of tissue remodelling that includes several steps occurring either concomitantly or successively during the process of repair: haemostasis, inflammation, angiogenesis, re-epithelialisation, granulation tissue formation, wound contraction and matrix remodelling. The main extracellular and cell surface proteases involved in wound healing are serine proteases, especially plasmin, and metalloproteases of the metzincin family (MMPs, ADAM(TS)s, tolloids, meprins, pappalysins) with cysteine proteases playing less prominent roles. Several regulatory proteins and hundreds of substrates have been identified for these proteases, either in vitro or in vivo. The aim of this review is not to present an exhaustive list of proteases and related molecules but to give an overview of the proteolytic events that are potentially relevant during tissue repair. New developments aimed at approaching a more integrative view of all the molecular events involved in tissue remodelling are also discussed.

Procollagen C-proteinase enhancers (PCPE-1 and -2) are extracellular glycoproteins that can stimulate the C-terminal processing of fibrillar procollagens by tolloid proteinases such as bone morphogenetic protein-1. They consist of two CUB domains (CUB1 and -2) that alone account for PCPE-enhancing activity and one C-terminal NTR domain. CUB domains are found in several extracellular and plasma membrane-associated proteins, many of which are proteases. We have modeled the structure of the CUB1 domain of PCPE-1 based on known three-dimensional structures of CUB-containing proteins. Sequence alignment shows conserved amino acids, notably two acidic residues (Asp-68 and Asp-109) involved in a putative surface-located calcium binding site, as well as a conserved tyrosine residue (Tyr-67). In addition, three residues (Glu-26, Thr-89, and Phe-90) are found only in PCPE CUB1 domains, in putative surface-exposed loops. Among the conserved residues, it was found that mutations of Asp-68 and Asp-109 to alanine almost completely abolished PCPE-1 stimulating activity, whereas mutation of Tyr-67 led to a smaller reduction of activity. Among residues specific to PCPEs, mutation of Glu-26 and Thr-89 had little effect, whereas mutation of Phe-90 dramatically decreased the activity. Changes in activity were paralleled by changes in binding of different PCPE-1 mutants to a mini-procollagen III substrate, as shown by surface plasmon resonance. We conclude that PCPE-stimulating activity requires a calcium binding motif in the CUB1 domain that is highly conserved among CUB-containing proteins but also that PCPEs contain specific sites that could become targets for the development of novel anti-fibrotic therapies.

Members of the bone morphogenetic protein-1/tolloid (BMP-1/Tld) family of metalloproteinases, also known as procollagen C-proteinases (PCPs), control multiple biological events (including matrix assembly, cross-linking, cell adhesion/migration and pattern formation) through enzymatic processing of several extracellular substrates. PCP activities on fibrillar procollagens can be stimulated by another family of extracellular proteins, PCP enhancers (PCPE-1, PCPE-2), which lack intrinsic enzymatic activity. While PCPs have multiple substrates, the extent to which PCPEs is involved in the processing of proteins other than fibrillar procollagens is unknown. In the experiments reported here, PCPE-1 was found to have no effect on the in vitro BMP-1 processing of procollagen VII, the procollagen V N-propeptide, the laminin 5 γ2 chain, osteoglycin, prolysyl oxidase, or chordin. In contrast, PCPE-1 enhanced C-terminal processing of human fibrillar procollagen III but only when this substrate was in its native, disulfide-bonded conformation. Surprisingly, processing of procollagen III continued to be enhanced when essentially all the triple-helical region was removed. These and previous results (Ricard-Blum, S., Bernocco, S., Font, B., Moali, C., Eichenberger, D., Farjanel, J., Burchardt, E. R., van der Rest, M., Kessler, E., and Hulmes, D. J. S. (2002) J. Biol. Chem. 277, 33864-33869; Bernocco, S., Steiglitz, B. M., Svergun, D. I., Petoukhov, M. V., Ruggiero, F., Ricard-Blum, S., Ebel, C., Geourjon, C., Deleage, G., Font, B., Eichenberger, D., Greenspan, D. S., and Hulmes, D. J. S. (2003) J. Biol. Chem. 278, 7199-7205) indicate that the mechanism of PCPE-1 action involves recognition sites in both the C-propeptide domain and in the C-telopeptide region of the procollagen molecule. PCPEs therefore define a new class of extracellular adaptor proteins that stimulate proteinase activity in a substrate-specific manner, thereby providing a new target for the selective regulation of PCP activity on fibrillar procollagen substrates.

Alpha-helical coiled-coils are widely occurring protein oligomerization motifs. Here we show that most members of the collagen superfamily contain short, repeating heptad sequences typical of coiled coils. Such sequences are found at the N-terminal ends of the C-propeptide domains in all fibrillar procollagens. When fused C-terminal to a reporter molecule containing a collagen-like sequence that does not spontaneously trimerize, the C-propeptide heptad repeats induced trimerization. C-terminal heptad repeats were also found in the oligomerization domains of the multiplexins (collagens XV and XVIII). N-terminal heptad repeats are known to drive trimerization in transmembrane collagens, whereas fibril-associated collagens with interrupted triple helices, as well as collagens VII, XIII, XXIII, and XXV, were found to contain heptad repeats between collagen domains. Finally, heptad repeats were found in the von Willebrand factor A domains known to be involved in trimerization of collagen VI, as well as in collagen VII. These observations suggest that coiled-coil oligomerization domains are widely used in the assembly of collagens and collagen-like proteins.

International audience; Purpose: To characterize the expression of the bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases (collectively called BTPs), which include BMP-1, mammalian tolloid (mTLD), and mammalian tolloid-like 1 (mTLL-1) and 2 (mTLL-2), as well as the associated proteins procollagen C-proteinase enhancers (PCPE-1 and -2), in corneal scarring. Methods: Using a mouse full-thickness corneal excision model, wound healing was followed for up to 28 days by transmission electron microscopy, immunohistology (BMP-1/mTLD and PCPE-1), and quantitative PCR (Q-PCR: collagen III, BMP-1/mTLD, mTLL-1, mTLL-2, PCPE-1, PCPE-2). Bone morphogenetic protein-1/mTLD and PCPE-1 were also immunolocalized in cases of human corneal scarring following injuries. Results: In the mouse model, throughout the follow-up period, there was a large increase in collagen III mRNA expression in the stroma. By transmission electron microscopy, there was marked cellular infiltration into the wound as well as disorganization of collagen fibrils, but no significant difference in fibril diameter. In control corneas, by Q-PCR, BMP-1/mTLD showed the highest expression, compared to low levels of mTLL-1 and undetectable levels of mTLL-2, in both epithelium and stroma. Following wounding, both BMP-1/mTLD and PCPE-1 mRNA and protein increased, while PCPE-2 mRNA decreased. Finally, by immunofluorescence, BMP-1/mTLD and PCPE-1 were strongly expressed in the scar region in both mouse and human corneas. Conclusions: Bone morphogenetic protein-1/mTLD and PCPE-1 are upregulated in corneal scars. Both proteins may therefore contribute to the process of corneal scarring.

International audience; The type I procollagen carboxyterminal(C-)propeptides are crucial in directing correct assembly of the procollagen heterotrimers. Defects in these domains have anecdotally been reported in patients with Osteogenesis Imperfecta (OI) and few genotype-phenotype correlations have been described. To gain insight in the functional consequences of C-propeptide defects, we performed a systematic review of clinical, molecular, and biochemical findings in all patients in whom we identified a type I procollagen C-propeptide defect, and compared this with literature data. We report 30 unique type I procollagen C-propeptide variants, 24 of which are novel. The outcome of COL1A1 nonsense and frameshift variants depends on the location of the premature termination codon. Those located prior to 50-55 nucleotides upstream of the most 3' exon-exon junction lead to nonsense-mediated mRNA decay (NMD) and cause mild OI. Those located beyond this boundary escape NMD, generally lead to production of stable, overmodified procollagen chains, which may partly be retained intracellularly, and are usually associated with severe-to-lethal OI. Proα1(I)-C-propeptide defects that permit chain association result in more severe phenotypes than those inhibiting chain association. We demonstrate that the crystal structure of the proα1(III)-C-propeptide is a reliable tool to predict phenotypic severity for most COL1A1-C-propeptide missense variants, whereas for COL1A2-C-propeptide variants, the phenotypic outcome is milder than predicted.

Recently several cDNAs have been described encoding lysyl oxidase-like proteins. Their deduced amino acid sequences are characterized by a strong similarity in the C-terminal region, corresponding to the lysyl oxidase family catalytic domain, and by marked differences in the N-terminal regions. Different biological functions have been described for lysyl oxidases in addition to their traditionally assumed cross-linking role. To answer the question of whether these different functions are carried out by different lysyl oxidases, purified and active forms of these enzymes are required. At present only the classical form of lysyl oxidase has been purified and characterized. The purpose of this study was to isolate and characterize the lysyl oxidase-like protein. In view of the strong sequence homology with the C-terminal domain of other lysyl oxidases, we chose to purify the protein from bovine aorta using antibodies specific to the N-terminal domain of the proenzyme. We have isolated a 56-kDa protein identified by amino acid sequencing as the bovine lysyl oxidase-like precursor, which is cleaved at the Arg-Arg-Arg sequence at positions 89-91 by a furin-like activity, as revealed after deblocking of the N-terminal residue. The immunopurified protein was largely inactive, but further processing in vitro by bone morphogenetic protein-1 led to an enzyme that was active on elastin and collagen substrates.

Recombinant human procollagen C-proteinase enhancer (rPCPE) was expressed using a baculovirus system and purified to homogeneity using a three-step procedure including heparin affinity chromatography. Heparin binding was dependent on the C-terminal netrin-like domain. The recombinant protein was found to be active, increasing the activity of procollagen C-proteinase/bone morphogenetic protein-1 on type I procollagen in a manner comparable to the native protein. Enhancing activity was dependent on intact disulfide bonding within the protein. By circular dichroism, the observed secondary structure of rPCPE was consistent with the known three-dimensional structures of proteins containing homologous domains.

The morphology and basic biochemical composition of articular cartilage from two strains of fowl were examined. Broiler breeder fowl are considered susceptible to degenerative joint disease (DJD); histological examination of one-year-old broiler breeders showed in some samples, articular cartilage thinning, fibrillation and chondrocyte cluster formation, features considered typical of DJD. Examination of similar samples from laying strain fowl showed only minor age-related changes such as some slight cartilage thinning and very mild fibrillation. The articular cartilage from the broiler breeder birds was significantly more hydrated with a higher uronic acid content than that of the laying strain birds. In addition, unloaded articular surfaces such as the proximal humerus had significantly higher amounts of uronic acid than the loaded cartilage surfaces of the proximal tarsometatarsus and the distal tibiotarsus; this suggested that the joint loading may have a role in any biochemical differences found between joints and between strains of fowl. These findings concur with other reports in mammals that showed increased hydration and uronic acid in association with early DJD and in models of osteoarthritis (OA). Thus, despite some differences between avian and mammalian articular cartilage, studies on avian DJD may give insights into mammalian disease.

Lysyl oxidase (LO) is a copper amine oxidase of the extracellular matrix which initiates covalent cross-linking in collagens and elastin. Human LO was expressed in Escherichia coli. At 37 degrees C, large amounts of protein were obtained, but in the form of insoluble aggregates. Lowering the growth temperature, and reducing the amount of inducer, resulted in the production of soluble LO, which was active on a degrees [3H]lysine-labeled elastin substrate. LO was also targeted to the periplasm as a fusion protein with the pelb signal peptide. The periplasmic enzyme was soluble, active and inhibited by beta-aminopropionitrile. Production of the carbonyl co-factor is therefore not a limitation in the expression of active LO in bacteria.Lysyl oxidase (LO) is a copper amine oxidase of the extracellular matrix which initiates covalent cross-linking in collagens and elastin. Human LO was expressed in Escherichia coli. At 37 degrees C, large amounts of protein were obtained, but in the form of insoluble aggregates. Lowering the growth temperature, and reducing the amount of inducer, resulted in the production of soluble LO, which was active on a degrees [3H]lysine-labeled elastin substrate. LO was also targeted to the periplasm as a fusion protein with the pelb signal peptide. The periplasmic enzyme was soluble, active and inhibited by beta-aminopropionitrile. Production of the carbonyl co-factor is therefore not a limitation in the expression of active LO in bacteria.

Biochemical determinations of the collagen and elastin content in 50 mm3 samples of human lung are presented in relation to morphometric measurements of lung structure, as the amount of alveolar wall surface area per unit volume (AWUV), on adjacent slices. There were no differences in AWUV values, collagen content (determined as hydroxyproline) or elastin content (determined as isodesmosine) between upper and lower lobes within a single lung. In a study of 102 samples from 9 smokers lungs with no evidence of macro- or microscopic emphysema (as estimated by AWUV measurement), there was a negative correlation between AWUV and the amounts of collagen or elastin per unit volume of inflated lung. The correlation was stronger when collagen and elastin content were expressed per unit area of alveolar wall. The negative correlation is interpreted as representing either the anatomical variation within the complex hierarchy of normal lung structure or possibly low levels of fibrosis in response to cigarette smoking.Biochemical determinations of the collagen and elastin content in 50 mm3 samples of human lung are presented in relation to morphometric measurements of lung structure, as the amount of alveolar wall surface area per unit volume (AWUV), on adjacent slices. There were no differences in AWUV values, collagen content (determined as hydroxyproline) or elastin content (determined as isodesmosine) between upper and lower lobes within a single lung. In a study of 102 samples from 9 smokers lungs with no evidence of macro- or microscopic emphysema (as estimated by AWUV measurement), there was a negative correlation between AWUV and the amounts of collagen or elastin per unit volume of inflated lung. The correlation was stronger when collagen and elastin content were expressed per unit area of alveolar wall. The negative correlation is interpreted as representing either the anatomical variation within the complex hierarchy of normal lung structure or possibly low levels of fibrosis in response to cigarette smoking.

A protein ( M r 24 K) that co-purifies with porcine skin lysyl oxidase ( M r 34 K) has been isolated and characterised. Five variants of the 24 K protein were identified by Mono Q ion-exchange FPLC, as were four variants of lysyl oxidase. Amino acid analysis and partial sequencing revealed near identity of a 36-residue CNBr peptide from porcine skin lysyl oxidase to corresponding regions of the putative lysyl oxidase precursor derived from rat and human cDNA. The 24 K protein was found to be unrelated to lysyl oxidase, but comparison with a protein sequence database showed it to be the same as a recently described protein from bovine skin that is associated with dermatan sulphate proteoglycans. The 24 K protein is relatively rich in tyrosine, and isoelectric focussing shows it to be acidic, with pI's in the range 4.1 to 4.4. In view of these properties, we propose the name TRAMP (Tyrosine Rich Acidic Matrix Protein) to identify this protein. Though TRAMP appears not to be glycosylated, several experiments indicate the presence of sulphotyrosine residues. When assayed using an elastin substrate, the activity of lysyl oxidase is unaffected by TRAMP.

Using antibodies to the procollagen C-proteinase enhancer of mouse fibroblast culture medium, we have screened by immunoblotting extracts of several post natal mouse and rat tissues for the presence of the enhancer antigen. All rodent connective tissues were relatively rich in enhancer; lower amounts were found in skeletal muscle and heart and essentially no enhancer was detected in kidney, liver or brain. The amounts of enhancer in mouse tendon and calvaria extracts were age related, with highest amounts in 11 and 19 d tendons and in 1 d calvaria-the times of rapid growth of these organs. The results suggest that procollagen C-proteinase enhancer is a specific connective tissue glycoprotein that is likely to regulate procollagen processing in vivo.Using antibodies to the procollagen C-proteinase enhancer of mouse fibroblast culture medium, we have screened by immunoblotting extracts of several post natal mouse and rat tissues for the presence of the enhancer antigen. All rodent connective tissues were relatively rich in enhancer; lower amounts were found in skeletal muscle and heart and essentially no enhancer was detected in kidney, liver or brain. The amounts of enhancer in mouse tendon and calvaria extracts were age related, with highest amounts in 11 and 19 d tendons and in 1 d calvaria-the times of rapid growth of these organs. The results suggest that procollagen C-proteinase enhancer is a specific connective tissue glycoprotein that is likely to regulate procollagen processing in vivo.

A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a [4,5-3H]-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware.A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a [4,5-3H]-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware.

International audience; We report the reconstruction and characterization of a hemicornea (epithelialized stroma), using primary human cells, for use in research and as an alternative to the use of animals in pharmacotoxicology testing. To create a stromal equivalent, keratocytes from human corneas were cultured in collagen-glycosaminoglycan-chitosan foams. Limbal stem cell-derived epithelial cells were seeded on top of these, giving rise to hemi-corneas. The epithelium appeared morphologically similar to its physiological counterpart, as shown by the basal cell expression of p63 isoforms including, in some cases, the stem cell marker p63DeltaNalpha, and the expression of keratin 3 and 14-3-3sigma in the upper cell layers. In addition, the cuboidal basal epithelial cells were anchored to a basement membrane containing collagen IV, laminin 5, and hemidesmosomes. In the stromal part, the keratocytes colonized the porous scaffold, formed a network of interconnecting cells, and synthesized an ultrastructurally organized extracellular matrix (ECM) containing collagen types I, V, and VI. Electron microscopy showed the newly synthesized collagen fibrils to have characteristic periodic striations, with diameters and interfibril spacings similar to those found in natural corneas. Compared to existing models for corneal pharmacotoxicology testing, this new model more closely approaches physiological conditions by including the inducing effects of mesenchyme and cell-matrix interactions on epithelial cell morphogenesis.We report the reconstruction and characterization of a hemicornea (epithelialized stroma), using primary human cells, for use in research and as an alternative to the use of animals in pharmacotoxicology testing. To create a stromal equivalent, keratocytes from human corneas were cultured in collagen-glycosaminoglycan-chitosan foams. Limbal stem cell-derived epithelial cells were seeded on top of these, giving rise to hemi-corneas. The epithelium appeared morphologically similar to its physiological counterpart, as shown by the basal cell expression of p63 isoforms including, in some cases, the stem cell marker p63DeltaNalpha, and the expression of keratin 3 and 14-3-3sigma in the upper cell layers. In addition, the cuboidal basal epithelial cells were anchored to a basement membrane containing collagen IV, laminin 5, and hemidesmosomes. In the stromal part, the keratocytes colonized the porous scaffold, formed a network of interconnecting cells, and synthesized an ultrastructurally organized extracellular matrix (ECM) containing collagen types I, V, and VI. Electron microscopy showed the newly synthesized collagen fibrils to have characteristic periodic striations, with diameters and interfibril spacings similar to those found in natural corneas. Compared to existing models for corneal pharmacotoxicology testing, this new model more closely approaches physiological conditions by including the inducing effects of mesenchyme and cell-matrix interactions on epithelial cell morphogenesis.

International audience; The proteolytic processing of procollagen V is complex and depends on the activity of several enzymes among which the BMP-1 (bone morphogenetic protein-1)/tolloid metalloproteinase and the furin-like proprotein convertases. Few of these processing interactions could have been predicted by analysing the presence of conserved consensus sequences in the proalpha1(V) chain. In the present study we opted for a cell approach that allows a straightforward identification of processing interactions. A construct encompassing the complete N-terminal end of the proalpha1(V) chain, referred to as Nalpha1, was recombinantly expressed to be used for enzymatic assays and for antibody production. Structural analysis showed that Nalpha1 is a monomer composed of a compact globule and an extended tail, which correspond respectively to the non-collagenous Nalpha1 subdomains, TSPN-1 (thrombospondin-1 N-terminal domain-like) and the variable region. Nalpha1 was efficiently cleaved by BMP-1 indicating that the triple helix is not required for enzyme activity. By mutating residues flanking the cleavage site, we showed that the aspartate residue at position P2' is essential for BMP-1 activity. BMP-1 activity at the C-terminal end of the procollagen V was assessed by generating a furin double mutant (R1584A/R1585A). We showed that, in absence of furin activity, BMP-1 is capable of processing the C-propeptide even though less efficiently than furin. Altogether, our results provide new relevant information on this complex and poorly understood mechanism of enzymatic processing in procollagen V function.The proteolytic processing of procollagen V is complex and depends on the activity of several enzymes among which the BMP-1 (bone morphogenetic protein-1)/tolloid metalloproteinase and the furin-like proprotein convertases. Few of these processing interactions could have been predicted by analysing the presence of conserved consensus sequences in the proalpha1(V) chain. In the present study we opted for a cell approach that allows a straightforward identification of processing interactions. A construct encompassing the complete N-terminal end of the proalpha1(V) chain, referred to as Nalpha1, was recombinantly expressed to be used for enzymatic assays and for antibody production. Structural analysis showed that Nalpha1 is a monomer composed of a compact globule and an extended tail, which correspond respectively to the non-collagenous Nalpha1 subdomains, TSPN-1 (thrombospondin-1 N-terminal domain-like) and the variable region. Nalpha1 was efficiently cleaved by BMP-1 indicating that the triple helix is not required for enzyme activity. By mutating residues flanking the cleavage site, we showed that the aspartate residue at position P2' is essential for BMP-1 activity. BMP-1 activity at the C-terminal end of the procollagen V was assessed by generating a furin double mutant (R1584A/R1585A). We showed that, in absence of furin activity, BMP-1 is capable of processing the C-propeptide even though less efficiently than furin. Altogether, our results provide new relevant information on this complex and poorly understood mechanism of enzymatic processing in procollagen V function.

International audience; The lethal disease anthrax is propagated by spores of Bacillus anthracis, which can penetrate into the mammalian host by inhalation, causing a rapid progression of the disease and a mostly fatal outcome. We have solved the three-dimensional structure of the major surface protein BclA on B. anthracis spores. Surprisingly, the structure resembles C1q, the first component of complement, despite there being no sequence homology. Although most assays for C1q-like activity, including binding to C1q receptors, suggest that BclA does not mimic C1q, we show that BclA, as well as C1q, interacts with components of the lung alveolar surfactant layer. Thus, to better recognize and invade its hosts, this pathogenic soil bacterium may have evolved a surface protein whose structure is strikingly close to a mammalian protein.The lethal disease anthrax is propagated by spores of Bacillus anthracis, which can penetrate into the mammalian host by inhalation, causing a rapid progression of the disease and a mostly fatal outcome. We have solved the three-dimensional structure of the major surface protein BclA on B. anthracis spores. Surprisingly, the structure resembles C1q, the first component of complement, despite there being no sequence homology. Although most assays for C1q-like activity, including binding to C1q receptors, suggest that BclA does not mimic C1q, we show that BclA, as well as C1q, interacts with components of the lung alveolar surfactant layer. Thus, to better recognize and invade its hosts, this pathogenic soil bacterium may have evolved a surface protein whose structure is strikingly close to a mammalian protein.

The effects of strain selection and body weight on proteoglycan metabolism and the onset of degenerative joint disease (DJD) were investigated in avian articular cartilage. Samples from the hock joint (proximal tarsometatarsus, PTM; distal tibiotarsus, DTT) of rapidly growing broiler fowl, fed either ad libitum or on a restricted-diet, were compared with those from a slow growing, light and non-selected strain (J-line). Synthesis and degradation of proteoglycans were investigated by radioactive pulse-chase studies, determination of total sulphated glycosaminoglycans and electrophoretic analysis. By gross morphology, degenerative changes in articular cartilage occurred solely in the DTT from ad libitum-fed broiler fowl, after 13 weeks. Differences in proteoglycan metabolism were also observed, most markedly in the DTT, where the rate of proteoglycan synthesis in the ad libitum-fed group was less than in age-matched J-line cartilage, and the proportions of both newly synthesised and resident proteoglycans released into the culture medium were greater. Results with the feed-restricted group were intermediate between ad libitum-fed and J-line. Electrophoretic analysis of proteoglycans in the culture media showed evidence of degradation solely in the ad libitum-fed group, with earliest onset in the DTT. The results indicate that proteoglycan metabolism in avian articular cartilage is similar to that in mammalian cartilage during the development of DJD, and that the onset of cartilage degeneration is linked with excessive load bearing.The effects of strain selection and body weight on proteoglycan metabolism and the onset of degenerative joint disease (DJD) were investigated in avian articular cartilage. Samples from the hock joint (proximal tarsometatarsus, PTM; distal tibiotarsus, DTT) of rapidly growing broiler fowl, fed either ad libitum or on a restricted-diet, were compared with those from a slow growing, light and non-selected strain (J-line). Synthesis and degradation of proteoglycans were investigated by radioactive pulse-chase studies, determination of total sulphated glycosaminoglycans and electrophoretic analysis. By gross morphology, degenerative changes in articular cartilage occurred solely in the DTT from ad libitum-fed broiler fowl, after 13 weeks. Differences in proteoglycan metabolism were also observed, most markedly in the DTT, where the rate of proteoglycan synthesis in the ad libitum-fed group was less than in age-matched J-line cartilage, and the proportions of both newly synthesised and resident proteoglycans released into the culture medium were greater. Results with the feed-restricted group were intermediate between ad libitum-fed and J-line. Electrophoretic analysis of proteoglycans in the culture media showed evidence of degradation solely in the ad libitum-fed group, with earliest onset in the DTT. The results indicate that proteoglycan metabolism in avian articular cartilage is similar to that in mammalian cartilage during the development of DJD, and that the onset of cartilage degeneration is linked with excessive load bearing.

Fibromodulin belongs to the family of small, leucine-rich proteoglycans which have been reported tointeract with collagens and to inhibit type I collagen fibrillogenesis. Decorin and fibromodulin exhibit anoticeable degree of sequence similarity. However, as previously reported [Font, B., Eichenberger, D.,Rosenberg, L. M. & van der Rest, M. (1996) Matrix Biol. 15,3412348] the domains of these moleculesimplicated in the interactions with type XII and type XIV collagens are different, these being the dermatansulphate/chondroitin sulphate chain for decorin and the core protein for fibromodulin. At the present timethe fibromodulin domains implicated in the interactions with fibrillar collagens remain unknown. Inexperiments reported here, we have sought to identify the structural requirements for fibromodulin in-teraction with collagen and for the control of type I collagen fibrillogenesis. Circular dichroism spectraand fibrillogenesis inhibition studies show that fibromodulin structure and its collagen fibrillogenesiscontrol function are strictly dependent on the presence of intact disulphide bridge(s). In addition, weshow that the binding of fibromodulin (or fibromodulin-derived fragments) to type I collagen is notnecessarily correlated with fibrillogenesis inhibition. To isolate fibromodulin domains, the native proteog-lycan was submitted to mild proteolysis. We have isolated an A-chymotrypsin2resistant fragment whichcontains the bulk of the N-terminal and central region of the molecule including the leucine-rich repeats4 and 6 reported for decorin to be involved in type I collagen binding. This fragment does not bind totype I collagen. Using enzymes with different specificities, a number of large fragments of fibromodulinwere obtained, suggesting a compact structure for this molecule which is relatively resistant to proteolysis.None of these N-glycosylated fragments were able to bind to type I collagen in co-sedimentation experi-ments. Taken together these results suggest that fibromodulin-type I collagen interactions leading tofibrillogenesis inhibition require more than one binding domain. One of these domains could be the C-terminal end of the molecule containing the disulphide loop which is absent in the chymotrypsin-resistantfragment.Keywords: collagen fibrillogenesis; fibromodulin; proteoglycan domain.In connective tissues, proteoglycans play important roles due cysteine residues, with at least one disulphide bridge, (b) ato their interactions with other components of the extracellular central region with a variable number (10212) of asparagine-matrix [1]. Among proteoglycans, several members of this group containing leucine-rich repeats containing N-linked keratan sul-are structurally related and constitute the small proteoglycan phate (fibromodulin and lumican) or N-linked oligosaccharidesfamily, namely biglycan, decorin, fibromodulin and lumican [2]. (biglycan and decorin) distributed among up to five potentialThese molecules are characterised by a core protein of about sites; and (c) a C-terminal region with a conserved disulphide40 kDa, and by the existence of three distinct regions: (a) a neg- loop.In vivo, small proteoglycans have been shown to associateatively charged N-terminal region with either covalently linked with fibrillar collagens [3]. In recent experiments, it has beendermatan sulphate/chondroitin sulphate (DS/CS) chains (usually reported that targeted disruption of the decorin gene in miceone in decorin and two in biglycan), or tyrosine sulphate in fi- leads to skin with markedly reduced tensile strength and to ab-bromodulin and lumican, and a highly conserved cluster of four normal morphology of collagen fibrils, indicating that in vivodecorin plays a major role in the regulation of fibril formation

The carboxyl-terminal propeptide of procollagen type I (CPP-I) plays a key role in the regulation of collagen fibrillogenesis. In addition, it has been reported that, after cleavage from procollagen, CPP-I exerts feedback control of collagen biosynthesis. To further elucidate the mechanisms involved in each of these processes, we have investigated the nature of cell surface receptors for CPP-I. CPP-I affinity chromatography, using detergent extracts of iodinated HT1080 cells and EDTA elution, resulted in the isolation of two polypeptides of molecular mass 160 and 110 kDa. Since the migratory behavior of these polypeptides under nonreducing and reducing conditions was characteristic of a subset of integrin receptors, their reactivity with anti-integrin monoclonal antibodies was tested. Antibodies directed against the alpha 2 and beta 1 subunits specifically immunoprecipitated both CPP-I-binding polypeptides, indicating that the CPP-I receptor is the integrin alpha 2 beta 1. CPP-I was found to support the attachment and spreading of HT1080 cells, demonstrating that it can function as an adhesion protein. Two other approaches supported the identification of alpha 2 beta 1 as the CPP-I receptor. First, anti-functional anti-integrin monoclonal antibodies directed against the alpha 2 and beta 1 subunits completely abrogated the adhesive activity of CPP-I and, second, highly purified CPP-I bound specifically to alpha 2 beta 1-containing integrin preparations in a solid-phase receptor-ligand binding assay. These findings have important implications for the function of fibrillar collagen carboxyl-terminal propeptides and for the role played by integrins in the regulation of cellular phenotype.The carboxyl-terminal propeptide of procollagen type I (CPP-I) plays a key role in the regulation of collagen fibrillogenesis. In addition, it has been reported that, after cleavage from procollagen, CPP-I exerts feedback control of collagen biosynthesis. To further elucidate the mechanisms involved in each of these processes, we have investigated the nature of cell surface receptors for CPP-I. CPP-I affinity chromatography, using detergent extracts of iodinated HT1080 cells and EDTA elution, resulted in the isolation of two polypeptides of molecular mass 160 and 110 kDa. Since the migratory behavior of these polypeptides under nonreducing and reducing conditions was characteristic of a subset of integrin receptors, their reactivity with anti-integrin monoclonal antibodies was tested. Antibodies directed against the alpha 2 and beta 1 subunits specifically immunoprecipitated both CPP-I-binding polypeptides, indicating that the CPP-I receptor is the integrin alpha 2 beta 1. CPP-I was found to support the attachment and spreading of HT1080 cells, demonstrating that it can function as an adhesion protein. Two other approaches supported the identification of alpha 2 beta 1 as the CPP-I receptor. First, anti-functional anti-integrin monoclonal antibodies directed against the alpha 2 and beta 1 subunits completely abrogated the adhesive activity of CPP-I and, second, highly purified CPP-I bound specifically to alpha 2 beta 1-containing integrin preparations in a solid-phase receptor-ligand binding assay. These findings have important implications for the function of fibrillar collagen carboxyl-terminal propeptides and for the role played by integrins in the regulation of cellular phenotype.

The interactions of [3H]procollagen I with various phospholipids were studied by density gradient centrifugation. At physiological conditions of pH, ionic strength, and temperature, there was no evidence for procollagen binding to phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, or phosphatidylserine liposomes. In contrast, procollagen I bound strongly to sphingomyelin liposomes in a reversible and saturable manner, with an apparent dissociation constant (Kd) of 2.6 nM. Binding occurred over a range of temperatures (4-37 degrees C) and was relatively unaffected by salt concentrations up to 1.2 M NaCl. Binding was observed in phosphate buffers, but not in the presence of high concentrations of Tris or Hepes. Bovine serum albumin had no effect on procollagen binding to sphingomyelin, and neither did unlabeled type I collagen, with or without the nonhelical telopeptides. Procollagen II and denatured procollagen I also bound to sphingomyelin. Procollagen binding to sphingomyelin at 35 degrees C was considerably reduced when small amounts of phosphatidylcholine were present, although binding was partially restored when the temperature was reduced below the corresponding phase transition temperature. Purified unlabeled procollagen COOH-terminal propeptides successfully competed for binding, and 125I-labeled COOH-terminal propeptides bound to sphingomyelin in the absence of procollagen. Weaker binding to sphingomyelin, mediated by the collagen triple helical region, was also observed; but this was dominated by the sphingomyelin-COOH-terminal propeptide interaction. The data suggest a novel mechanism for matrix vesicle-mediated biomineralization.The interactions of [3H]procollagen I with various phospholipids were studied by density gradient centrifugation. At physiological conditions of pH, ionic strength, and temperature, there was no evidence for procollagen binding to phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, or phosphatidylserine liposomes. In contrast, procollagen I bound strongly to sphingomyelin liposomes in a reversible and saturable manner, with an apparent dissociation constant (Kd) of 2.6 nM. Binding occurred over a range of temperatures (4-37 degrees C) and was relatively unaffected by salt concentrations up to 1.2 M NaCl. Binding was observed in phosphate buffers, but not in the presence of high concentrations of Tris or Hepes. Bovine serum albumin had no effect on procollagen binding to sphingomyelin, and neither did unlabeled type I collagen, with or without the nonhelical telopeptides. Procollagen II and denatured procollagen I also bound to sphingomyelin. Procollagen binding to sphingomyelin at 35 degrees C was considerably reduced when small amounts of phosphatidylcholine were present, although binding was partially restored when the temperature was reduced below the corresponding phase transition temperature. Purified unlabeled procollagen COOH-terminal propeptides successfully competed for binding, and 125I-labeled COOH-terminal propeptides bound to sphingomyelin in the absence of procollagen. Weaker binding to sphingomyelin, mediated by the collagen triple helical region, was also observed; but this was dominated by the sphingomyelin-COOH-terminal propeptide interaction. The data suggest a novel mechanism for matrix vesicle-mediated biomineralization.

International audience; The solubility limit of purified chick type I procollagen, incubated at 37 degrees C in phosphate-buffered saline, was found to be in the range 1 to 1.5 mg/ml. At higher concentrations large aggregates formed. These comprised: (1) D-periodic assemblies; (2) narrow filaments with no apparent periodicity; and (3) segment-long-spacing-like aggregates. The D-periodic assemblies, which predominated at high concentrations, were separated from the other types of aggregate and found to be ribbon-like. Ribbons were uniform in thickness (approximately 8 nm) and up to 1 micron wide. Staining patterns showed features similar to those in native-type collagen fibrils. Immunolabelling indicated that the carboxyl-terminal propeptide domains were close to the carboxyl-terminal gap-overlap junction, and that the amino-terminal propeptide domains were folded over into the amino-terminal side of the overlap zone. Both propeptide domains appeared to be located on the surface of the assemblies. These observations show that intact propeptide domains hinder, but do not prevent, the formation of D-periodic assemblies. The presence of the propeptide domains on the surface of a growing assembly could restrict its lateral growth and limit its final thickness.The solubility limit of purified chick type I procollagen, incubated at 37 degrees C in phosphate-buffered saline, was found to be in the range 1 to 1.5 mg/ml. At higher concentrations large aggregates formed. These comprised: (1) D-periodic assemblies; (2) narrow filaments with no apparent periodicity; and (3) segment-long-spacing-like aggregates. The D-periodic assemblies, which predominated at high concentrations, were separated from the other types of aggregate and found to be ribbon-like. Ribbons were uniform in thickness (approximately 8 nm) and up to 1 micron wide. Staining patterns showed features similar to those in native-type collagen fibrils. Immunolabelling indicated that the carboxyl-terminal propeptide domains were close to the carboxyl-terminal gap-overlap junction, and that the amino-terminal propeptide domains were folded over into the amino-terminal side of the overlap zone. Both propeptide domains appeared to be located on the surface of the assemblies. These observations show that intact propeptide domains hinder, but do not prevent, the formation of D-periodic assemblies. The presence of the propeptide domains on the surface of a growing assembly could restrict its lateral growth and limit its final thickness.

The urinary excretion of 6 beta-hydroxycortisol (6 beta-OHF), free cortisol, and estriol was studied at 4-hourly intervals in 9 subjects between the 36th and 38th week of pregnancy. The ratio of 6 beta-OHF to creatinine showed a significant diurnal rhythm with a 40% fall from a peak at 06.00-10.00 h to a trough at 22.00-02.00 h. There was a significant positive correlation between the excretion of 6 beta-OHF and free cortisol. Estrogen excretion showed no diurnal variation. No increase was observed in the 6 beta-OHF:creatinine ratio in early morning urine samples from 5 subjects followed daily prior to the onset of spontaneous labour at term. Normal nonpregnant females were also found to excrete significant quantities of 6 beta-OHF. These findings suggest that the clinical significance of 6 beta-OHF levels measured in a single void sample of urine has to be reassessed in the face of such a marked diurnal variation.

Objective: The purpose of this study was to determine the effects of intra-articular injections of high molecular weight (2000kDa) sodium hyaluronate (HA) on the progression of articular cartilage degeneration in a rabbit partial medial meniscectomy model of osteoarthritis.Design: Six experimental groups included normal, sham operated, and operated and injected animals, the latter injected once-weekly (for two weeks or twelve weeks, beginning four weeks after surgery) with either 1% (w/v) HA or phosphate buffered saline (PBS). Following assessment of gross morphology, serial adjacent blocks of full-depth articular cartilage were prepared from the tibial condyle for analysis of total water, hydroxyproline, DNA and proteoglycan (uronic acid) content, as well as the ratio of galactosamine to glucosamine. Samples were sub-divided into inner (medial) and outer (lateral) regions.Results: No morphological differences were recognized between joints injected with PBS and those receiving HA. When analysed biochemically, there were no significant differences in hydration, hydroxyproline or DNA content between the experimental groups. In contrast, HA injection did affect changes in proteoglycan content. Expressed per tissue dry weight, uronic acid content in the operated group injected with PBS for two weeks was lower than normal (P

Tyrosine-rich acidic matrix protein (TRAMP; 22 kDa extracellular matrix protein; dermatopontin) is a protein that co-purifies with lysyl oxidase and with dermatan sulphate proteoglycans, with possible functions in cell-matrix interactions and matrix assembly. Using a rabbit polyclonal antiserum raised against porcine TRAMP, which cross-reacts with both the human and murine forms of the protein, we show by immunoblotting that TRAMP has a widespread tissue distribution, including skin, skeletal muscle, heart, lung, kidney, cartilage and bone. In cultures of human skin fibroblasts, TRAMP incorporates both [35S]sulphate and [3H]tyrosine and is secreted into the medium, as shown by immunoprecipitation. Amino acid analysis of immunoprecipitated TRAMP demonstrates that many of the tyrosine residues in TRAMP are sulphated.Tyrosine-rich acidic matrix protein (TRAMP; 22 kDa extracellular matrix protein; dermatopontin) is a protein that co-purifies with lysyl oxidase and with dermatan sulphate proteoglycans, with possible functions in cell-matrix interactions and matrix assembly. Using a rabbit polyclonal antiserum raised against porcine TRAMP, which cross-reacts with both the human and murine forms of the protein, we show by immunoblotting that TRAMP has a widespread tissue distribution, including skin, skeletal muscle, heart, lung, kidney, cartilage and bone. In cultures of human skin fibroblasts, TRAMP incorporates both [35S]sulphate and [3H]tyrosine and is secreted into the medium, as shown by immunoprecipitation. Amino acid analysis of immunoprecipitated TRAMP demonstrates that many of the tyrosine residues in TRAMP are sulphated.

The polymorphic forms of ordered collagen aggregation in vitro and in vivo are reviewed. The axially projected structures of a class of fibrils known as fibrous long spacing (FLS) collagen are solved using simulated positively stained banding patterns based on the amino acid sequence. This method is also used to solve the axial projection of a 670 A ( D ) periodic structure with a symmetrical banding pattern (DPS) re-precipitated from skin collagen. The relation between the obliquely striated and 110 A periodic forms of collagen is discussed. The specificity for the formation of FLS, DPS and segment long spacing (SLS) collagen is shown to be in the distributions of various amino acids in the sequence. Different residues are important for each type of structure, their importance being dependent on the chemical conditions and the presence of other macromolecules. The interaction of collagen fibrils with proteoglycans in vivo is discussed in terms of the amino acid sequence. Also the factors which affect collagen morphology in the presence of mucopolysaccharides and proteoglycans in vitro and in vivo are discussed. Some insight is gamed into the principles which govern the self-assembly of molecules into ordered fibrous aggregates.