Undersulfated chondroitin sulfate in the cartilage matrix of brachymorphic mice.

Homozygous brachymorphic ($\text{bm}\text{bm}$) mice have a disproportionately short stature, similar to human achondroplasia. We previously showed that each zone of growth in young $\text{bm}\text{bm}$ epiphyseal cartilages is smaller than normal and that the extracellular matrix appears to contain normal collagen fibrils, but smaller and reduced numbers of proteoglycan matrix granules. Our studies reported here indicate that mutant, like normal cartilage, synthesizes type II collagen and contains normal quantities of glycosaminoglycans as judged by uronic acid content. However, the glycosaminoglycans from the mutant differ from the normal in their chromatographic and electrophoretic properties. Further studies established that glycosaminoglycans from cartilages of brachymorphic animals were undersulfated. Whereas chondroitinase digests of glycosaminoglycans from cartilage of normal $\text{C57Bl}\text{6J}$ 5-day-old mice contained predominantly disaccharides sulfated in the 4-position, that of the mutant contained appreciable unsulfated disaccharides as well.     

Aberrant metabolism of matrix components in neonatal fibular cartilage of brachypod (bpH) mice.

Collagen and the acid mucopolysaccharides (AMPS) were studied in the fibular cartilage of brachypod ($\text{bp}{}^{\mathrm{<mtext>H</mtext>}}\text{bp}{}^{\mathrm{<mtext>H</mtext>}}$) and normal (+/+) newborn mice. At this age the mutant fibulae are still cartilaginous and are comprised of closely packed chondrocytes, homogenous in size and shape.Brachypod fibular chondrocytes synthesized a normal cartilage-type collagen molecule but at half the normal rate. Incorporation of tryptophan indicated this was related to a depression of general protein synthesis rather than being specific for collagen. Pulse-chase experiments showed that collagen degradation over a 3-day culture period was 15% slower than normal thus accounting for the higher collagen content in mutant fibulae.AMPS synthesis in normals and brachypods was nearly equal; however, in pulse-chase experiments radioactivity could not be chased out of the mutant tissue. The failure of AMPS degradation also accounted for greater than normal quantity of AMPS in the mutant cartilage. Characterization of the AMPS led to the discovery of a small population of unsulfated chondroitin molecules in normal, but not brachypod cartilage. The importance of a coordinated metabolism of matrix products during limb development is discussed.     

A method for the determination of lysine-derived collagen crosslinks

A rapid and inexpensive method was devised for the determination of lysine-derived aldimine crosslink contents in collagen. The aldimines were converted to their secondary amine derivatives by NaBH₄ reduction, and the acid or base collagen hydrolysates analysed $\text{directly}$ for these derivatives (HLHNL and HLNL). It was found that in native bone, dentin and cartilage collagen fibres, every two tropocollagen molecules are joined by a minimum of one aldimine crosslink. Negligible amounts of HLNL and HNHNL were found in unreduced collagens, indicating that maturation does not involve a simple $\text{in vivo}$ reduction of the aldimine crosslinks.     

The collagen of chick embryonic notochord

Notochords, isolated from 2 $\text{1}\text{2}$ day chick embryos, were cultured in the presence of ³H proline and the labeled proteins co-purified with chick skin carrier collagen. The purified material, most of which eluted from CM-cellulose as a single peak in the region of the carrier collagen α1 chain, contained 41% of the incorporated proline as hydroxyproline and from gel filtration measurements had a molecular weight of approximately 100,000 daltons. When the material was chromatographed on DEAE-cellulose with carrier α1 chains from both skin [α1 (I)] and cartilage [α1 (II)], it eluted predominantly with the cartilage chains.     

Studies on glycosaminoglycans of regenerating rabbit ear cartilage

Cartilage regeneration in the adult rabbit ear was examined with respect to glycosaminoglycan (GAG) synthesis at various stages of the regeneration process. Increased hyaluronic acid and chondroitin sulfate synthesis was first seen 31 days after wounding, when a metachromatic cartilage matrix could be distinguished from blastemal cells. Analysis of cartilage and the overlying skin separately showed that 90% of the labeled chondroitin sulfate was found in the cartilage being regenerated. DEAE-cellulose chromatography of GAG preparations from 35-day regenerating cartilages showed hyaluronic acid and chondroitin sulfate peaks eluting in the same position as those isolated from normal cartilages. The identity of the hyaluronic acid and chondroitin sulfate peaks was confirmed by their susceptibility to Streptomyces hyaluronidase and chondroitinase ABC, respectively. Although the degree of sulfation in normal and regenerated cartilages was similar, the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate was increased in regenerated cartilages. GAG preparations from unlabeled cartilages were digested with chondroitinase ABC and the disaccharide digestive products were identified and quantitiated. Normal cartilage had a $\text{Δ}\text{Di-6S}\text{Δ}\text{Di-4S}$ ratio of 0.27; the same ratio for the regenerated cartilage was 1.58.     

Human aorta collagens: evidence for three distinct species

Three different molecular species of collagen and a soluble form of elastin were obtained by digestion of human aortas with pepsin. Two of the three collagens contain $\text{1}\text{2}$ cystine, present in interchain disulfide crosslinkages, and appear to represent type IV collagen previously described in basement membranes and type III collagen, recently found in fetal skin. The third collagen species is type I, the molecule found in a wide variety of connective tissues including skin, bone, tendon and ligaments.     

Degradation of cartilage proteoglycan and collagen by synovial cells: Stimulation by macrophages under activation by phagocytosis,lymphocyte factors,bacterial products or other inflammatory stimuli

When cultured together with dead ³⁵S-labelled cartilage discs or at the surface of $\text{[}{}^3\text{H]proteoglycan}\text{[}{}^{14}\text{C]collagen-coated}$ plates, synovial cells from either arthritic or normal rabbit joints digested both the proteoglycan and the collagen of the substrates after a lag-period of 1–2 days. These digestions were inversely related to the age (number of subculture passages) of the synovial cells and they could be modulated by serum components that were either inhibitory or stimulatory. They were dependent on a protein synthesis by the cells and were paralleled, in young cultures, by the release of collagenase and of a proteoglycan-degrading neutral proteinase. The co-culture of synovial cells with macrophages or their culture with macrophage-conditioned culture media caused a more rapid and more extensive degradation of collagen and proteoglycan due to the stimulation of the synovial cells by a nondialysable macrophage factor. The production of this synovial cell-activating ‘matrix regulatory monokine’ by the macrophage was enhanced by several immunological or inflammatory stimuli such as lymphocyte factors, phagocytosis, asbestos fibres, endotoxin, adjuvant muramyl dipeptide or chemotactic formyl-methionyl peptide, as well as by other membrane-active agents (phorbol myristate acetate, concanavalin A). It is presumed that these interactions are of importance in the development of cartilage destruction in rheumatoid and other chronic inflammatory arthritis.     

Defective PAPS-synthesis in epiphyseal cartilage from brachymorphic mice

Activity levels of sulfotransferases, requisite for the sulfation of chondroitin sulfate proteoglycan, were measured in cell-free homogenates prepared from neonatal epiphyseal cartilage of normal C57B1/6J or homozygous brachymorphic mice. In the presence of [³⁵S]-PAPS only or [³⁵S]-PAPS plus an exogenous sulfate acceptor, comparable amounts of ${}^{35}\text{SO}{}_4{}^{\mathrm{2?}}$ were incorporated into chondroitin sulfate by the normal and mutant types of cartilage. In contrast, the mutant cartilage catalyzed the conversion of only 30% of the ${}^{35}\text{SO}{}_4{}^{\mathrm{2?}}$ into chondroitin sulfate as compared to normal mouse cartilage when synthesis was initiated from ATP and H₂³⁵SO₄. These results suggest that the production of an undersulfated proteoglycan which has previously been reported in brachymorphic mice (Orkin, R.W. $\text{et}\text{al}$. (1976) Devel. Biol. $\text{50}$, 82–94) may result from a defect in the synthesis of the sulfate donor PAPS.     

Biosynthesis of collagen crosslinks in rabbit articular cartilage in vivo

The biosynthesis in vivo of the two reducible aldimine crosslinks of immature rabbit articular collagen, hydroxylysinohydroxynorleucine and hydroxylysinonorleucine, is demonstrated. The peak amount of crosslink was detected 1–2 weeks following labeling of the cartilage with [¹⁴C]lysine. The subsequent diminution which occurred was due primarily to a decrease in the amount of hydroxylysinohydroxynorleucine. Natural reduction of the aldimine crosslinks in vivo did not occur. Glucosylgalactosyl hydroxylysine and galactosylhydroxylysine, in a $\text{1.45}\text{1.00}$ ratio, were synthesized. Seventy-three percent of the hydroxylysine residues were glycosylated. [³H]NaBH₄ reduction of non-¹⁴C-labeled cartilage showed diminished amounts of reducible crosslink with time and the presence of hexosyl lysines and hexosyl hydroxylysines in mature articular cartilage.     

Activation of human breast carcinoma collagenase through plasminogen activator

Latent collagenase activity was detected in the media of a well-characterized line of human breast carcinoma cells maintained for over two years in culture. The media also contained sufficient plasminogen activator to convert extrinsically added plasminogen to plasmin which in turn activated the collagenase. During culture of the breast carcinoma in serum-free medium, collagenase activity was maximum on day 12 whereas plasminogen activator activity changed little with time. Using type I collagen as a substrate, the activated breast tumor collagenase produced $\text{3}\text{4}\text{?}\text{1}\text{4}$ fragments consistent with a mammalian collagenase. These findings suggest a pathologic role of plasminogen activator in the activation of latent collagenase during tumor invasion.A number of investigators have postulated that proteases may play a role in tumor invasion (1–5). Collagenase is one such protease which is active at neutral pH and specifically cleaves triple helical collagen into two ($\text{3}\text{4}\text{?}\text{1}\text{4}$ fragments (6). Secretion of collagenase by tumor cells migrating from the primary mass provides an attractive hypothesis for the mechanism of tumor invasion of surrounding host connective tissue—since the local environment would likely be at neutral pH. Consequently, a number of investigators have reported significant levels of collagenase activity in a wide variety of tumors (7–14). Abramson (13) has correlated aggressive $\text{in vivo}$ growth in carcinomas of the head and neck with collagenase activity, and Kuettner et al. (14) have postulated that inhibitors of collagenase may prevent tumors from invading cartilage.Collagenase is produced in both latent and active forms (6). The latent form can be activated with brief protease treatment (15). Since one of the proteases capable of activating collagenase is plasmin (15), the possibility arose that tumor cells could activate collagenase through plasminogen activator. Plasminogen activator secreted by tumor cells (4, 5) could convert plasminogen zymogen to plasmin which would in turn activate latent tumor collagenase. Testing this hypothesis $\text{in vitro}$ was the subject of the present study.Previous studies on collagenase from human carcinoma (7, 13, 14) have suffered from the drawback that contaminating inflammatory cells and fibroblasts may have been the source of the collagenase. Therefore, we have studied collagenase production from cultured human breast carcinoma cells which have been well characterized to be mammary epithelial in origin, malignant in karyotype, and able to grow in nude mice. Production of collagenase from these cells is therefore unequivocally of human carcinoma origin. The time course of latent collagenase and plasminogen activator secretion by these cultured tumor cells was studied following withdrawal of serum. To test whether plasminogen activator was secreted in sufficient amounts to indirectly activate latent collagenase, collagenase activity of the culture media was studied after the extrinsic addition of plasminogen. Finally, to verify that the tumor-secreted collagenase cleaved type I collagen at a single locus, enzyme degradation products were studied by gel electrophoresis.     

Analysis of cartilage differentiation from skeletal muscle grown on bone matrix: II. Chondroitin sulfate synthesis and reaction to exogenous glycosaminoglycans

In the first paper in this series (Nathanson, M. A., and Hay, E. D. (1980). Develop. Biol. 78, 301–331), we described the ultrastructural alterations that take place when embryonic skeletal muscle is induced to form hyaline cartilage by demineralized bone matrix in vitro. In this paper, we analyze the pattern of appearance of chondroitin sulfates and dermatan sulfate in injured muscle in situ and in explants of muscle cultured either on bone matrix or on collagen gel. We also investigate the effects of exogenous glycosaminoglycans on the cultures to determine whether chondroitin sulfate (Ch-S) and hyaluronic acid (HA) can enhance or inhibit the biochemical differentiation of cartilage under these conditions. Our results indicate that during the first morphological phase, 1–3 days in vitro, there is an increased sulfate uptake, a shift in the relative abundance of Ch-S, and an increase in the ratio of chondroitin-4-sulfate (Ch-4-S) to chondroitin-6-sulfate (Ch-6-S); this change is correlated with the transformation of myoblasts to fibroblast-like cells in both types of cultures. A similar increase in the $\text{Ch-4-S}\text{Ch-6-S}$ ratio occurs in injured muscle in situ, suggesting that phase I is a regenerative response. Explants on bone matrix sustain Ch-4-S levels between 4 and 5 days (phase II) and show a large increase in Ch-4-S and sulfate incorporation when they form cartilage at 6–10 days (phase III). Explants on collagen gels regenerate muscle at 4–10 days with decreasing $\text{Ch-4-S}\text{Ch-6-S}$ ratios and decreasing sulfate incorporation. The data demonstrate that an environmental influence, such as trauma, is sufficient to alter the biosynthetic expression of skeletal muscle and that under appropriate conditions (such as the presence of bone matrix) this response may be augmented, leading to the synthesis of extracellular matrix components at ratios characteristic of cartilage. Exogenous Ch-S and HA did not significantly effect this overall pattern. These results are discussed in relation to the morphological observations presented in the preceding paper.     

A comparative study of the distribution of the stable crosslink,pyridinoline, in bone collagens from normal,osteoblastoma, and vitamin D-deficient chicks

Tryptic peptides of bone collagens from 4-week-old normal, osteoblastoma and vitamin D-deficient chicks were studied using gel filtration chromatography. Absorbance at 230 nm and fluorescence (excitation at 330 nm, emission at 390 nm) of $\text{each}\text{fraction}$ were measured. The relative quantities of each peak from the absorbance and fluorescence patterns were semiquantified by planimetry. Osteoblastoma bone collagen had a prominent, fluorescent, crosslinked peptide that contained pyridinoline. Fluorescence of this pyridinoline-containing peak in AO collagen was much greater than in the vitamin D-deficient and normal bone collagen counterparts. A comparison of fluorescence patterns clearly showed that the distribution of pyridinoline in collagen from normal and diseased bone was totally dissimilar.The dissimilarities in distribution of pyridinoline in these bone collagens may be attributed to differences in the degree of lysine hydroxylation, to the degree of mineralization, or some other factor.     

Elevation of maternal glucocorticoid hormones alters neurotransmitter phenotypic expression in embryos

The tissue interaction between the notochord and the somites of the vertebrate embryo establishes the proper shape and constitution of the vertebral cartilage. Soon after somite formation, the somite differentiates into a cartilage-forming part, the sclerotome, and a muscle and skin-forming part, the dermamyotome. These components of the somite were dissected from $\text{3}\text{1}\text{2}\text{-}\text{day-old}$ chick embryos (stage $\text{18}\text{1}\text{2}\text{–19}$ and cultured in vitro in the presence or absence of notochord. It was found that the sclerotome cells respond to the notochord by an increased incidence of hyaline cartilage nodules, greater accumulation of sulfated glycosaminoglycans, synthesis of larger aggregates of proteoglycans, increased DNA accumulation, and accelerated DNA synthesis. The dermamyotome did not show these changes. These results indicate that the notochord enhances cartilage differentiation in the sclerotome. Under these conditions, the notochord did not elicit cartilage formation in the dermamyotome.     

Type-specific collagenolysis: a type V collagen-degrading enzyme from macrophages

An enzymatic activity capable of degrading type V collagen at neutral pH was found in the medium from cultured rabbit pulmonary alveolar macrophages which had been “activated” $\text{in}\text{vivo}$ by injection of complete Freund's Adjuvant. This enzyme was characterized as a metalloproteinase by virtue of its inhibition by EDTA but not by phenylmethylsulfonyl fluoride or N-ethyl maleimide. Ion-exchange chromatography on DEAE-cellulose was successful in separating the type V collagen-degrading activity from the type I collagenase which is also secreted by these cells. These observations suggest that the degradation of type V collagen is independent of the degradation of the interstitial collagens and may require the action of its own “specific collagenase”.     

Cell surface antigens on erythroid cells: A comparison of normal and anemic (WW) mice

Cell surface antigens of normal and anemic ($\text{W}\text{W}$) mouse erythroid cells have been examined in cytotoxicity assays with two rat antisera. When tested on fetal liver cells, a rat anti-erythroblast serum recognized antigen(s) present on erythroid cells early in development, while rat anti-adult red blood cell serum recognized antigen(s) present on mature erythroid cells. Each of these sera had different activity on normal (+/+ or $\text{W}\text{+}$) as compared to anemic ($\text{W}\text{W}$) erythroid cells.     

Skunks have gene sequences in their cellular DNA related to squirrel monkey retrovirus: transmission between species of a new world primate endogenous type D retrovirus.

The squirrel monkey ($\text{Saimiri}$,$\text{sciureus}$), a New World primate, contains multiple copies of endogenous type D retroviral gene sequences in the cellular DNA of all its tissues. Gene sequences partially homologous to these type D virus genes are also found in the cellular DNA of normal tissues of the New World carnivore, the skunk ($\text{Mephitis}$,$\text{mephitis}$ and $\text{Spilogale}$,$\text{putorius}$). We there-fore conclude that this class of viruses has, under natural conditions, been transmitted between the germ lines of these evolutionarily distant species. The example of interspecies transmission described here is the first that has been described among New World species and also the first that has been demonstrated for retroviruses other than type C viruses.     

Comparative analysis of the sequences of the three collagen chains α1(I), α2 and α1(III): Functional and genetic aspects

The amino acid sequences of type I collagen containing α1(I) and α2 chains at a ratio of 2:1, and of type III collagen consisting of α1 (III) chains are known. A statistical analysis of the sequences of these α chains is presented. The inter-chain comparison showed a high level of homology between the three α chains. The interactive amino acids, such as the polar charged and part of the hydrophobic residues responsible for the assembly of the molecules, are strongly conserved. The intra-chain analysis revealed that the α chains are divided into four related D units, each with a length of 234 residues. Between the D units within a chain the polar residues show a higher variability than the hydrophobic amino acids.Besides the D units, other periodicities such as $\text{D}\text{3}$ (78 residues), $\text{D}\text{6}$ (39 residues), $\text{solD}\text{11}$ (21 residues) and $\text{solD}\text{13}$ (18 residues) were observed, particularly in α1 (I) and α1 (III). The D unit is a functional repeat that is formed by the interactive polar charged and hydrophobic residues and which determines the aggregation of the molecules. The $\text{solD}\text{3}$ unit is mainly pronounced by the non-interactive residues such as proline and alanine and appears to be a reminiscence of a primordial gene. The smaller periodic repeating units may be considered as additional genetic units or as structural units, which determine the triplehelical pitch and thus the lateral aggregation of the molecules.In contrast to α1 (I) and α1 (III), the α2 chain shows less regularity in its internal structure.     

Maturation of collagenous tissue: Specific invivo proteolytic cleavage of only α1(I) chains

A partially cleaved α1(I) chain, α1_χ, has been isolated from earlier synthesized or older (acid-extracted) guinea pig skin collagen. The α1_χ component is shown to be absent from the newly synthesized (neutral salt-extracted) collagen. This degradation is a result of specific $\text{in}\text{vivo}$ proteolytic sission of α1(I) chain since the soluble collagen has no corresponding product from the α2 chain. The $\text{in}\text{vivo}$ proteolytic cleavage is believed to result from processes related to natural physiological maturation of collagenous tissue.     

Molecular structure and physical properties of type IV collagen in solution

The physical properties of intact type IV collagen from the mouse EHS sarcoma were studied in acid solution using laser light scattering and viscometry. The experimentally observed values of molecular weight, translational diffusion coefficient, particle scattering factor at 175.5° and a wavelength of 633 nm and intrinsic viscosity at 22°C were 532000, 0.66 × 10⁻⁷cm²s⁻¹, 0.492 and 74.7 ml/g respectively. Plots of $\text{Kc/}\text{R}{}_0$ versus collagen concentration were linear with a slope of approximately 0, indicating that under the conditions studied, type IV collagen molecules do not form supra-molecular aggregates. Experimentally determined translational diffusion coefficients closely approximated the calculated value for a rod-like molecule 424 nm long and 1.5 nm in diameter. Based on this observation, it is concluded that the type IV collagen molecule translates like a bent rigid rod similar to the interstitial collagens. However, the low intrinsic viscosity and larger value of the particle scattering factor for type IV collagen molecules in comparison with the interstitial collagens indicate that type IV collagen is considerably more flexible. Physical measurements on molecules in solution are consistent with a model of the type IV molecule containing numerous flexible bends with bend angles less than 125°. It is concluded that the type IV collagen molecule behaves like a worm-like rod in solution.     

Preferential synthesis of yeast mitochondrial DNA in alpha factor-arrested cells

α factor is a diffusible substance produced by $\text{S. cerevisiae}$ cells of the $\text{α}$ mating type which inhibits cell division (1) and the initiation of nuclear DNA synthesis (2) in cells of the $\text{a}$ mating type. In this report, it is shown that mitochondrial DNA synthesis continues at a normal rate in $\text{a}$ cells for at least 6 hours in the presence of α factor, resulting in a 5-fold increase in the amount of mitochondrial DNA per cell. The continued synthesis of mitochondrial DNA in the absence of nuclear DNA synthesis allows specific labeling of yeast mitochondrial DNA.