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Contemporary scientific instruments, synchrotrons for example, which are powerful sources of X-rays, reveal a wealth of amazing shapes of matter, invisible to the naked eye. We have thus discovered the remarkable aesthetic properties of the structures of the macromolecules which make up living beings. Analyses of fossilised organisms, carried out in the very matrix which preserved them, allow their morphology to be determined down to the smallest detail. The high-quality visualisation of the very inside of artefacts and objets d'art produced by man, reveals the secrets of their fabrication and helps us better understand and preserve them., Résumé : Les instruments scientifiques contemporains, comme les synchrotrons, sources puissantes de rayons-X, révèlent de nombreuses formes admirables de la matière, invisibles à l'œil nu. Nous avons ainsi découvert les propriétés esthétiques remarquables des structures des macromolécules constitutives des êtres vivants. Les analyses des organismes fossilisés, effectuées dans la matrice même qui les a conservés, permettent de déterminer leur morphologie dans les moindres détails. La visualisation, sans dégradation, de l'intérieur même des artefacts et objets d'art produits par l'homme, révèle les secrets de leur fabrication et nous aide à mieux les connaître et les conserver., Van der Rest Michel. Les formes au-delà du visible. In: Les Cahiers du Musée des Confluences. Revue thématique Sciences et Sociétés du Musée des Confluences, tome 10, 2013. Formes. pp. 27-37.

A careful selection of the probiotic agent, standardization of the dose and detailed characterization of the beneficial effects are essential when considering use of a probiotic for the dietary management of serious diseases. However, changes in the manufacturing processes, equipment or facilities can result in differences in the product itself due to the live nature of probiotics. The need to reconfirm safety and/or efficacy for any probiotic product made at a different factory is therefore mandatory. Recently, under the brand VSL#3®, a formulation produced by a manufacturer different from the previous one, has been commercialized in some European countries (the UK and Holland). VSL#3 is a high concentration multi-strain preparation which has been recognized by the main Gastroenterology Associations for the dietary management of pouchitis as well as ulcerative colitis. We have compared the "original" VSL#3 produced in USA with the "newfound" VSL#3 produced in Italy. According to our results, the "newfound" VSL#3 has 130-150% more "dead bacteria" compared to the "original" product, raising concerns for the well-known association between dead microbes with adverse effects. The abilities of bacterial lysates from the two formulations to influence in vitro viability and proliferation of different tumor cell lines also resulted different. The repair of previously scratched monolayers of various adherent tumor cell lines (i.e. HT1080, and Caco-2 cells) was inhibited more significantly by the "original" VSL#3 when compared to the "newfound" VSL#3. Tumor cell cycle profile, in particular cell cycle arrest and apoptotic death of the cancer cells, further confirms that the "original" VSL#3 has a better functional profile than the "newfound" VSL#3, at least in in vitro. Our data stress the importance of the production conditions for the "newfound" VSL#3 considering that this product is intended to be used for the dietary management of patients with very serious diseases, such as chronic inflammatory bowel diseases.

Endostatin (20 kDa) is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds zinc, heparin/heparan sulfate, laminin, and sulfatides and inhibits angiogenesis and tumor growth. Here we determined the kinetics and affinity of the interaction of endostatin with heparin/heparan sulfate and investigated the effects of divalent cations on these interactions and on the biological activities of endostatin. The binding of human recombinant endostatin to heparin and heparan sulfate was studied by surface plasmon resonance using BIAcore technology and further characterized by docking and molecular dynamics simulations. Kinetic data, evaluated using a 1:1 interaction model, showed that heparan sulfate bound to and dissociated from endostatin faster than heparin and that endostatin bound to heparin and heparan sulfate with a moderate affinity (K(D) approximately 2 microm). Molecular modeling of the complex between endostatin and heparin oligosaccharides predicted that, compared with mutagenesis studies, two further arginine residues, Arg(47) and Arg(66), participated in the binding. The binding of endostatin to heparin and heparan sulfate required the presence of divalent cations. The addition of ZnCl(2) to endostatin enhanced its binding to heparan sulfate by approximately 40% as well as its antiproliferative effect on endothelial cells stimulated by fibroblast growth factor-2, suggesting that this activity is mediated by the binding of endostatin to heparan sulfate. In contrast, no increase in the antiangiogenic and anti-proliferative activities of endostatin promoted by vascular endothelial growth factor was observed upon the addition of zinc.

Chicken alpha1(V) collagen cDNAs have been cloned by a variety of methods and positively identified. We present here the entire translated sequence of the chick polypeptide and compare selected regions to other collagen chains in the type V/XI family.

It is generally accepted that the folding of collagen triple helical domains occur from the C-terminus toward the N-terminus by a “zipper” mechanism. The regions at the C-terminus of the triple helices must therefore play a critical role in the processes of chain recognition and assembly to get the proper stoichiometries and of chain registration to align the chains for the folding of the triple helix. Examination of these regions reveals a broad diversity of structures and suggests that different mechanisms of assembly are used in the various collagen and collagen-like molecules. We review here three different mechanisms that have recently come to light. The collectins, a group of serum proteins containing collagen-like triple helical domains, are assembled through hydrophobic interactions in a tripleα helix. Collagens VIII and X, C1q and several related proteins contain homologous C-terminal domains that are characterized by aβ-pleated sheet structure. They assemble through very strong hydrophobic interactions that probably involve an “aromatic zipper”. Collagens IX, XII and XIV fibril associated collagen with interrupted triple helices (FACITs), are assembled by a mechanism in which both the C-terminal triple helix and a very short cysteine-containing sequence are involved.

We have shown previously that hydroxylation played a critical role in the trimer assembly and disulfide bonding of the three constituent α chains of a minicollagen composed of the extreme C-terminal collagenous (COL1) and noncollagenous (NC1) domains of type XII collagen in HeLa cells (Mazzorana, M., Gruffat, H., Sergeant, A., and van der Rest, M. (1993) J. Biol. Chem. 268, 3029-3032). We have further characterized the involvement of prolyl 4-hydroxylase in the assembly of the three α chains to form trimeric disulfide-bonded type XII minicollagen in an insect cell expression system. For this purpose, type XII minicollagen was produced in insect cells from baculovirus vectors, alone or together with wild-type human prolyl 4-hydroxylase or with the human enzyme mutated in the catalytic site of its α or β subunits or with the individual α or β subunits. When type XII minicollagen was produced alone, negligible amounts of disulfide-bonded trimers were found to be produced by the cells. However, coproduction of the collagen with the two subunits of the wild-type human enzyme dramatically increased the amount of disulfide-bonded trimeric type XII minicollagen molecules. In contrast, coproduction of the collagen with α subunits that had a mutation completely inactivating the human enzyme failed to enhance the trimer assembly. These results directly show that an active prolyl 4-hydroxylase is required for the assembly of disulfide-bonded trimers of type XII minicollagen.

Type XII and type XIV collagens are closely related homotrimeric and disulfide-bonded extracellular matrix constituents that contain two short triple helical domains near the C -terminus and a very large modular non-triple helical domain at the N -terminus. This domain contains multiple fibronectin type III repeats, von Willebrand factor A-like modules and a module homologous to the N-terminal region of thrombospondin. When visualized by rotary shadowing electron microscopy they have a cross shape with a thin collagenous tail, 75 nm in length, connected to a central globule from which three arms are sticking out (from 60 to 90 nm depending on the collagen type and the splicing variant). Collagens XII and XIV have been localized around collagen I-containing fibrils in dense connective tissues. Despite the presence in their primary structure of modules and sequences characteristic of adhesive glycoproteins, these molecules do not appear to have cell adhesion activity. They appear rather to play a role in modulating the biomechanical properties of collagen-containing tissues by mediating deformability of fibril bundles. Collagen XIV also serves as a ligand to procollagen N -proteinase and may contribute in this way to sequester this activity around the collagen fibrils.

Type XI collagen is mainly found as a minor constituent in type II-containing fibrils and presents a α1(XI)α2(XI)α3(XI) stoichiometry. This molecule was shown to be partially processed in its intact tissue form. Moreover, alternative splicing has been demonstrated in the variable region of the N-terminal domain of α1(XI) and α2(XI) chains. In this work, the processing of a major intact form of α1(XI) from matrix laid down by chick chondrocytes in culture was identified using N-terminal sequencing and antibodies to synthetic peptides corresponding to the N-terminal propeptide cDNA-derived sequence. The results show that the fully processed form of α1(XI) begins at Gln254 of the N-terminal propeptide, seven residues before the end of the proline/arginine-rich protein region encoded by exon I (Zhidkova, N. I., Justice, S. K., and Mayne, R. (1995) J. Biol. Chem. 270, 9486-9493). This sequence is immediately followed by a sequence encoded by exon III. The processing takes place at an Ala-Gln sequence that corresponds to a consensus sequence for procollagen N-proteinase. The antibody raised against a sequence located within the region corresponding to exon IV (anti-P8) fails to recognize this fully processed form of the α1(XI) chain. It recognizes, however, two minor bands of high molecular mass. These results suggest that a major cartilage form of α1(XI) is the product of alternative splicing in which sequences encoded by both exons II and IV are skipped. The presence of a highly acidic subdomain encoded by exon III at the N terminus of the major form of the α1(XI) chain, as predicted by these data, provides potential sites for interaction of collagen XI with other molecules.

The 3D structure of the COL1-NC1 junction of FACIT type XIV collagen was investigated using GYCDPSSCAG and (GPP*)3GYCDPSSCAG synthetic peptides, circular dichroism, and NMR. At -20 degrees C and under air oxidation catalyzed by Cu2+, the peptide (GPP*)3GYCDPSSCAG is able to self-associated with high yield into a stable triple disulfide bonded trimer. The presence of a triple helical conformation was confirmed by circular dichroism. The analysis of the trimer by 2D NMR provided a set of distance constraints for the noncollagenous part. Molecular models for the 3D structure of COL1-NC1 junction were calculated, using the NMR distance constraints in combination with the 3D structural data recently established by X-ray crystallography [Bella, J., Eaton, M., Brodsky, B., & Berman, H. M. (1994) Science 266, 75-81] for a collagenous triple helix. From the eight theoretically possible arrangements for the three interchain disulfide bonds, only two close disulfide conformers are compatible with the experimental data. The main feature of the trimer structure is the asymmetry of the molecule due to the disulfide bond pattern that induces a particular folding of one chain. This chain forms a turn-like structure locked by two disulfide bonds with the two other chains. The turn-like folding is close to that observed for the cyclized oxidized monomeric peptide. This is the first report of the 3D structure model for a junction between a collagenous triple helical domain and a noncollagenous domain.The 3D structure of the COL1-NC1 junction of FACIT type XIV collagen was investigated using GYCDPSSCAG and (GPP*)3GYCDPSSCAG synthetic peptides, circular dichroism, and NMR. At -20 degrees C and under air oxidation catalyzed by Cu2+, the peptide (GPP*)3GYCDPSSCAG is able to self-associated with high yield into a stable triple disulfide bonded trimer. The presence of a triple helical conformation was confirmed by circular dichroism. The analysis of the trimer by 2D NMR provided a set of distance constraints for the noncollagenous part. Molecular models for the 3D structure of COL1-NC1 junction were calculated, using the NMR distance constraints in combination with the 3D structural data recently established by X-ray crystallography [Bella, J., Eaton, M., Brodsky, B., & Berman, H. M. (1994) Science 266, 75-81] for a collagenous triple helix. From the eight theoretically possible arrangements for the three interchain disulfide bonds, only two close disulfide conformers are compatible with the experimental data. The main feature of the trimer structure is the asymmetry of the molecule due to the disulfide bond pattern that induces a particular folding of one chain. This chain forms a turn-like structure locked by two disulfide bonds with the two other chains. The turn-like folding is close to that observed for the cyclized oxidized monomeric peptide. This is the first report of the 3D structure model for a junction between a collagenous triple helical domain and a noncollagenous domain.

The fibril-associated-collagens-with-interrupted-triple-helices (FACITs) are devoid of large C-propeptides like those involved in the trimeric assembly of the fibrillar collagens. Under these conditions, the C-terminal non triple-helical domain (NC1) and the adjacent triple-helical domain (COL1) are likely to be responsible for the trimeric assembly of these collagen molecules. Using a recombinant minigene of one of the FACITs, collagen XII, we show that a deletion covering most of the NC1 domain, except the first seven residues containing a cysteine and constituting the main part of the conserved junction between the COL1 and NC1 domains, does not prevent the formation of trimeric disulfide-bonded assembly of truncated α chains. These results suggest that if the non triple-helical NC1 domain is involved in the initial events governing the trimeric assembly, it must be through its amino acid residues participating in the junction. Our data confirm also the results obtained in a previous paper (Mazzorana et al.: J. Biol. Chem. 268: 3029–3032, 1993) showing that the formation of disulfide bonds is dependent on hydroxylation and suggesting that the folding of the triple helix (or a part of it) precedes the formation of the disulfide bonds.

Laminin-5 (kalinin) was purified from spent cell culture media (SCC25 cells) by affinity chromatography on monoclonal antibody BM165. The protein was recovered as a mixture of the typical polypeptides of 165-155, 140, and 105 kDa as judged b SDS-polyacrylamide gel electrophoresis analysis under reducing conditions. The amino acid composition of purified laminin-5 was in agreement with that compiled from the recently published cDNA sequences of the alpha 3-, beta 3-, and gamma 2-laminin chains. Moreover, the content of half-cystine residues in laminin-5 was about two-thirds that in laminin-1, which confirms the prediction of a smaller number of epidermal growth factor-like repeats in the amino-terminal portion of the three chains. The content of coiled-coil alpha-helices (27%) determined by CD spectroscopy was comparable to that reported for laminin-1, which indicates that the long arm portion of laminin-5 is equivalent to that of other laminin isoforms. The melting temperature was recorded at 72 degrees C by CD monitoring of unfolding and refolding of the coiled-coil structures during thermal denaturation and renaturation, respectively. The thermal stability of laminin-5 is therefore significantly higher than that of laminin-1 or alpha 2-chain-containing laminins, which suggests higher ionic interactions between the three polypeptide chains of laminin-5. Cell adhesion-promoting activity of laminin-5 was found to be strictly and entirely dependent on the presence of coiled-coil structures. It decreased gradually after heat denaturation of the protein above 65 degrees C and was totally abrogated at 75 degrees C. This is in contrast to laminin-1, which contains both conformation-dependent and -independent cell-binding sites on the long and short arm domains, respectively.Laminin-5 (kalinin) was purified from spent cell culture media (SCC25 cells) by affinity chromatography on monoclonal antibody BM165. The protein was recovered as a mixture of the typical polypeptides of 165-155, 140, and 105 kDa as judged b SDS-polyacrylamide gel electrophoresis analysis under reducing conditions. The amino acid composition of purified laminin-5 was in agreement with that compiled from the recently published cDNA sequences of the alpha 3-, beta 3-, and gamma 2-laminin chains. Moreover, the content of half-cystine residues in laminin-5 was about two-thirds that in laminin-1, which confirms the prediction of a smaller number of epidermal growth factor-like repeats in the amino-terminal portion of the three chains. The content of coiled-coil alpha-helices (27%) determined by CD spectroscopy was comparable to that reported for laminin-1, which indicates that the long arm portion of laminin-5 is equivalent to that of other laminin isoforms. The melting temperature was recorded at 72 degrees C by CD monitoring of unfolding and refolding of the coiled-coil structures during thermal denaturation and renaturation, respectively. The thermal stability of laminin-5 is therefore significantly higher than that of laminin-1 or alpha 2-chain-containing laminins, which suggests higher ionic interactions between the three polypeptide chains of laminin-5. Cell adhesion-promoting activity of laminin-5 was found to be strictly and entirely dependent on the presence of coiled-coil structures. It decreased gradually after heat denaturation of the protein above 65 degrees C and was totally abrogated at 75 degrees C. This is in contrast to laminin-1, which contains both conformation-dependent and -independent cell-binding sites on the long and short arm domains, respectively.

Ehlers-Danlos syndrome type IV (EDS IV) is an autosomal dominant disorder characterized by fragile skin, blood vessels, and internal organs and associated with decreased production, secretion, or thermal stability of type III procollagen. Mutations in the gene for type III procollagen have been identified in patients exhibiting decreased secretion or thermal stability of the protein, but no defect has been elucidated to explain the decreased production of type III procollagen in some patients with EDS IV. We report on a patient with a moderate case of EDS IV who produced decreased amounts of type III procollagen despite normal levels of translatable type III procollagen mRNA. SI nuclease analysis of the type III procollagen mRNA indicated a defect in the region encoding exon 27. Sequence analysis of cDNA clones and genomic fragments generated by polymerase chpin reaction amplification revealed that sequences encoded by exon 27 were absent from 3 out of 5 cDNA clones and that a G at the +5 position of the splce donor site in intron 27 was changed to an A in one allcle of the patient's type III procollagen gene. Using a cDNA genomic DNA hybrid probe in Si nuclease analysis, fragments consistent with mRNA species containing and lacking exon 27 were detected in a 1:1 ratio. Pulse label and chase experiments in the presence or absenci: of brefeldin A indicated that most of the type III procollagen molecules synthesized by the patient'; fibroblasts were not secreted into the medium but were degraded in the endoplasmic reticulum-Golgi compartment by a nonlysosomal mechanism. © 1995 Wiley-Liss, Inc.

The secondary structure of a conserved non-collagenous module in alpha 1(V), alpha 1(XI), alpha 1(IX), alpha 1(XII), alpha 1(XIV) and alpha 1(XVI) collagen chains and in proline- and arginine-rich protein was analyzed using different algorithms. The results predict that a common anti-parallel beta-sheet structure composed of nine consensus beta-strands is present in these non-collagenous modules. A model for the packing of these beta-sheets is proposed which suggests that the predicted beta-sheet structure may be involved in molecular recognition functions.

The picture emerging from recent discoveries about collagens, other matrix macromolecules and their modes of organization is one of a complex modular system operating at all levels, molecular, supramolecular and whole tissue. This complexity provides multiple opportunities for interactions between structurally similar and dissimilar components. The analysis of these interactions will eventually help us to understand the regulatory principles underlying the morphogenesis of extracellular matrices.

It is generally accepted that the folding of collagen triple helical domains occur from the C-terminus toward the N-terminus by a “zipper” mechanism. The regions at the C-terminus of the triple helices must therefore play a critical role in the processes of chain recognition and assembly to get the proper stoichiometries and of chain registration to align the chains for the folding of the triple helix. Examination of these regions reveals a broad diversity of structures and suggests that different mechanisms of assembly are used in the various collagen and collagen-like molecules. We review here three different mechanisms that have recently come to light. The collectins, a group of serum proteins containing collagen-like triple helical domains, are assembled through hydrophobic interactions in a tripleα helix. Collagens VIII and X, C1q and several related proteins contain homologous C-terminal domains that are characterized by aβ-pleated sheet structure. They assemble through very strong hydrophobic interactions that probably involve an “aromatic zipper”. Collagens IX, XII and XIV fibril associated collagen with interrupted triple helices (FACITs), are assembled by a mechanism in which both the C-terminal triple helix and a very short cysteine-containing sequence are involved.

The biosynthesis of collagen by the A204 cell line was examined using polyclonal antibodies raised against collagen type V and type XI. The study of the pepsin-digested collagen showed that it is composed mainly of alpha 1(XI) and alpha 2(V) collagen chains in an apparent 2:1 ratio, suggesting the formation of heterotypic molecules [alpha 1(XI)]2 alpha 2(V). The existence of this chain stoichiometry was further demonstrated by immunoprecipitation of the molecule with an antibody recognizing alpha 2(V) but not alpha 1(XI) collagen chains. Electron microscopy analyses of 24-h cultures showed that this matrix is composed of thin fibrils, that can be decorated with immunogold-labelled anti-(type-V collagen) IgG, but not with anti-(type-XI collagen) IgG. The collagen matrix laid down by A204 cells is highly insoluble. In the presence of beta-aminopropionitrile, an inhibitor of lysyl oxidase, only a small proportion of intact collagen could be extracted without proteolytic treatment. Immunoblotting of intact medium collagen from cultures performed in the presence of beta-aminopropionitrile showed four distinct bands with each antibody. The migration of the bands, stained with anti-(type-V collagen) IgG, had apparent molecular masses of 127, 149, 161 and 198 kDa (compared to globular standards) while the bands stained with anti-(type-XI collagen) IgG had apparent masses of 145, 182, 207 and 225 kDa. These data indicate that type-V and type-XI collagen chains can assemble in heterotypic isoforms. In this system, the synthesized isoforms are able to aggregate into a highly cohesive matrix and they undergo a proteolytic processing closely similar to that of other fibrillar collagens.

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.

Human collagen X contains a highly conserved 161-amino acid C-terminal non-triple helical domain that is homologous to the C-terminal domain of collagen VIII and to the C1q module of the human C1 enzyme. We have expressed this domain (residues 545-680) in Escherichia coli as a glutathione S-transferase fusion protein. The purified fusion protein trimerizes spontaneously in vitro, and after thrombin cleavage, the purified C-terminal domain trimer (46.2 kDa) is extremely stable and trypsin-resistant. Mutations within the C-terminal domain have been observed in patients with Schmid's metaphyseal chondrodysplasia (SMCD). Some of these mutations (Y598D, G618V, W651X, or H669X; X is the stop codon) were constructed by site-directed mutagenesis. Each mutation had identical consequences regarding the fusion protein: 1) absence of trimeric formation, 2) copurification of the approximately 60-kDa GroEL chaperone protein, and 3) sensitivity of the monomeric fusion protein to trypsin digestion. These results show that the C-terminal domain of collagen X is sufficient to produce a very stable and compact trimer in the absence of collagen Gly-X-Y repeats. Moreover, mutations causing SMCD interfere in this system with the correct folding of the C-terminal domain. The existence of a similar mechanism in chondrocytes might explain the relative homogeneity of phenotypes in SMCD despite the diversity of mutations.

Collagen XI is found mainly as a component of cartilage fibrils. Among the different transcripts identified by RT-PCR for the alpha1 (XI) chain, the major tissue form has been reported to be the splicing product of exons I, III and V. In this study, two other splice isoforms of the alpha1(XI) chain were identified using N-terminal sequencing. Like the major alpha1(XI) chain, the fully processed isoforms begin at Gln254 within the N-terminal domain encoded by exon I. This sequence is followed by sequences encoded by exon IIA or III. An anti-peptide antibody allowed the identification of the exon IV encoded sequence within both isoforms. Therefore, these isoforms of the alpha1(XI) chain correspond to the splicing of exons I, IIA, III, IV and V or of exons I, III, IV and V, thus presenting larger acidic sequences than the major form. They could mediate strong ionic interactions within the cartilage matrix.

International audience; Collagens XII and XIV localize near the surface of collagen fibrils and may be involved in epithelial-mesenchymal interactions as well as in the modulation of tissue biomechanical properties. Moreover, human skin fibroblasts cultured in monolayer are known to lose their ability to produce collagen XIV and to switch the transcription of collagen XII from the small splice variant (220 kDa) to the large (320 kDa), whereas the small form is the main form found in human skin. We have investigated the expression patterns of these two molecules in human skin as a function of donor age and anatomic site, by using immunohistology with specific monoclonal antibodies. We demonstrated changes in the expression patterns of collagens XII and XIV in human skin after birth. Moreover, in adult scalp skin, very strong staining of collagen XII fibril bundles was observed around hair follicles, in association with very low expression of collagen XIV. We also investigated the expression of collagens XII and XIV by fibroblasts and keratinocytes cultured in a reconstructed skin. In these culture conditions, fibroblasts recovered their ability to produce collagen XIV and re-expressed the small splice variant of collagen XII. These results could be explained by the deposition of large amounts of collagen fibrils by fibroblasts in this culture system. Thus, the re-expression of these collagens suggests that the deposition of banded collagen fibrils is a pre-requisite for the expression of collagen XIV and small variant of collagen XII.Collagens XII and XIV localize near the surface of collagen fibrils and may be involved in epithelial-mesenchymal interactions as well as in the modulation of tissue biomechanical properties. Moreover, human skin fibroblasts cultured in monolayer are known to lose their ability to produce collagen XIV and to switch the transcription of collagen XII from the small splice variant (220 kDa) to the large (320 kDa), whereas the small form is the main form found in human skin. We have investigated the expression patterns of these two molecules in human skin as a function of donor age and anatomic site, by using immunohistology with specific monoclonal antibodies. We demonstrated changes in the expression patterns of collagens XII and XIV in human skin after birth. Moreover, in adult scalp skin, very strong staining of collagen XII fibril bundles was observed around hair follicles, in association with very low expression of collagen XIV. We also investigated the expression of collagens XII and XIV by fibroblasts and keratinocytes cultured in a reconstructed skin. In these culture conditions, fibroblasts recovered their ability to produce collagen XIV and re-expressed the small splice variant of collagen XII. These results could be explained by the deposition of large amounts of collagen fibrils by fibroblasts in this culture system. Thus, the re-expression of these collagens suggests that the deposition of banded collagen fibrils is a pre-requisite for the expression of collagen XIV and small variant of collagen XII.

International audience; Mutational analyses of genes that encode components of the anchoring complex underlying the basolateral surface of external epithelia indicate that this structure is the major element providing for resistance to external friction. Ultrastructurally, laminin 5 (alpha3beta3gamma2; a component of the anchoring filament) appears as a thin filament bridging the hemidesmosome with the anchoring fibrils. Laminin 5 binds the cell surface through hemidesmosomal integrin alpha6beta4. However, the interaction of laminin 5 with the anchoring fibril (type VII collagen) has not been elucidated. In this study we demonstrate that monomeric laminin 5 binds the NH2-terminal NC-1 domain of type VII collagen. The binding is dependent upon the native conformation of both laminin 5 and type VII collagen NC-1. Laminin 6 (alpha3beta1gamma1) has no detectable affinity for type VII collagen NC-1, indicating that the binding is mediated by the beta3 and/or gamma2 chains of laminin 5. Approximately half of the laminin 5 solubilized from human amnion or skin is covalently complexed with laminins 6 or 7 (alpha3beta2gamma1). The adduction occurs between the NH2 terminus of laminin 5 and the branch point of the short arms of laminins 6 or 7. The results are consistent with the presumed orientation of laminin 5, having the COOH-terminal G domain apposed to the hemidesmosomal integrin, and the NH2-terminal domains within the lamina densa. The results also support a model predicting that monomeric laminin 5 constitutes the anchoring filaments and bridges integrin alpha6beta4 with type VII collagen, and the laminin 5-6/7 complexes are present within the interhemidesmosomal spaces bound at least by integrin alpha3beta1 where they may mediate basement membrane assembly or stability, but contribute less significantly to epithelial friction resistance.Mutational analyses of genes that encode components of the anchoring complex underlying the basolateral surface of external epithelia indicate that this structure is the major element providing for resistance to external friction. Ultrastructurally, laminin 5 (alpha3beta3gamma2; a component of the anchoring filament) appears as a thin filament bridging the hemidesmosome with the anchoring fibrils. Laminin 5 binds the cell surface through hemidesmosomal integrin alpha6beta4. However, the interaction of laminin 5 with the anchoring fibril (type VII collagen) has not been elucidated. In this study we demonstrate that monomeric laminin 5 binds the NH2-terminal NC-1 domain of type VII collagen. The binding is dependent upon the native conformation of both laminin 5 and type VII collagen NC-1. Laminin 6 (alpha3beta1gamma1) has no detectable affinity for type VII collagen NC-1, indicating that the binding is mediated by the beta3 and/or gamma2 chains of laminin 5. Approximately half of the laminin 5 solubilized from human amnion or skin is covalently complexed with laminins 6 or 7 (alpha3beta2gamma1). The adduction occurs between the NH2 terminus of laminin 5 and the branch point of the short arms of laminins 6 or 7. The results are consistent with the presumed orientation of laminin 5, having the COOH-terminal G domain apposed to the hemidesmosomal integrin, and the NH2-terminal domains within the lamina densa. The results also support a model predicting that monomeric laminin 5 constitutes the anchoring filaments and bridges integrin alpha6beta4 with type VII collagen, and the laminin 5-6/7 complexes are present within the interhemidesmosomal spaces bound at least by integrin alpha3beta1 where they may mediate basement membrane assembly or stability, but contribute less significantly to epithelial friction resistance.

Type XIV collagen is a newly described member of the fibril-associated collagens with interrupted triple helices (FACITs). Expression of this collagen has been localized to various embryonic tissues, suggesting that it has a functional role in development. All FACITs thus far described (types IX, XII, XIV, and XVI) contain a highly homologous carboxyl-terminal triple helical domain designated COL1. We have studied the capacity of various matrix metalloproteinases (interstitial collagenase, stromelysin, matrilysin, and 92-kDa gelatinase) to degrade the COL1 domain of collagen XIV. We found that only 92-kDa gelatinase cleaves COL1. Furthermore, digestion of whole native collagen XIV by the 92-kDa gelatinase indicates that this enzyme specifically attacks the carboxyl-terminal triple helix-containing region of the molecule. COL1 is cleaved by 92-kDa gelatinase at 30 degrees C, a full 5-6 degrees C below the melting temperature (Tm) of this domain; native collagen XIV is also degraded at 30 degrees C. In comparison to interstitial collagenase degradation of its physiologic native type I collagen substrate, the 92-kDa enzyme cleaved COL1 (XIV) with comparable catalytic efficacy. Interestingly, following thermal denaturation of the COL1 fragment, its susceptibility to 92-kDa gelatinase increases, but only to a degree that leaves it several orders of magnitude less sensitive to degradation than denatured collagens I and III. These data indicate that native COL1 and collagen XIV are readily and specifically cleaved by 92-kDa gelatinase. They also suggest a role for 92-kDa gelatinase activity in the structural tissue remodeling of the developing embryo.

International audience; We have previously shown that SV40 large T oncogene is able to induce mouse chondrocyte proliferation without loss of expression of types II, IX, and XI collagen, as well as cartilage aggrecan and link protein. The cell line obtained (termed MC 615) also expressed some type I collagen in monolayer and we have investigated if anchorage-independent conditions could inhibit type I collagen synthesis and promote hypertrophy and type X collagen synthesis. The MC 615 cells were grown in agarose in the presence of serum, and GAG accumulation, DNA content, and matrix synthesis rates were monitored after incubation with [35S]sulfate and [3H]- or [14C]proline. SDS-PAGE analysis of pepsin-extracted samples showed that type I collagen was still synthesized by the MC 615 cells, from the beginning of the culture and at low or high density. Type II collagen synthesis was demonstrated by immunoblotting, but type X collagen synthesis was not detected, indicating that the MC 615 chondrocytes immortalized by large T were still blocked in their maturation pathway. The cells were also grown over agarose and electron microscopy (E. M.) analysis of the cell aggregates showed an extracellular matrix rich in proteoglycans and in type II-containing collagen fibrils. To gain insight into the role of type IX collagen in cartilage collagen assembly and/or matrix organization, we also immortalized embryonic chondrocytes isolated from mice lacking alpha 1 (IX) collagen and obtained a clone termed 4KO 91. As found for the MC 615 cells, the 4KO 91 cells synthesized type II collagen as demonstrated by Western blotting and some type I collagen identified by the presence of alpha 2(I) chains after electrophoretic analysis of pepsin-digested collagen chains. E. M. analysis of the extracellular matrices synthesized by the two cell lines revealed differences in collagen structure and organization. In the absence of alpha 1 (IX) collagen chains, the collagen fibrils seemed to fuse laterally, suggesting that collagen IX acts as a "spacer" between fibrils, to keep them apart.We have previously shown that SV40 large T oncogene is able to induce mouse chondrocyte proliferation without loss of expression of types II, IX, and XI collagen, as well as cartilage aggrecan and link protein. The cell line obtained (termed MC 615) also expressed some type I collagen in monolayer and we have investigated if anchorage-independent conditions could inhibit type I collagen synthesis and promote hypertrophy and type X collagen synthesis. The MC 615 cells were grown in agarose in the presence of serum, and GAG accumulation, DNA content, and matrix synthesis rates were monitored after incubation with [35S]sulfate and [3H]- or [14C]proline. SDS-PAGE analysis of pepsin-extracted samples showed that type I collagen was still synthesized by the MC 615 cells, from the beginning of the culture and at low or high density. Type II collagen synthesis was demonstrated by immunoblotting, but type X collagen synthesis was not detected, indicating that the MC 615 chondrocytes immortalized by large T were still blocked in their maturation pathway. The cells were also grown over agarose and electron microscopy (E. M.) analysis of the cell aggregates showed an extracellular matrix rich in proteoglycans and in type II-containing collagen fibrils. To gain insight into the role of type IX collagen in cartilage collagen assembly and/or matrix organization, we also immortalized embryonic chondrocytes isolated from mice lacking alpha 1 (IX) collagen and obtained a clone termed 4KO 91. As found for the MC 615 cells, the 4KO 91 cells synthesized type II collagen as demonstrated by Western blotting and some type I collagen identified by the presence of alpha 2(I) chains after electrophoretic analysis of pepsin-digested collagen chains. E. M. analysis of the extracellular matrices synthesized by the two cell lines revealed differences in collagen structure and organization. In the absence of alpha 1 (IX) collagen chains, the collagen fibrils seemed to fuse laterally, suggesting that collagen IX acts as a "spacer" between fibrils, to keep them apart.

Collagens are the major proteinaceous constituents of cartilage. Three collagen types participate in the formation of striated fibrils of cartilage, collagens II, IX, and XI. Collagen II and XI belong to the subgroup of fibrillar collagens and are structurally closely related, differing mainly in their N-propeptides. Collagen IX has a very different structure but is nevertheless an essential constituent of the striated fibrils. Two other collagen types are also found in cartilage but form distinct structures. Collagen VI, found mainly in the periphery of the chondrocytes, forms beaded filaments. These filaments are probably formed by interaction of collagen VI with hyaluronan. Collagen X is expressed by hypertrophic chondrocytes. It has been shown to form in vitro hexagonal lattices and in vivo to be associated either with striated fibrils or with mats which may correspond to the lattices. The functional role of the collagen diversity in cartilage is discussed.

The processing of human collagen type-V chains was studied using anti-peptide polyclonal antibodies raised against peptide sequences at the N-terminal non-triple-helical region of pro-alpha 1(V) and pro-alpha 2(V) chains. The anti-peptide polyclonal antibody raised against positions 48-57 of the N-terminal alpha 2(V) sequence recognized the mature form of the human alpha 2(V) chain extracted without any proteolytic treatment from several tissues in the presence of a mixture of protease inhibitors. It also recognized the pro-alpha 2(V) and pN-alpha 2(V) collagen chains secreted in the cell-culture media of the rhabdomyosarcoma A204 cell line. The pN-alpha 2(V) collagen chain from this cell line migrated during electrophoresis with the alpha 2(V) chain obtained from tissues. This demonstrates that the alpha 2(V) chain in tissues is incompletely processed and is present as the pN-alpha 2(V) collagen chain which lacks the C-propeptide. In comparison, an anti-peptide polyclonal antibody raised against residues at positions 284-299 of the N-terminal alpha 1(V) human sequence failed to recognize the mature form of the alpha 1(V) chain while it reacted with the pN-alpha 1(V) collagen chain form. These results suggest that the alpha 1(V) chain undergoes a processing event in the N-terminal region that involves the removal of at least the first 284 residues. Amino acid sequence analysis was performed on cyanogen-bromide-generated or trypsin-generated peptides of the two electrophoretic bands obtained for the tissue form of collagen V. The slower-migrating band corresponding to the intact alpha 1(V) chain gave, as expected, only sequences corresponding to the alpha 1(V) chain. However, the band previously considered to be the intact alpha 2(V) chain also gave sequences for the alpha 1(V) chain in addition to the alpha 2(V) chain. This result indicates the presence in tissue extracts of a further processed form of alpha 1(V) chain which migrates with the intact alpha 2(V) chain. On further analysis, we observed that the two bands of the tissue form of collagen V occurred in a 1:1 ratio whereas, after the pepsin digestion to remove non-collagenous regions, two bands were observed with an alpha 1(V)/alpha 2(V) chain ratio of 3:1. These results indicate that the alpha 1(V) chain exists in an additional stoichiometry, different from [alpha 1(V)]2 alpha 2(V).(ABSTRACT TRUNCATED AT 400 WORDS)

This study has examined the frequency and distribution of polymorphisms in the type I collagen coding sequences. RNA from a group of human skin fibroblast cell lines was analyzed by the chemical cleavage mismatch detection method using hydroxylamine, a reagent specific for C-base mismatches, and overlapping cDNA probes covering the entire prepro alpha 1(I) and prepro alpha 2(I) coding regions. Mismatches were detected at only two nucleotide positions, one in each of the type I collagen sequences, suggesting that polymorphisms are relatively rare within these cDNAs. cDNA sequence analysis demonstrated that the prepro alpha 1(I) mismatch, detected in only one cell line, was due to a sequence polymorphism involving the wobble position of the codon for arginine residue 59 within the amino-propeptide globular subdomain of the pro alpha 1(I) chain and not resulting in a change in the polypeptide primary structure. In contrast, the prepro alpha 2(I) mismatch, detected in 6 of the 16 cell lines, was shown to arise from a sequence polymorphism affecting the identity of Y-position residue 459 of the alpha 2(I) triple helical domain, resulting in an alanine/proline dimorphism at this position. This study is the first to identify a type I collagen coding sequence polymorphism resulting in an alteration at the level of the amino acid sequence. The data suggest that at least some alpha 1(I) and alpha 2(I) helix Y positions may be tolerant of sequence variation, particularly if the replacing amino acid is proline, a residue involved in stabilizing the collagen triple helix.

We have completed the analysis of a genomic clone, G238, that contains most of the coding region of the sponge COLF1 fibrillar collagen gene. The main triple helical domain is encoded by 31 exons. Except for the 5′ junction exon and the two last 3′ exons (126 and 18 base pairs), all these exons are related to a 54-bp unit and begin with an intact glycine codon. A good correlation can be made between this sponge gene and a vertebrate fibrillar collagen gene, revealing the high conservation of the members of this family during evolution. The reconstitution of an ancestral collagen gene can be made by considering all the exon/intron junctions of these genes. We suggest that such an ancestral gene arose from multiple duplications of a 54-bp exon and a (54 + 45)-bp module.

Publisher Summary Many of the recently identified molecules are very different from the originally described type I collagen molecule. The generally accepted current definition of a collagen is that it is a structural protein of the extracellular matrix that contains one or more domains having the conformation of a triple helix. This definition implies that collagen molecules are multidomain proteins and that some domain(s) are involved in the multimolecular aggregates forming the architecture of the extracellular matrix. The triple-helical domains of collagens are thus the main characteristic of this family of proteins to which they confer unique properties of aggregation. This chapter provides a brief overview of thecollagen family of proteins and discusses that particular protein conformation, how it is formed, and how triple-helical domains can interact to form multimolecular aggregates. The nontriple-helical domains of molecules containing triple helices of the collagen type could be of any nature, and they are indeed highly diverse in structure and function. The chapter discusses the domains separately.

International audience; Type XIV collagen belongs to the subclass of fibril-associated collagens with interrupted triple helices, which are composed of alternative triple helical and non-collagenous domains. Structural data show that these molecules interact with collagen fibrils and suggest that they might interact with cells. We have investigated the expression of type XIV collagen in bovine skin during development. Fetuses from 9 to 37 weeks were examined. Anti-type XIV collagen monoclonal antibody was produced, characterized, and used for immunofluorescence detection of the molecule. The localization of immunolabeling was analyzed by comparison with light and electron microscopic observations. In 9-week-old fetus, no type XIV collagen was found in the skin. From 19 weeks to birth, extensive immunofluorescence was observed on bundles of collagen fibrils in deep dermis. As shown by electron microscopy, this area exhibited bundles of collagen fibrils and cells with an abundant rough endoplasmic reticulum. In the upper dermis, a delicate fibrillar network of type XIV collagen was revealed by immunofluorescence around growing hair follicles at 19 and 24 weeks. Double labeling for type XIV collagen and fibronectin shows a more restricted pattern of expression of type XIV collagen in this area. The electron microscopic examination of skin of fetuses at these stages shows that the whole upper dermis is composed by a loose connective tissue containing scattered small bundles of collagen fibrils. Type XIV collagen was synthesized in the upper dermis between 24 weeks and birth. From this study, it appears that type XIV collagen expression is distinct from that of fibrillar collagens, at least during some developmental events. The prominent localization of type XIV collagen around growing hair follicles suggests a role for this molecule in epithelial-mesenchymal interactions.Type XIV collagen belongs to the subclass of fibril-associated collagens with interrupted triple helices, which are composed of alternative triple helical and non-collagenous domains. Structural data show that these molecules interact with collagen fibrils and suggest that they might interact with cells. We have investigated the expression of type XIV collagen in bovine skin during development. Fetuses from 9 to 37 weeks were examined. Anti-type XIV collagen monoclonal antibody was produced, characterized, and used for immunofluorescence detection of the molecule. The localization of immunolabeling was analyzed by comparison with light and electron microscopic observations. In 9-week-old fetus, no type XIV collagen was found in the skin. From 19 weeks to birth, extensive immunofluorescence was observed on bundles of collagen fibrils in deep dermis. As shown by electron microscopy, this area exhibited bundles of collagen fibrils and cells with an abundant rough endoplasmic reticulum. In the upper dermis, a delicate fibrillar network of type XIV collagen was revealed by immunofluorescence around growing hair follicles at 19 and 24 weeks. Double labeling for type XIV collagen and fibronectin shows a more restricted pattern of expression of type XIV collagen in this area. The electron microscopic examination of skin of fetuses at these stages shows that the whole upper dermis is composed by a loose connective tissue containing scattered small bundles of collagen fibrils. Type XIV collagen was synthesized in the upper dermis between 24 weeks and birth. From this study, it appears that type XIV collagen expression is distinct from that of fibrillar collagens, at least during some developmental events. The prominent localization of type XIV collagen around growing hair follicles suggests a role for this molecule in epithelial-mesenchymal interactions.

A 1.8-kb cDNA encoding portion of a novel collagenous chain was isolated from a human rhabdomyosarcoma cell line by cross-hybridization using a chicken type V collagen probe. Sequence analysis suggests that this chain belongs to the recently discovered group of collagens, termed the FACIT class of macromolecules. This cDNA was used to locate the corresponding gene (D6S228E) to chromosome 6, notably at position 6q12–q14. Interestingly, within this region of human chromosome 6 resides the α1(IX) collagen gene (COL9A1), a member of the FACIT group.

cDNA from embryonic chick skin has been isolated and characterized which encodes a novel member of the FACIT (fibril-associated collagen with interrupted triple helices) group whose other known members are collagen types IX and XII. Nucleotide sequence analysis of the cDNA, combined with characterization of a pepsin-resistant fragment of the protein from embryonic chick skin, demonstrates that the collagen chain is more closely related to the chain of type XII collagen than to those of type IX. It is most similar to a collagen, type XIV, recently identified in bovine skin. It is possible, therefore, that the cDNA codes for a chain of chicken type XIV collagen. From the additional data on molecular structure obtained by sequencing the cDNA, the FACIT family appears to consist of at least two classes of molecules: one of which contains the three chains of type IX collagen, and a second which includes the chains of collagen types XII and XIV.

De nombreuses proteines comportent des motifs homologues, appeles modules. Les macromolecules secretees, malgre leur grande diversite, sont construites a partir d'un nombre relativement restreint de modules differents. La modularite des proteines de la matrice extracellulaire est importante, tant par les assemblages supramoleculaires que par son role physiologique. Les modules peuvent, en effet, etre impliques a la fois dans la structure de ces proteines et dans les interactions qu'elles menagent entre elles ou avec des recepteurs cellulaires qui leur sont propres, ce qui contribue a la coherence des matrices extracellulaires. Les mutations affectant la structure d'un module et/ou la fonction qui peut lui etre associee sont a l'origine de plusieurs maladies hereditaires des matrices extracellulaires

Isolated photosynthetic reaction center from the bacterium Rhodospirillum rubrum was extracted with acetone-methanol. Its main pigments were identified as bacteriochlorophyll, bacteriopheophytin, and spirilloxanthin. The extinction coefficients of these pigments in acetone-methanol were determined. Quantitative spectroscopic analysis of the dry acetone-methanol extracts indicated a bacteriochlorophyll to bacteriopheophytin mole ratio of 2 and led to an extinction coefficient at 868 nm of 71.3, 142.6, or 214 mm-1 cm-1, depending on whether the photosynthetic center was assumed to contain 2, 4, or 6 bacteriochlorophyll molecules. The extinction coefficient of P870 was determined by mixing photo-oxidized photosynthetic center with ferrocytochrome c in a stopped flow spectrophotometer. Complete reaction was observed when the reactants were mixed in equimolar ratio calculated on the basis of an extinction coefficient of 143 mm-1 cm-1 at 868 nm. This photosynthetic center is proposed to contain 4 moles of bacteriochlorophyll, 2 moles of bacteriopheophytin, and 1 mole of spirilloxanthin per equivalent of P870.

Rhodospirillum rubrum chromatophores were solubilized by various detergent treatments and fractionated. The main fractions were a particle enriched in P870 photochemical activity, a purified photochemical reaction center, and AUT “photoreceptor” particles. These fractions and others were compared to the chromatophore preparation by immunodiffusion in the presence of deoxycholate, in order to solubilize the antigenic components of the membrane. They were compared also by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea. These techniques show that each particle has different components. The purified reaction center preparation contains two antigens and three electrophoretic components with apparent molecular weights of 28,000, 25,500, and 21,000. These three components are present in all the fractions studied, although in small amounts in the pellet obtained after solubilization with dodecyl dimethylamine N-oxide. This fraction contains, as a major component, a polypeptide of about 10,000 apparent molecular weight which is thought to be associated with light-harvesting pigments. This polypeptide is virtually absent from purified photochemical reaction center. The reaction center protein is estimated to represent 15% of the chromatophore preparation on a protein basis.

✓ It has been suggested that a deficiency in the expression of type III collagen may play a role in the pathogenesis of cerebral aneurysms. To test this hypothesis in cases of familial cerebral aneurysms, fibroblast cell cultures were established and the expression of collagen types I and III was studied in a patient with three cerebral aneurysms whose mother and sister also had cerebral aneurysms. Cultured skin fibroblasts were labeled with tritiated proline. The collagens and procollagens were precipitated and run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction to analyze procollagen chains. Control cell lines were analyzed simultaneously. Quantitation of the ratios of type III to type I procollagen synthesis was achieved by integration of the intensities of the pro-α1 (III), pro-α1(I), and pro-α2(I) bands on fluorograms of electrophoretic gels of medium proteins. There was no difference in type I and III procollagen levels observed between the cells from the aneurysm patient and those from the control cell lines. These data do not support the hypothesis that familial cerebral aneurysms are caused by a deficiency of type III collagen.

Five specimens were taken from the uterine cervix immediately post partum, one specimen was taken before onset of labor, and one was taken at 16 weeks' pregnancy for comparison with five surgical specimens taken from the cervix during the proliferative phase of the menstrual cycle. Cervical collagen, including the reducible cross-links in the acetic acid-soluble and insoluble fractions, was studied. Samples taken from nonpregnant women and from women during delivery could not be differentiated using the ratio of hydroxyproline/total protein as an index of collagen in the soluble fractions. However, after electrophoresis, intense bands corresponding to collagen chains were seen in the proliferative phase and during early pregnancy, whereas only small amounts of intact collagen chains could be detected ante- and intrapartum, indicating the prevalence of collagen breakdown products. In the insoluble fractions the hydroxyproline/total protein ratio drops from 0.75 in the samples from the nonpregnant group to 0.35 in the intrapartum samples. The typing of collagen in the insoluble fraction showed the predominance of type I collagen. Twenty to 38 per cent of collagen present was type III. No striking difference was seen in the analysis of reducible cross-links between the groups. These data show that cervical collagen undergoes significant biochemical changes that might play an important role in cervical dilatation.

The bacteriochlorophyll ( P -800 and P -870) of the carotenoidless photoreaction center isolated from Rhodospirillum rubrum (strain G9) is bleached irreversibly when the preparations are exposed to intense near infrared light in the presence of oxygen. This effect is much smaller in preparations, extracted from the wild type, which contain, as shown earlier, 1 mol of spirilloxanthin per mol of P -870. This photodynamic effec is shown to be due to singlet O 2 . The oxidation of adrenaline in the presence of superoxide dismutase and the oxidation of 1,3-diphenylisobenzofuran are used as reporter reactions. Singlet oxygen is presumably generated by the triplet-triplet energy transfer 3 bacteriochlorophyll → O 2 ( 3 Σ ). Four purified bacterial carotenoids, spirilloxanthin, sphaeroidene, sphaeroidenone and chloroxanthin were attached onto the carotenoidless photoreaction center from strain G9 in nearly 1 : 1 mol ratios with respect to P -870. Once fixed, these carotenoids confer protection against the photodynamic bleaching of bacteriochlorophyll. The relative photoprotection efficiency was 1.0 for spirilloxanthin and sphaeroidene, 0.4 for chloroxanthin and 0.2 for sphaeroidenone. The fixed carotenoids display optical activity and their molar ellipticity appears to be correlated with their relative photoprotection efficiency. The efficiency of energy transfer to P -870 is 0.90 for sphaeroidene, 0.35 for sphaeroidenone, 0.30 for chloroxanthin and 0.20 for spirilloxanthin. The energy transfer efficiency from the carotenoids to bacteriochlorophyll is suggested to be governed by the rate of the internal conversion processes of the excited singlet state of the carotenoids. A study of the difference absorption and CD spectra of the reconstituted minus carotenoidless preparations leads to the interpretation that the fixed carotenoids are in a central monocis conformation.

1. 1. The major collagen in lamprey notochord is type II, as determined by its amino acid composition and solubility properties. 2. 2. This collagen has a distribution of charged residues indistinguishable from higher vertebrate Type II collagens as judged by its SLS banding pattern. 3. 3. Lamprey type II collagen has a higher thermal stability than lamprey skin collagen, in contrast to the identical melting temperatures for these types in mammals. 4. 4. A minor collagen in lamprey notochord has solubility properties, amino acid composition, and electrophoretic mobility similar to that of 1α, 2α, 3α collagen in human cartilage.

Cultured skin fibroblasts from a variant of osteogenesis imperfecta were previously shown to synthesize a type I procollagen which was a homotrimer of pro alpha 1(I) chains. Trimers of alpha 1(I) collagen were isolated by pepsin digestion of culture medium from these fibroblasts. The amino acid composition of the isolated protein indicated that it contained an increased amount of hydroxylysine, apparently because of post-translational over-modification. The thermal stability of the alpha 1(I) trimers was examined by circular dichroism. We found no consistent difference in the melting curve of the alpha 1(I) trimers compared to control type I collagen. We next examined the thermal stability of the alpha 1(I) trimers using digestion with a combination of trypsin and alpha-chymotrypsin as an alternative probe of helical stability. When enzymatic digestions were carried out at 36 degrees to 40 degrees C, the alpha 1(I) chains in the trimers were cleaved to polypeptides which were shortened by approximately 100 amino acids. Vertebrate collagenase digestion of the shortened molecules indicated that the 100 amino acid segment removed from each alpha 1(I) chain was located at the carboxyl-terminus. The decreased thermal stability of the alpha 1(I) trimers was probably explained by the absence of alpha 2(I) chains in the molecules. The results, however, did not exclude the possibility that the post-translational over-modification of the alpha 1(I) chains contributed to the altered helical structure.