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We report the effects of quercetin, a flavonoid present in human diet, on early stage beta-amyloid (Aβ) aggregation – a seminal event in Alzheimer’s disease. Molecular level changes in Aβ arrangements are monitored by time-resolved emission spectral (TRES) measurements of the fluorescence of Aβ’s single tyrosine intrinsic fluorophore (Tyr). The results suggest that quercetin binds beta-amyloid oligomers at early stages of their aggregation, which leads to the formation of modified oligomers and hinders the creation of beta-sheet structures, potentially preventing the onset of Alzheimer’s disease.

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.

The procollagen C-propeptides of the fibrillar collagens play key roles in the intracellular assembly of procollagen molecules from their constituent polypeptides chains, and in the extracellular assembly of collagen molecules into fibrils. Here we review recent advances in understanding the molecular mechanisms controlling C-propeptide trimerization which have revealed the importance of inter-chain disulphide bonding and a small number of charged amino acids in the stability and specificity of different types of chain association. We also show how the crystal structure of the complex between the C-propeptide trimer of procollagen III and the active fragment of procollagen C-proteinase enhancer-1 leads to a detailed model for accelerating release of the C-propeptides from procollagen by bone morphogenetic protein-1 and related proteinases. We then discuss the effects of disease-related missense mutations in the C-propeptides in relation to the sites of these mutations in the three-dimensional structure. While in general there is a good correlation between disease severity and structure-based predictions, there are notable exceptions, suggesting new interactions involving the C-propeptides yet to be characterized. Mutations affecting proteolytic release of the C-propeptides from procollagen are discussed in detail. Finally, the roles of recently discovered interaction partners for the C-propeptides are considered during fibril assembly and cross-linking.

The defence collagens C1q and mannose-binding lectin (MBL) are immune recognition proteins that associate with the serine proteinases C1r/C1s and MBL-associated serine proteases (MASPs) to trigger activation of complement, a major innate immune system. Bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases (BTPs) are metalloproteinases with major roles in extracellular matrix assembly and growth factor signalling. Despite their different functions, C1r/C1s/MASPs and BTPs share structural similarities, including a specific CUB-EGF-CUB domain arrangement found only in these enzymes that mediates interactions with collagen-like proteins, suggesting a possible functional relationship. Here we investigated the potential interactions between the defence collagens C1q and MBL and the BTPs BMP-1 and mammalian tolloid-like-1 (mTLL-1). C1q and MBL bound to immobilized BMP-1 and mTLL-1 with nanomolar affinities. These interactions involved the collagen-like regions of the defence collagens and were inhibited by pre-incubation of C1q or MBL with their cognate complement proteinases. Soluble BMP-1 and mTLL-1 did not inhibit complement activation and the defence collagens were neither substrates nor inhibitors of BMP-1. Finally, C1q co-localized with BMP-1 in skin biopsies following melanoma excision and from patients with recessive dystrophic epidermolysis bullosa. The observed interactions provide support for a functional link between complement and BTPs during inflammation and tissue repair.

Development of a pseudoaneurysm of the ascending aorta is an uncommon complication of aortic surgery. Several nonsurgical techniques are available for treatment of ascending aortic pseudoaneurysms (AAPs). This report outlines a single-center retrospective experience with 14 nonsurgical procedures for treatment of AAPs in 10 patients. Modified stent grafts, septal defect occlusion devices, coil embolics, and liquid embolics were deployed by transthoracic and endovascular approaches. Complete stasis of the AAP was achieved in 7 of 10 patients (70%). Mean postprocedural recoveries occurred within 3.5 days. Nonsurgical techniques for repair of AAPs offer a comparatively safe and effective alternative to open surgical repair.

Mutations in the type I procollagen C-propeptide occur in ~6.5% of Osteogenesis Imperfecta (OI) patients. They are of special interest because this region of procollagen is involved in α chain selection and folding, but is processed prior to fibril assembly and is absent in mature collagen fibrils in tissue. We investigated the consequences of seven COL1A1 C-propeptide mutations for collagen biochemistry in comparison to three probands with classical glycine substitutions in the collagen helix near the C-propeptide and a normal control. Procollagens with C-propeptide defects showed the expected delayed chain incorporation, slow folding and overmodification. Immunofluorescence microscopy indicated that procollagen with C-propeptide defects was mislocalized to the ER lumen, in contrast to the ER membrane localization of normal procollagen and procollagen with helical substitutions. Notably, pericellular processing of procollagen with C-propeptide mutations was defective, with accumulation of pC-collagen and/or reduced production of mature collagen. In vitro cleavage assays with BMP-1 ± PCPE-1 confirmed impaired C-propeptide processing of procollagens containing mutant proα1(I) chains. Overmodified collagens were incorporated into the matrix in culture. Dermal fibrils showed alterations in average diameter and diameter variability and bone fibrils were disorganized. Altered ER-localization and reduced pericellular processing of defective C-propeptides are expected to contribute to abnormal osteoblast differentiation and matrix function, respectively.

Fibrillar collagens of the extracellular matrix are critical for tissue structure and physiology; however, excessive or abnormal deposition of collagens is a defining feature of fibrosis. Regulatory mechanisms that act on collagen fibril assembly potentially offer new targets for antifibrotic treatments. Tissue weakening, altered collagen fibril morphologies, or both, are shared phenotypes of mice lacking matricellular thrombospondins. Thrombospondin-1 (TSP1) plays an indirect role in collagen homeostasis through interactions with matrix metalloproteinases and transforming growth factor–1 (TGF-1). We found that TSP1 also affects collagen fibril formation directly. Compared to skin from wild-type mice, skin from Thbs1-/- mice had reduced collagen cross-linking and reduced prolysyl oxidase (proLOX) abundance with increased conversion to catalytically active LOX. In vitro, TSP1 bound to both the C-propeptide domain of collagen I and the highly conserved KGHR sequences of the collagen triple-helical domain that participate in cross-linking. TSP1 also bound to proLOX and inhibited proLOX processing by bone morphogenetic protein-1. In human dermal fibroblasts (HDFs), TSP1 and collagen I colocalized in intracellular vesicles and on extracellular collagen fibrils, whereas TSP1 and proLOX colocalized only in intracellular vesicles. Inhibition of LOX-mediated collagen cross-linking did not prevent the extracellular association between collagen and TSP1; however, treatment of HDFs with KGHR-containing, TSP1-binding, triple-helical peptides disrupted the collagen-TSP1 association, perturbed the collagen extracellular matrix, and increased myofibroblastic differentiation in a manner that depended on TGF- receptor 1. Thus, the extracellular KGHR-dependent interaction of TSP1 with fibrillar collagens contributes to fibroblast homeostasis.

Purpose To evaluate the utility of dynamic, contrast-enhanced magnetic resonance imaging (MRI) in combination with single-shot T2-weighted (ssT2) sequences in the differentiation of lipid-poor adrenal adenomas from non-adenomas. Materials and methods This retrospective study was approved by the institutional review board and is HIPAA compliant. Between January 2007 and December 2010, 46 patients with MRI demonstrating a lipid-poor adrenal lesion who underwent either surgical resection or a minimum of 24 months of imaging follow-up were identified retrospectively. All images were retrospectively reviewed in blinded fashion by two radiologists. Each adrenal lesion was categorized by dynamic enhancement features and qualitative signal on ssT2 images and was categorized as an adenoma if it demonstrated homogenous enhancement in the arterial phase, washout with capsule enhancement in the delayed phase, and T2 signal isointense to normal adrenal tissue. Any lesion that did not fulfill all the criteria was classified as a non-adenoma. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for characterization of adenoma were calculated for each reader with 95% confidence intervals. A κ test assessed level of agreement between readers. Results Application of our criteria lead to an MRI diagnosis of lipid-poor adrenal adenoma with a sensitivity of 84.2–89.5% (16/19–17/19), specificity of 96.3% (26/27), positive predictive value of 94.1–94.4% (16/17–17/18), negative predictive value of 89.7–92.9% (26/29–26/28), and accuracy of 91.3–93.5% (42/46–43/46). Agreement between the two readers showed substantial κ agreement for the differentiation of adenoma from non-adenoma. Conclusions Dynamic, contrast-enhanced T1-weighted three-dimensional gradient echo sequences in combination with ssT2 images can accurately differentiate lipid-poor adrenal adenomas from non-adenomas.

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 objective of our study was to determine patterns and cost of imaging tumor surveillance in patients after a benign fine-needle aspiration (FNA) biopsy of the thyroid in a large teaching hospital as well as the rate of subsequent cancer detection.This cohort study was approved by the appropriate institutional review board and complied with HIPAA. All patients who had a benign thyroid FNA biopsy between January 1, 1999, and December 31, 2003, were identified from an institutional pathology database. We gathered information from electronic medical records on imaging tumor surveillance and subsequent cancer detection. Cost was determined using the facility total relative value unit and the 2014 Hospital Outpatient Prospective Payment System conversion factor.Between January 1, 1999, and December 31, 2003, 1685 patients had a benign thyroid FNA biopsy, 800 (47.5%) of whom underwent follow-up imaging. These patients underwent 2223 thyroid ultrasound examinations, 606 ultrasound-guided thyroid FNA biopsies, 78 thyroid scintigraphy examinations, 168 neck CTs, and 53 neck MRIs at a cost of $529,874, $176,157, $39,622, $80,580, and $53,114, respectively, for a total cost of $879,347 or $1099 per patient. The mean length of follow-up was 7.3 years, during which time 19 (2.4%) patients were diagnosed with thyroid cancer at a cost of $46,281 per cancer. Seventeen (89.5%) were diagnosed with papillary carcinoma and two (10.5%) with Hurthle cell carcinoma.Over a 5-year period, about half of the patients who had a benign thyroid FNA biopsy underwent follow-up imaging at considerable cost with a small rate of subsequent malignancy.

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.

We recently showed that the highly organized architecture of the corneal stroma could be reproduced using scaffolds consisting of orthogonally aligned multilayers of collagen fibrils prepared using a high magnetic field. Here we show that such scaffolds permit the reconstruction in vitro of human hemi-corneas (stroma + epithelium), using primary human keratocytes and limbal stem cell derived human keratinocytes. On the surface of these hemi-corneas, a well-differentiated epithelium was formed, as determined both histologically and ultrastructurally and by the expression of characteristic markers. Within the stroma, the keratocytes aligned with the directions of the fibrils in the scaffold and synthesized a new extracellular matrix with typical collagen markers and small, uniform diameter fibrils. Finally, in vivo experiments using a rabbit model showed that these orthogonally oriented multi-layer scaffolds could be used to repair the anterior region of the stroma, leading to re-epithelialization and recovery of both transparency and ultrastructural organization.

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.

Procollagen C-proteinase enhancers (PCPE-1 and -2) specifically activate bone morphogenetic protein-1 (BMP-1) and other members of the tolloid proteinase family during C-terminal processing of fibrillar collagen precursors. PCPEs consist of two CUB domains (CUB1 and CUB2) and one NTR domain separated by one short and one long linker. It was previously shown that PCPEs can strongly interact with procollagen molecules, but the exact mechanism by which they enhance BMP-1 activity remains largely unknown. Here, we used a series of deletion mutants of PCPE-1 and two chimeric constructs with repetitions of the same CUB domain to study the role of each domain and linker. Out of all the forms tested, only those containing both CUB1 and CUB2 were capable of enhancing BMP-1 activity and binding to a mini-procollagen substrate with nanomolar affinity. Both these properties were lost by individual CUB domains, which had dissociation constants at least three orders of magnitude higher. In addition, none of the constructs tested could inhibit PCPE activity, although CUB2CUB2NTR was found to modulate BMP-1 activity through direct complex formation with the enzyme, resulting in a decreased rate of substrate processing. Finally, increasing the length of the short linker between CUB1 and CUB2 was without detrimental effect on both activity and substrate binding. These data support the conclusion that CUB1 and CUB2 bind to the procollagen substrate in a cooperative manner, involving the short linker that provides a flexible tether linking the two binding regions.

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.

Bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases, here called BTPs, include the proteases originally identified for their roles in the C-terminal maturation of fibrillar procollagens ("procollagen C-proteinase"). Though numerous other substrates have since been discovered, the BTPs remain the main proteases involved in extracellular matrix assembly with little or no implication in matrix degradation. During the same period however, the BTPs have also become established as important proteases in the activation of growth factors, including TGF-β1, BMP-2/-4, GDF-8/-11 and IGFs, as well as the release of anti-angiogenic fragments from parent proteins. The BTPs are therefore key players in many pathophysiological processes such as morphogenesis, tissue repair and tumor progression. This mini-review summarizes our current knowledge of the functions of BTPs, their substrates and unusual mechanisms of regulation, and discusses their potential as new targets for future therapies.

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.

Type I fibrillar collagen is the most abundant protein in the human body, crucial for the formation and strength of bones, skin, and tendon. Proteolytic enzymes are essential for initiation of the assembly of collagen fibrils by cleaving off the propeptides. We report that Mep1a −/− and Mep1b −/− mice revealed lower amounts of mature collagen I compared with WT mice and exhibited significantly reduced collagen deposition in skin, along with markedly decreased tissue tensile strength. While exploring the mechanism of this phenotype, we found that cleavage of full-length human procollagen I heterotrimers by either meprin α or meprin β led to the generation of mature collagen molecules that spontaneously assembled into collagen fibrils. Thus, meprin α and meprin β are unique in their ability to process and release both C- and N-propeptides from type I procollagen in vitro and in vivo and contribute to the integrity of connective tissue in skin, with consequent implications for inherited connective tissue disorders.

International audience; Tight regulation of collagen fibril deposition in the extracellular matrix is essential for normal tissue homeostasis and repair, defects in which are associated with several degenerative or fibrotic disorders. A key regulatory step in collagen fibril assembly is the C-terminal proteolytic processing of soluble procollagen precursors. This step, carried out mainly by bone morphogenetic protein-1/tolloid-like proteinases, is itself subject to regulation by procollagen C-proteinase enhancer proteins (PCPEs) which can dramatically increase bone morphogenetic protein-1/tolloid-like proteinase activity, in a substrate-specific manner. Although it is known that this enhancing activity requires binding of PCPE to the procollagen C-propeptide trimer, identification of the precise binding site has so far remained elusive. Here, use of small-angle X-ray scattering provides structural data on this protein complex indicating that PCPE binds to the stalk region of the procollagen C-propeptide trimer, where the three polypeptide chains associate together, at the junction with the base region. This is supported by site-directed mutagenesis, which identifies two highly conserved, surface-exposed lysine residues in this region of the trimer that are essential for binding, thus revealing structural parallels with the interactions of Complement C1r/C1s, Uegf, BMP-1 (CUB) domain-containing proteins in diverse biological systems such as complement activation, receptor signaling, and transport. Together with detailed kinetics and interaction analysis, these results provide insights into the mechanism of action of PCPEs and suggest clear strategies for the development of novel antifibrotic therapies.

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.

The C-propeptide domains of the fibrillar procollagens, which are present throughout the Metazoa in the form of ∼90 kDa trimers, play crucial roles in both intracellular molecular assembly and extracellular formation of collagen fibrils. The first crystallization of a C-propeptide domain, that from human procollagen III, is described. Following transient expression in mammalian 293T cells of both the native protein and a selenomethionine derivative, two crystal forms of the homotrimer were obtained: an orthorhombic form (P2(1)2(1)2(1)) that diffracted to 1.7 Å resolution and a trigonal form (P321) that diffracted to 3.5 Å resolution. Characterization by MALDI-TOF mass spectrometry allowed the efficiency of selenomethionine incorporation to be determined.

To measure diagnostic performance in the detection of hepatocellular carcinoma (HCC) by using the most recent technology and multiphase gadolinium-enhanced magnetic resonance (MR) imaging and to compare with earlier results at the same institution.This retrospective study was institutional review board approved and HIPAA compliant. Informed consent was obtained. Between January 2008 and April 2010, 101 patients underwent liver transplantation and pretransplantation abdominal MR imaging within 90 days. Prospective image interpretations from the clinical record were reviewed for documentation of HCC, including size, number, and location. Liver explant histologic examination provided the reference standard for lesion analysis and was performed in axial gross slices in conjunction with the MR imaging report for direct comparison. Tumors were categorized according to size (≥ 2 cm or2 cm), and MR imaging detection sensitivity, specificity, predictive values, and accuracy were calculated according to category. The Fisher exact test was used to compare results from this study against prior reported results.Thirty-five (34.7%) of 101 patients had HCC at explant analysis. Patient-based analysis of all lesions showed a sensitivity and specificity of 97.1% (34 of 35) and 100% (66 of 66), respectively. For lesions 2 cm or larger, MR imaging had a sensitivity and specificity of 100% (23 of 23) and 100% (78 of 78), respectively. For lesions smaller than 2 cm, MR imaging had a sensitivity and specificity of 82.6% (19 of 23) and 100% (78 of 78), respectively. Lesion-based sensitivity for all tumors was 91.4% (53 of 58) in the current study, compared with 77.8% in 2007 (P = .07). For lesions smaller than 2 cm, the sensitivity was 87.5% (28 of 32) in the current study, compared with 55.6% previously (P = .02).MR imaging remains a highly accurate diagnostic method for the preoperative evaluation of HCC, and detection of small (2 cm) tumors has been significantly improved compared with that of earlier studies.

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.

Meprins α and β, a subgroup of zinc metalloproteinases belonging to the astacin family, are known to cleave components of the extracellular matrix, either during physiological remodeling or in pathological situations. In this study we present a new role for meprins in matrix assembly, namely the proteolytic processing of procollagens. Both meprins α and β release the N- and C-propeptides from procollagen III, with such processing events being critical steps in collagen fibril formation. In addition, both meprins cleave procollagen III at exactly the same site as the procollagen C-proteinases, including bone morphogenetic protein-1 (BMP-1) and other members of the tolloid proteinase family. Indeed, cleavage of procollagen III by meprins is more efficient than by BMP-1. In addition, unlike BMP-1, whose activity is stimulated by procollagen C-proteinase enhancer proteins (PCPEs), the activity of meprins on procollagen III is diminished by PCPE-1. Finally, following our earlier observations of meprin expression by human epidermal keratinocytes, meprin α is also shown to be expressed by human dermal fibroblasts. In the dermis of fibrotic skin (keloids), expression of meprin α increases and meprin β begins to be detected. Our study suggests that meprins could be important players in several remodeling processes involving collagen fiber deposition.

Objective To characterize the background and outcomes of tonsillectomy malpractice claims. Methods Review of 69 New York State insurance claims (Part I) and 87 national court trials (Part II) alleging injury after tonsillectomy. Results Part I. New York State insurance cases were most commonly discontinued (44%) or settled (42%) before trial. Compensations with a settlement or verdict were made in 48 percent of cases. Part II. Death or major injury occurred in 52 percent of insurance cases, with a mean award of $403,656 being made to plaintiffs. Of cases reaching trial, 60 percent of plaintiffs were compensated. Awards against anesthesiologists were more frequent and higher than against surgeons ($5 million vs $839,650). Death or major injury occurred in 52 percent of court cases, resulting in mean indemnity of $3.8 million. Most cases of death or major injury were attributable to airway complications. Conclusions Approximately half of both New York state claims and court cases involved death or devastating morbidity, mostly related to airway complications, resulting in large awards. Tonsillectomy is a source of uncommon but potentially high-dollar–value litigation exposure to the surgeon, often attributable to non-surgical complications.

Dialysis related amyloidosis (DRA) is a progressive and serious complication in patients under long-term hemodialysis and mainly leads to osteo-articular diseases. Although beta(2)-microglobulin (beta2-m) is the major structural component of beta2-m amyloid fibrils, the initiation of amyloid formation is not clearly understood. Here, we have identified procollagen C-proteinase enhancer-1 (PCPE-1) as a new interacting protein with beta2-m by screening a human synovium cDNA library. The interaction of beta2-m with full-length PCPE-1 was confirmed by immunoprecipitation, solid-phase binding and pull-down assays. By yeast two-hybrid analysis and pull-down assay, beta2-m appeared to interact with PCPE-1 via the NTR (netrin-like) domain and not via the CUB (C1r/C1s, Uegf and BMP-1) domain region. In synovial tissues derived from hemodialysis patients with DRA, beta2-m co-localized and formed a complex with PCPE-1. beta2-m did not alter the basal activity of bone morphogenetic protein-1/procollagen C-proteinase (BMP-1/PCP) nor BMP-1/PCP activity enhanced by PCPE-1. PCPE-1 did not stimulate beta2-m amyloid fibril formation from monomeric beta2-m in vitro under acidic and neutral conditions as revealed by thioflavin T fluorescence spectroscopy and electron microscopy. Since PCPE-1 is abundantly expressed in connective tissues rich in type I collagen, it may be involved in the initial accumulation of beta2-m in selected tissues such as tendon, synovium and bone. Furthermore, since such preferential deposition of beta2-m may be linked to subsequent beta2-m amyloid fibril formation, the disruption of the interaction between beta2-m and PCPE-1 may prevent beta2-m amyloid fibril formation and therefore PCPE-1 could be a new target for the treatment of DRA.

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.

International audience; Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that can stimulate the action of tolloid metalloproteinases, such as bone morphogenetic protein-1, on a procollagen substrate, by up to 20-fold. The PCPE molecule consists of two CUB domains followed by a C-terminal NTR (netrin-like) domain. In order to obtain structural insights into the function of PCPE, the recombinant protein was characterized by a range of biophysical techniques, including analytical ultracentrifugation, transmission electron microscopy, and small angle x-ray scattering. All three approaches showed PCPE to be a rod-like molecule, with a length of approximately 150 A. Homology modeling of both CUB domains and the NTR domain was consistent with the low-resolution structure of PCPE deduced from the small angle x-ray scattering data. Comparison with the low-resolution structure of the procollagen C-terminal region supports a recently proposed model (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) for the mechanism of action of PCPE.

Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that binds to the C-propeptide of procollagen I and can enhance the activities of procollagen C-proteinases up to 20-fold. To determine the molecular mechanism of PCPE activity, the interactions of the recombinant protein with the procollagen molecule as well as with its isolated C-propeptide domain were studied using surface plasmon resonance (BIAcore) technology. Binding required the presence of divalent metal cations such as calcium and manganese. By ligand blotting, calcium was found to bind to the C-propeptide domains of procollagens I and III but not to PCPE. By chemical cross-linking, the stoichiometry of the PCPE/C-propeptide interaction was found to be 1:1 in accordance with enzyme kinetic data. The use of a monoclonal antibody directed against the N-terminal region of the C-propeptide suggested that this region is probably not involved in binding to PCPE. Association and dissociation kinetics of the C-propeptide domains of procollagens I and III on immobilized PCPE were rapid. Extrapolation to saturation equilibrium yielded apparent equilibrium dissociation constants in the range 150-400 nM. In contrast, the association/dissociation kinetics of intact procollagen molecules on immobilized PCPE were relatively slow, corresponding to a dissociation constant of 1 nM. Finally, pN-collagen (i.e. procollagen devoid of the C-terminal propeptide domain) was also found to bind to immobilized PCPE, suggesting that PCPE binds to sites on either side of the procollagen cleavage site, thereby facilitating the action of procollagen C-proteinases.Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that binds to the C-propeptide of procollagen I and can enhance the activities of procollagen C-proteinases up to 20-fold. To determine the molecular mechanism of PCPE activity, the interactions of the recombinant protein with the procollagen molecule as well as with its isolated C-propeptide domain were studied using surface plasmon resonance (BIAcore) technology. Binding required the presence of divalent metal cations such as calcium and manganese. By ligand blotting, calcium was found to bind to the C-propeptide domains of procollagens I and III but not to PCPE. By chemical cross-linking, the stoichiometry of the PCPE/C-propeptide interaction was found to be 1:1 in accordance with enzyme kinetic data. The use of a monoclonal antibody directed against the N-terminal region of the C-propeptide suggested that this region is probably not involved in binding to PCPE. Association and dissociation kinetics of the C-propeptide domains of procollagens I and III on immobilized PCPE were rapid. Extrapolation to saturation equilibrium yielded apparent equilibrium dissociation constants in the range 150-400 nM. In contrast, the association/dissociation kinetics of intact procollagen molecules on immobilized PCPE were relatively slow, corresponding to a dissociation constant of 1 nM. Finally, pN-collagen (i.e. procollagen devoid of the C-terminal propeptide domain) was also found to bind to immobilized PCPE, suggesting that PCPE binds to sites on either side of the procollagen cleavage site, thereby facilitating the action of procollagen C-proteinases.

Procollagen C-propeptide domains direct chain association during intracellular assembly of procollagen molecules. In addition, they control collagen solubility during extracellular proteolytic processing and fibril formation and interact with cell surface receptors and extracellular matrix components involved in feedback inhibition, mineralization, cell growth arrest, and chemotaxis. At present, three-dimensional structural information for the C-propeptides, which would help to understand the underlying molecular mechanisms, is lacking. Here we have carried out a biophysical study of the recombinant C-propeptide trimer from human procollagen III using laser light scattering, analytical ultracentrifugation, and small angle x-ray scattering. The results show that the trimer is an elongated molecule, which by modeling of the x-ray scattering data appears to be cruciform in shape with three large lobes and one minor lobe. We speculate that each of the major lobes corresponds to one of the three component polypeptide chains, which come together in a junction region to connect to the rest of the procollagen molecule.Procollagen C-propeptide domains direct chain association during intracellular assembly of procollagen molecules. In addition, they control collagen solubility during extracellular proteolytic processing and fibril formation and interact with cell surface receptors and extracellular matrix components involved in feedback inhibition, mineralization, cell growth arrest, and chemotaxis. At present, three-dimensional structural information for the C-propeptides, which would help to understand the underlying molecular mechanisms, is lacking. Here we have carried out a biophysical study of the recombinant C-propeptide trimer from human procollagen III using laser light scattering, analytical ultracentrifugation, and small angle x-ray scattering. The results show that the trimer is an elongated molecule, which by modeling of the x-ray scattering data appears to be cruciform in shape with three large lobes and one minor lobe. We speculate that each of the major lobes corresponds to one of the three component polypeptide chains, which come together in a junction region to connect to the rest of the procollagen molecule.

Naturally occurring segment-long-spacing crystallites of procollagen collagen have been found in the culture medium of fibroblasts from chick embryo tendon, human skin, and dermatosparaxic calf skin; in whole-mount preparations of cultured human skin fibroblasts; in homogenates of lathyritic chick embryo tendon, cartilage, and cornea; and in a partially purified preparation of procollagen. Bundles of similar aggregates occurred within secretory vacuoles of collagen-synthesizing fibroblasts and chondrocytes. These observations suggest that fibroblasts and chondrocytes secrete procollagen assemblies that are stable in the extracellular environment. We propose that subsequent enzymatic processing is accompanied by direct incorporation of such structures into the assembling fibril, which then may be considered as an n X 67 nm staggered array of segment-long-spacing crystallites.

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.

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.

A cross-sectional analytic survey of 92 patients permitted to attempt vaginal delivery after previous lower segment cesarean section ("trial of scar") is reported. Variables which may predict mode of delivery were assessed. Fifty patients (54.3%) were delivered vaginally; 42 patients (45.7%) had repeat cesarean sections in labor. There were three cases of scar dehiscence (3.2%). There were no maternal or fetal mortality. When the cervix was less than 3 cm dilated at initial examination in labor, 10 of 37 patients (27%) were delivered vaginally, compared to 38 of 55 patients (69%) who were delivered vaginally when the cervix was greater than 3 cm dilated. Assessment of cervical dilatation on admission in labor proved to be the most significant prognostic factor at the onset of labor, with regard to successful vaginal delivery in a patient with a lower segment cesarean section scar.

The perinatal mortality rate among very low-birth weight infants has been decreased by 20% during the last 4 years of the 1973 to 1980 period here reported. The concurrent increase in the cesarean section rate from 11.9% to 49.1% during the same time frames has been assumed to be responsible for the improved outcome. The changes were most marked in the extremely low-birth weight group (less than 1,000 gm). The survival rates and cesarean section rates were examined among infants of similar birth weight and gestational age in the vertex presentation, in the same time frames. A similar or greater reduction in mortality rate (from 85% to 45%) was noted in the very low-birth weight vertex infants, whereas the cesarean section rate remained minimally and not significantly increased (14.2% to 22.2%). The interpretation of this finding is by no means clear but must include the hypothesis that the increased cesarean section rate may be incidental and in no way related to the improved outcome. The most statistically significant determinants of outcome remain birth weight and gestational age strata, with no significant difference in outcomes when the extremely low-birth weight group is analyzed separately from the entire very low-birth weight group. As yet unidentified perinatal care practices, other than cesarean section, may be more likely to affect outcome in this high-risk group.

International audience; The assembly of type I collagen and type I pN-collagen was studied in vitro using a system for generating these molecules enzymatically from their immediate biosynthetic precursors. Collagen generated by C-proteinase digestion of pC-collagen formed D-periodically banded fibrils that were essentially cylindrical (i.e. circular in cross-section). In contrast, pN-collagen generated by C-proteinase digestion of procollagen formed thin, sheet-like structures that were axially D-periodic in longitudinal section, of varying lateral widths (up to several microns) and uniform in thickness (approximately 8 nm). Mixtures of collagen and pN-collagen assembled to form a variety of pleomorphic fibrils. With increasing pN-collagen content, fibril cross-sections were progressively distorted from circular to lobulated to thin and branched structures. Some of these structures were similar to fibrils observed in certain heritable disorders of connective tissue where N-terminal procollagen processing is defective. The observations are considered in terms of the hypothesis that the N-propeptides are preferentially located on the surface of a growing assembly. The implications for normal diameter control of collagen fibrils in vivo are discussed.The assembly of type I collagen and type I pN-collagen was studied in vitro using a system for generating these molecules enzymatically from their immediate biosynthetic precursors. Collagen generated by C-proteinase digestion of pC-collagen formed D-periodically banded fibrils that were essentially cylindrical (i.e. circular in cross-section). In contrast, pN-collagen generated by C-proteinase digestion of procollagen formed thin, sheet-like structures that were axially D-periodic in longitudinal section, of varying lateral widths (up to several microns) and uniform in thickness (approximately 8 nm). Mixtures of collagen and pN-collagen assembled to form a variety of pleomorphic fibrils. With increasing pN-collagen content, fibril cross-sections were progressively distorted from circular to lobulated to thin and branched structures. Some of these structures were similar to fibrils observed in certain heritable disorders of connective tissue where N-terminal procollagen processing is defective. The observations are considered in terms of the hypothesis that the N-propeptides are preferentially located on the surface of a growing assembly. The implications for normal diameter control of collagen fibrils in vivo are discussed.

A descriptive study of 300 consecutive spontaneous labors in primigravid patients whose pregnancies were of 37 or more weeks' gestation with a singleton fetus in the vertex presentation, showed a cesarean section rate of 13%, a forceps delivery rate of 49%, and a spontaneous delivery rate of 38%. Oxytocin was used in 17% and epidural analgesia was used in 75% of the patients. The median rate for cervical dilatation for those women with spontaneous deliveries was 2 cm/hr (interquartile range = 1.5 to 3.3 cm/hr) and for those delivered with forceps, 1.2 cm/hr (interquartile range = 0.9 to 1.8 cm/hr). When labor was prolonged by 4 hours or more, the cesarean section rate rose to 34%. Oxytocin was used in only 41% of these patients. Of 23 women delivered by cesarean section for dystocia/disproportion, only nine received oxytocin. From the low incidence of low Apgar scores in all labor groups from this series, there would not appear to be a fetal advantage to earlier intervention. Although the suggestion from this study is that oxytocin administration when labor is prolonged by 4 hours will reduce the need for cesarean section, the true value of such an intervention can be tested only by a randomized controlled trial.