Identification of cross-linking sites in bovine cartilage type IX collagen reveals an antiparallel type II-type IX molecular relationship and type IX to type IX bonding.

Type IX collagen functions in covalent cross-linkage to type II collagen in cartilage (Eyre, D. R., Apone, S., Wu, J. J., Ericsson, L. H., and Walsh, K. A. (1987) FEBS Lett. 220, 337-341). To understand this molecular relationship better, an analysis of all cross-linking sites labeled by [3H]borohydride was undertaken using the protein prepared from fetal bovine cartilage. Sequence analysis of tryptic peptides containing the 3H-labeled cross-links showed that each of the chains of type IX collagen, alpha 1(IX), alpha 2(IX), and alpha 3(IX), contained a site of cross-linking at the amino terminus of the COL2 triple-helix to which the alpha 1(II)N-telopeptide could bond. The alpha 3(IX)COL2 domain alone also had an attachment site for the alpha 1(II)C-telopeptide. The distance between the alpha 1(II)N-telopeptide and alpha 1(II)C-telopeptide interaction sites, 137 residues, is equal to the length of the hole zone (0.6D) in a type II collagen fibril. This implies an antiparallel type II to type IX cross-linking relationship. Peptide analysis also revealed an unknown amino acid sequence linked to the COL2 cross-linking domains in both the alpha 1(IX) and alpha 3(IX) chains. Using antibodies to this novel peptide, its origin in the collagen alpha 3(IX)NC1 domain was established. In summary, the results confirm extensive covalent cross-linking between type IX and type II collagen molecules and reveal the existence of type IX-type IX bonding. These data provide a molecular basis for the proposed function of type IX collagen as a critical contributor to the mechanical stability and resistance to swelling of the collagen type II fibril framework of cartilage.     

Localization of binding sites within human von Willebrand factor for monomeric type III collagen

Purified human plasma von Willebrand factor (vWf) binds to pepsin-digested monomeric type III collagen in a saturable (KD = 1 X 10(-8) M), specific, and rapid manner with a stoichiometry of approximately 1:15 [vWf subunit (Mr 270,000):collagen trimer (Mr 300,000)]. Two reduced and alkylated CNBr peptides of vWf, termed M11 residues 542-622 and M20 residues 948-998 [Titani, K., Kumar, S., Takio, K., Ericsson, L. H., Wade, R. D., Ashida, K., Walsh, K. A., Chopek, M. W., Sadler, J. E., & Fujikawa, K. (1986) Biochemistry 25, 3171-3184], inhibited vWf binding to collagen. With 125I-vWf (2 X 10(-9) M) as ligand, M11, M20, fragment III (a dimeric, V8 protease, NH2-terminal fragment, Mr 320,000 referenced above), and unlabeled vWf inhibited binding to collagen with EC50 values of 4.8 X 10(-7), 9.4 X 10(-7), 1.1 X 10(-7), and 0.2 X 10(-7) M, respectively. M11 and M20 bind to collagen directly when 125I-labeled peptides are used as ligands. Other CNBr fragments of vWf were less effective as inhibitors (5-fold or less) and bound less avidly to collagen (5-fold or less) compared to M11 and M20. A murine anti-human vWf monoclonal antibody (MR5), which blocks the binding of vWf to collagen, bound selectively to both M11 and M20 when tested in an enzyme-linked immunoadsorbent assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR studies of the active site of isopenicillin N synthase, a non-heme iron(II) enzyme.

The active site structure of isopenicillin N synthase (IPNS) has been previously studied by the use of M?ssbauer, EPR, electronic absorption, and NMR spectroscopies [Chen, V.J., Frolik, C.A., Orville, A.M., Harpel, M.R., Lipscomb, J.D., Surerus, K.K., & Münck, E. (1989) J. Biol. Chem. 264, 21677-21681; Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C.A., & Chen, V.J. (1990) Inorg. Chem. 26, 1111-1112]. These studies have revealed three coordinated His residues along with three sites for substrate [delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine, ACV], NO, and water binding on the active Fe(II) of IPNS. We report here NMR studies of Fe(II)IPNS and its Co(II)-substituted derivative [Co(II)IPNS]. By the use of NOE techniques on the Co(II)IPNS-ACV complex, we have recognized a -CH2-CH less than spin system at 14.6, 24.3, and 38.6 ppm that is assigned to the alpha and beta protons of a coordinated Asp residue. Corresponding solvent nonexchangeable features are found near 40 ppm in Fe(II)IPNS and the Fe(II)IPNS-ACV complex, but the peaks are too broad for NOE effects to be observed. The binding of NO to the Fe(II) center results in a significant change in the configuration of the metal site: (a) The C beta H2 resonances due to the coordinated Asp residue disappear. The loss of the signal may indicate a change of the carboxylate configuration from syn-like to anti-like or, less likely, its displacement by NO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concerted modulation of alpha 1(XI) and alpha 2(V) collagen mRNAs in bovine vascular smooth muscle cells.

We have isolated a partial cDNA for alpha 1(XI) collagen from a bovine smooth muscle cell (SMC) library. Previously, this collagen was not known to be expressed in SMCs. Comparison of the nucleotide and deduced amino acid sequence of the 2.7-kilobase bovine clone and the human alpha 1(XI) sequence indicates 92 and 98% homology, respectively. Bovine SMCs in culture were found to produce alpha 1(XI) mRNA. However, alpha 2(XI) and alpha 1(II) collagen RNA were not detectable; therefore, SMCs cannot synthesize the same type XI collagen as found in cartilage. Since type XI collagen is structurally related to type V collagen, the expression of alpha 1(XI) and alpha 2(V) collagen mRNA in SMCs was characterized. Levels of alpha 1(XI) and alpha 2(V) collagen mRNAs were low in exponentially growing SMCs and increased 3-4-fold as cells became confluent. Increased mRNA levels were also observed when exponentially growing subconfluent SMCs were incubated in medium containing 0.5% fetal bovine serum for 24 h, similar to the effects of serum deprivation on the expression of types I and III collagen genes (Kindy, M. S., Chang, C.-J., and Sonenshein, G. E. (1988) J. Biol. Chem. 263, 11426-11430). However, as cell density increased, serum deprivation resulted in very different responses for these collagen genes. Serum deprivation caused a decrease in expression of alpha 1(XI) and alpha 2(V) collagen mRNAs in cultures as they approached confluence. In contrast, at confluence alpha 1(I) and alpha 2(I) mRNA levels no longer responded to serum concentration whereas expression of alpha 1(III) mRNA remained inducible by serum deprivation. These results suggest concerted regulation of alpha 1(XI) and alpha 2(V) collagen gene expression, which is distinct from that for the chains of type I and type III collagen with respect to cell density and serum.     

Type V collagen controls the initiation of collagen fibril assembly

Vertebrate collagen fibrils are heterotypically composed of a quantitatively major and minor fibril collagen. In non-cartilaginous tissues, type I collagen accounts for the majority of the collagen mass, and collagen type V, the functions of which are poorly understood, is a minor component. Type V collagen has been implicated in the regulation of fibril diameter, and we reported recently preliminary evidence that type V collagen is required for collagen fibril nucleation (Wenstrup, R. J., Florer, J. B., Cole, W. G., Willing, M. C., and Birk, D. E. (2004) J. Cell. Biochem. 92, 113-124). The purpose of this study was to define the roles of type V collagen in the regulation of collagen fibrillogenesis and matrix assembly. Mouse embryos completely deficient in pro-alpha1(V) chains were created by homologous recombination. The col5a1-/- animals die in early embryogenesis, at approximately embryonic day 10. The type V collagen-deficient mice demonstrate a virtual lack of collagen fibril formation. In contrast, the col5a1+/- animals are viable. The reduced type V collagen content is associated with a 50% reduction in fibril number and dermal collagen content. In addition, relatively normal, cylindrical fibrils are assembled with a second population of large, structurally abnormal collagen fibrils. The structural properties of the abnormal matrix are decreased relative to the wild type control animals. These data indicate a central role for the evolutionary, ancient type V collagen in the regulation of fibrillogenesis. The complete dependence of fibril formation on type V collagen is indicative of the critical role of the latter in early fibril initiation. In addition, this fibril collagen is important in the determination of fibril structure and matrix organization.     

The thrombospondin motif of aggrecanase-1 (ADAMTS-4) is critical for aggrecan substrate recognition and cleavage

Aggrecanase-1 (ADAMTS-4) is a member of the a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) protein family that was recently identified. Aggrecanase-1 is one of two ADAMTS cartilage-degrading enzymes purified from interleukin-1-stimulated bovine nasal cartilage (Tortorella, M. D., Burn, T. C., Pratta, M. A. , Abbaszade, I., Hollis, J. M., Liu, R., Rosenfeld, S. A., Copeland, R. A., Decicco, C. P., Wynn, R., Rockwell, A., Yang, F., Duke, J. L., Solomon, K., George, H., Bruckner, R., Nagase, H., Itoh, Y., Ellis, D. M., Ross, H., Wiswall, B. H., Murphy, K., Hillman, M. C., Jr., Hollis, G. F., and Arner, E.C. (1999) Science 284, 1664-1666; 2 Abbaszade, I., Liu, R. Q., Yang, F., Rosenfeld, S. A., Ross, O. H., Link, J. R., Ellis, D. M., Tortorella, M. D., Pratta, M. A., Hollis, J. M., Wynn, R., Duke, J. L., George, H. J., Hillman, M. C., Jr., Murphy, K., Wiswall, B. H., Copeland, R. A., Decicco, C. P., Bruckner, R., Nagase, H., Itoh, Y., Newton, R. C., Magolda, R. L., Trzaskos, J. M., and Burn, T. C. (1999) J. Biol. Chem. 274, 23443-23450). The aggrecan products generated by this enzyme are found in cartilage cultures stimulated with cytokines and in synovial fluid from patients with arthritis, suggesting that aggrecanase-1 may be important in diseases involving cartilage destruction. Here we demonstrate that the thrombospondin type-1 (TSP-1) motif located within the C terminus of aggrecanase-1 binds to the glycosaminoglycans of aggrecan. Data from several studies indicate that this binding of aggrecanase-1 to aggrecan through the TSP-1 motif is necessary for enzymatic cleavage of aggrecan. 1) A truncated form of aggrecanase-1 lacking the TSP-1 motif was not effective in cleaving aggrecan. 2) Several peptides representing different regions of the TSP-1 motif effectively blocked aggrecanase-1 cleavage of aggrecan by preventing the enzyme from binding to the substrate. 3) Aggrecanase-1 was not effective in cleaving glycosaminoglycan-free aggrecan. Taken together, these data suggest that the TSP-1 motif of aggrecanase-1 is critical for substrate recognition and cleavage.     

Glycine to serine substitution in the triple helical domain of pro-alpha 1 (II) collagen results in a lethal perinatal form of short-limbed dwarfism

Previous biochemical studies on cartilage tissue from a proband with Type II achondrogenesis-hypochondrogenesis (Godfrey, M., and Hollister, D. W. (1988) Am. J. Hum. Genet. 43, 904-913) indicated heterozygosity for a structural abnormality in the triple helical domain of pro-alpha 1 (II) collagen. Here we demonstrate that the mutation in the type II procollagen gene is a single base change that converts the codon for glycine (GGC) at amino acid 943 of the alpha 1 (II) chain to a codon for serine (AGC). The substitution disrupts the invariant Gly-X-Y structural motif necessary for perfect triple helix formation and leads to extensive overmodification, intracellular retention, and reduced secretion of type II collagen. These findings confirm the proposal that new dominant mutations in the type II procollagen gene may account for some cases of Type II achondrogenesis-hypochondrogenesis. Since recent studies (Lee, B., Vissing, H., Ramirez, F., Rogers, D., and Rimoin, D. (1989) Science 244, 978-980) have identified a dominantly inherited type II procollagen gene deletion in a non-lethal form of skeletal dysplasia, namely spondyloepiphyseal dysplasia, the data more generally demonstrate that different type II procollagen gene mutations eventuate in a wide and diverse spectrum of clinical phenotypes.     

Mapping the collagen-binding site in the I domain of the glycoprotein Ia/IIa (integrin alpha(2)beta(1))

The I domain present within the alpha2 chain of the integrin alpha(2)beta(1) (GPIa/IIa) contains the principal collagen-binding site. Based on the crystal structure of the alpha2-I domain, a hypothetical model was proposed in which collagen binds to a groove on the upper surface of the I domain (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517). We have introduced point mutations into 13 residues on the upper surface of the domain. Recombinant mutant proteins were assayed for binding to monoclonal antibodies 6F1 and 12F1, to collagen under static conditions, and for the ability to retain adhesive activity under flow conditions. The mutations to residues surrounding the metal ion-dependent adhesion site that caused the greatest loss of collagen binding under both static and flow conditions are N154S in the betaA-alpha1 turn, N190D in the betaB-betaC turn, D219R in the alpha3-alpha4 turn, and E256V and H258V in the betaD-alpha5 turn. Mutation in one of the residues that coordinate the metal binding, S155A, completely lost the adhesive activity under flow but bound normally under static conditions, whereas the mutation Y285F had the converse effect. We conclude that the upper surface of the domain, including the metal ion-dependent adhesion site motif, defines the collagen recognition site.     

Characterization of collagen types XII and XIV from fetal bovine cartilage.

The structurally related type XII-like collagen molecules TL-A and TL-B were recently identified in fetal bovine epiphyseal cartilage and subsequently shown to be collagen types XII and XIV, respectively. By indirect immunofluorescent staining of cartilage using monoclonal antibodies to the NC3 domains of each molecule, it was shown that type XII collagen was present predominantly around cartilage canals, the articular surface, subperichondrial margins, and the perichondrium, was less so in the remaining cartilage matrix, and was absent from the growth plate region. In the permanent cartilage of trachea, type XII stained somewhat more intensely in the margins beneath the loose connective tissue. Type XIV collagen localized more uniformly throughout the articular cartilage and was also absent from the growth plate region, whereas in tracheal cartilage, its distribution was similar to type XII. We have characterized the structure of these cartilage molecules and compared them with those from fetal bovine skin. Extraction of cartilage with 1 M NaCl and differential NaCl precipitation yields a fraction enriched for these two collagens. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with monoclonal antibodies to the large amino-terminal non-triple-helical domain, NC3, revealed the presence in cartilage of two forms of type XII collagen: type XIIB, the molecule previously identified in chick and bovine tissues, and type XIIA, a much larger form equivalent to the molecule recently identified in WISH-transformed epithelial cell culture medium (Lunstrum, G. P., McDonough, A. M., Marinkovich, M. P., Keene, D. R., Morris, N. P., and Burgeson, R. E. (1992) J. Biol. Chem. 267, 20087-20092). Digestion with bacterial collagenase shows that the increased mass is present in the NC3A domain. Additional purification by velocity sedimentation and observation of rotary-shadowed images demonstrates molecules with extended non-triple-helical arms approximately 80 nm in length analogous to the WISH cell molecules. Electrophoretic mobilities of bands corresponding to type XIIA, but not type XIIB, are sensitive to chondroitinase ABC, indicating that type XIIA is a chondroitin sulfate proteoglycan and that modification occurs predominantly within the NC3A domain distal to NC3B. Neither type XIIB from skin nor type XIIA from WISH cells are chondroitinase-sensitive. By similar analysis, a portion of the type XIV collagen chains in cartilage was also sensitive to chondroitinase digestion. Chondroitin sulfate is apparently not located on its NC3 domain. As in skin, collagen types XII and XIV have subtly different distributions within cartilage and type XII may have a tissue-specific structure.     

X-ray absorption spectroscopic studies of the high-spin iron(II) active site of isopenicillin N synthase: evidence for Fe-S interaction in the enzyme-substrate complex.

Isopenicillin N synthase from Cephalosporium acremonium (IPNS; M(r) 38.4K) is an Fe(2+)-requiring enzyme which catalyzes the oxidative conversion of (L-alpha-amino-delta-adipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N, with concomitant reduction of O2 to 2H2O. Chemical and spectroscopic data have suggested that catalysis proceeds via an enzyme complex of ACV bound to the iron through its cysteinyl thiolate [Baldwin, J. E., & Abraham, E. P. (1988) Nat. Prod. Rep. 5, 129-145; Chen, V. J., Orville, A. M., Harpel, M. R., Frolik, C. A., Surerus, K. K., Münck, E., & Lipscomb, J. D. (1989) J. Biol. Chem. 264, 21677-21681; Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C. A., & Chen, V. J. (1991) Biochemistry 30, 11653-11659]. Here we have employed the technique of Fe K-edge extended X-ray absorption fine structure (EXAFS) to characterize the iron site and to seek direct evidence for or against the formation of an Fe-S interaction upon ACV binding. Our data collected in the absence of substrate and O2 are consistent with the iron center of IPNS being coordinated by only (N,O)-containing ligands in an approximately octahedral arrangement and with an average Fe-(N,O) distance of 2.15 +/- 0.02 A. Upon anaerobic binding of ACV, the iron coordination environment changes considerably, and the associated Fe EXAFS cannot be adequately simulated without incorporating an Fe-S interaction at 2.34 +/- 0.02 A along with four or five Fe-(N,O) interactions at 2.15 +/- 0.02 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of the Goodpasture epitope to a novel chain of basement membrane collagen

The chain origins of subunits M1, M2*, and M3 previously described (Butkowski, R. L., Wieslander, J., Wisdom, B.J., Barr, J.F., Noelken, M.E., and Hudson, B.G. (1985) J. Biol. Chem. 260, 3739-3747) of the globular domain of basement membrane collagen were identified, by amino-terminal amino acid sequence analysis, with respect to their relationship to the chains of collagen IV. M1 comprises two polypeptides which correspond to the noncollagenous segments (NC1) of the alpha 1 ad alpha 2 chains of collagen IV. M2*, containing the Goodpasture epitope, and M3 are distinct from these two constituents and from each other but have Gly-X-Y triplets and hydroxyproline at their amino terminus, reflecting the fact that each has a collagen chain origin. These results indicate the presence of two new collagen chains in basement membrane. These new chains appear to be integral components of collagen IV molecules. Alternatively, they could represent new molecular species of basement membrane collagen containing a globular domain, comprising M2* and M3, with physicochemical properties very similar to those of collagen IV.     

Calcium binding to the low affinity sites in troponin C induces conformational changes in the high affinity domain. A possible route of information transfer in activation of muscle contraction

Residues 89-100 of troponin C (C89-100) and 96-116 of troponin I (I96-116) interact with each other in the troponin complex (Dalgarno, D.C., Grand, R.J.A., Levine, B.A. Moir, A., J.G., Scott, G.M.M., and Perry, S.V. (1982) FEBS Lett. 150, 54-58) and are necessary for the Ca2+ sensitivity of actomyosin ATPase (Syska, H., Wilkinson, J.M., Grand, R.J.A., and Perry, S.V. (1976) Biochem. J. 153, 375-387 and Grabarek, Z., Drabikowski, W., Leavis, P.C., Rosenfeld, S.S., and Gergely, J. (1981) J. Biol. Chem. 256, 13121-13127). We have studied Ca2+-induced changes in the region C89-100 by monitoring the fluorescence of troponin C (TnC) labeled at Cys-98 with 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid. Equilibrium titration of the labeled TnC with Ca2+ indicates that the probe is sensitive to binding to both classes of sites in free TnC as well as in its complex with TnI. When Mg2 X TnC is mixed with Ca2+ in a stopped flow apparatus, there is a rapid fluorescence increase related to Ca2+ binding to the unoccupied sites I and II followed by a slower increase (k = 9.9 s-1) that represents Mg2+-Ca2+ exchange at sites III and IV. In the TnC X TnI complex, the fast phase is much larger and the Mg2+-Ca2+ exchange at sites III and IV results in a small decrease rather than an increase in the fluorescence of the probe. The possibility is discussed that the fast change in the environment of Cys-98 upon Ca2+ binding to sites I and II may be instrumental in triggering activation of the thin filament by facilitating a contact between C89-100 and I96-116.     

New pepsin-solubilized low molecular weight collagenous component possibly unique to periodontal ligament

Limited pepsin digestion of bovine periodontal ligament releases genetic types I, III, and V collagen and a high cystine containing low molecular weight collagenous component. Salt fractionation and molecular sieve chromatography allowed the isolation of the latter as an apparently pure homogeneous moiety which had an approximate molecular mass of 30 000 daltons. Reduction with mercaptoethanol yielded a single 10 000-dalton band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This led us to conclude that the newly isolated low molecular weight collagen fragment consists of three similar molecular weight chains. Unreduced collagen-like glycoprotein (CGP) [Jander, R., Troyer, D., & Rauterberg, J. (1984) Biochemistry 23, 3675-3681] after extraction from tissues with collagen denaturing solvents yields the GP140 glycoprotein upon reduction and does not release any collagen fragment below 90 000 daltons upon mild or vigorous pepsin digestion. The GP140 glycoprotein [Heller-Harrison, R. A., & Carter, W. G. (1984) J. Biol. Chem. 259, 6858-6864] isolated by extraction under reducing and collagen denaturing solvent conditions did not yield a collagen fragment below 40 000 daltons after pepsin treatment. It was clearly shown that both CGP and GP140 yield type VI collagen fragments in the above-cited reports. Since this report demonstrates that the Mr 30 000 collagen fragment is only released by pepsin treatment of nondenaturing solvent treated periodontal ligament and that only very small peptides are found in denaturing solvent treated tissue after pepsin digestion, it is concluded that the newly isolated Mr 30 000 collagen fragment reported here is not derived from type VI collagen.(ABSTRACT TRUNCATED AT 250 WORDS)     

The superoxide dismutase activity of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774.

Desulfoferrodoxin (Dfx), a small iron protein containing two mononuclear iron centres (designated centre I and II), was shown to complement superoxide dismutase (SOD) deficient mutants of Escherichia coli [Pianzzola, M.J., Soubes M. & Touati, D. (1996) J. Bacteriol. 178, 6736-6742]. Furthermore, neelaredoxin, a protein from Desulfovibrio gigas containing an iron site similar to centre II of Dfx, was recently shown to have a significant SOD activity [Silva, G., Oliveira, S., Gomes, C.M., Pacheco, I., Liu, M.Y., Xavier, A.V., Teixeira, M., Le Gall, J. & Rodrigues-Pousada, C. (1999) Eur. J. Biochem. 259, 235-243]. Thus, the SOD activity of Dfx isolated from the sulphate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 was studied. The protein exhibits a SOD activity of 70 U x mg-1, which increases approximately 2.5-fold upon incubation with cyanide. Cyanide binds specifically to Dfx centre II, yielding a low-spin iron species with g-values at 2.27 (g perpendicular) and 1.96 (g parallel). Upon reaction of fully oxidized Dfx with the superoxide generating system xanthine/xanthine oxidase, Dfx centres I and II become partially reduced, suggesting that Dfx operates by a redox cycling mechanism, similar to those proposed for other SODs. Evidence for another SOD in D. desulfuricans is also presented - this enzyme is inhibited by cyanide, and N-terminal sequence data strongly indicates that it is an analogue to Cu,Zn-SODs isolated from other sources. This is the first indication that a Cu-containing protein may be present in a sulphate-reducing bacterium.     

Extracellular matrix formation by chondrocytes in monolayer culture

In previous studies were have reported on the secretion and extracellular deposition of type II collagen and fibronectin (Dessau et al., 1978, J. Cell Biol., 79:342-355) and chondroitin sulfate proteoglycan (CSPG) (Vertel and Dorfman, 1979, Proc. Natl. Acad. Sci. U. S. A. 76:1261-1264) in chondrocyte cultures. This study describes a combined effort to compare sequence and pattern of secretion and deposition of all three macromolecules in the same chondrocyte culture experiment. By immunofluorescence labeling experiments, we demonstrate that type II collagen, fibronectin, and CSPG reappear on the cell surface after enzymatic release of chondrocytes from embryonic chick cartilage but develop different patterns in the pericellular matrix. When chondrocytes spread on the culture dish, CSPG is deposited in the extracellular space as an amorphous mass and fibronectin forms fine, intercellular strands, whereas type II collagen disappears from the chondrocyte surface and remains absent from the extracellular space in early cultures. Only after cells in the center of chondrocyte colonies shape reassume spherical shape does the immunofluorescence reveal type II collagen in the refractile matrix characteristic of differentiated cartilage. By immunofluorescence double staining of the newly formed cartilage matrix, we demonstrate that CSPG spreads farther out into the extracellular space that type II collagen. Fibronectin finally disappears from the cartilage matrix.     

Involvement of HMGB1 and HMGB2 proteins in exogenous DNA integration reaction into the genome of HeLa S3 cells

Electrophoretic and Western blot studies were conducted on collagen fractions extracted from Sepia officinalis (cuttlefish) cartilage using a modified salt precipitation method developed for the isolation of vertebrate collagens. The antibodies used had been raised in rabbit against the following types of collagen: Sepia I-like; fish I; human I; chicken I, II, and IX; rat V; and calf IX and XI. The main finding was that various types of collagen are present in Sepia cartilage, as they are in vertebrate hyaline cartilage. However, the main component of Sepia cartilage is a heterochain collagen similar to vertebrate type I, and this is associated with minor forms similar to type V/XI and type IX. The cephalopod type I-like heterochain collagen can be considered a first step toward the evolutionary development of a collagen analogous to the typical collagen of vertebrate cartilage (type II homochain). The type V/XI collagen present in molluscs, and indeed all phyla from the Porifera upwards, may represent an ancestral collagen molecule conserved relatively unchanged throughout evolution. Type IX-like collagen seems to be essential for the formation of cartilaginous tissue.     

Identification, using sera from exposed animals, of putative viral antigens in livers of primates with callitrichid hepatitis.

Callitrichid hepatitis (CH) is an acute, frequently fatal viral hepatitis which affects members of the primate family Callitrichidae (R. J. Montali, E. C. Ramsay, C. B. Stephensen, M. Worley, J. A. Davis, and K. V. Holmes, J. Infect. Dis. 160:759-765, 1989; E. C. Ramsay, R. J. Montali, M. Worley, C. B. Stephensen, and K. V. Holmes, J. Zoo Wildlife Med. 20:178-183, 1989). Outbreaks of the disease occur in zoos and animal parks. In this study, CH-specific antigens were identified in the livers of infected animals by using immune sera from primates with CH and CH-exposed asymptomatic animals. Three CH-specific antigens with apparent molecular masses of 34, 54, and 65 kDa were identified. A polyclonal antiserum was raised against the 54-kDa antigen. These antigens were not found in the livers of uninfected animals and may be viral proteins. Our results suggest that at least five of the six outbreaks of CH considered here were caused by the same virus or by an antigenically related virus.     

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