Canine COL1A2 mutation resulting in C-terminal truncation of pro-alpha2(I) and severe osteogenesis imperfecta.

RNA and type I collagen were analyzed from cultured skin fibroblasts of a Beagle puppy with fractures consistent with type III osteogenesis imperfecta (OI). In a nonisotopic RNAse cleavage assay (NIRCA), the proband's RNA had a unique cleavage pattern in the region of COL1A2 encoding the C-propeptide. DNA sequence analyses identified a mutation in which nucleotides 3991-3994 ("CTAG") were replaced with "TGTCATTGG." The first seven bases of the inserted sequence were identical to nucleotides 4002-4008 of the normal canine COL1A2 sequence. The resulting frameshift changed 30 amino acids and introduced a premature stop codon. Reverse-transcription polymerase chain reaction (RT-PCR) with primers flanking the mutation site amplified two complementary DNA (cDNA) fragments for the proband and a single product for the control. Restriction enzyme digestions also were consistent with a heterozygous mutation in the proband. Type I procollagen labeled with [3H]proline was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Increased density of pC-alpha2(I) suggested comigration with the similarly sized pro-alpha2(I) derived from the mutant allele. Furthermore, a-chains were overhydroxylated and the ratio of alpha1(I):alpha2(I) was 3.2:1, consistent with the presence of alpha1(I) homotrimers. Analyses of COL1A2 and type I collagen were both consistent with the described heterozygous mutation affecting the pro-alpha2(I) C-propeptide and confirmed a diagnosis of OI.

Acetylcholine receptor regulation in L5 muscle cells is independent of increases in collagen secretion induced by ascorbic acid.

Ascorbic acid is the active component of fetal brain extract that induces increased acetylcholine receptor (AChR) expression in L5 rat clonal muscle cell cultures. The induction of AChR expression, as determined by 125I-alpha-bungarotoxin binding, occurs with a delay of 20-25 h. We report that the delayed increase in AChR can be triggered by a 5-h exposure to ascorbic acid. These studies suggest that intermediary processes may be involved. Ascorbic acid treatment also causes a threefold increase in collagen secretion in L5 cultures by 3 h. The rapid increase in collagen secretion and the delayed induction of surface AChR suggested that there may be a link between these two responses. However, although bacterial collagenase eliminates secreted collagen, it had no effect on the increase in surface AChR. Thus, the ascorbic acid effect on elevating AChR expression is independent of its effect on collagen secretion.

Effects of exercise and polysulfated glycosaminoglycan on repair of articular cartilage defects in the equine carpus.

Our aim was to determine if mild to moderate postoperative exercise and intra-articular polysulfated glycosaminoglycan result in improved repair of large, experimentally induced osteochondral defects in a weight-bearing surface of equine joints. Arthroscopic debridement was used to produce full-thickness defects in a weight-bearing area of the radial carpal bones in 18 ponies. The ponies were randomly assigned to two groups balanced for age: nine animals in the exercise and nine in the no exercise group. Six ponies in each group were medicated weekly for 5 weeks with an intra-articular injection of polysulfated glycosaminoglycan in one middle carpal joint beginning at the time of operation. Walking (twice daily) was begun 6 days postoperatively, and by the twelfth week postoperatively the ponies were trotting for 25 min and walking for 15 min twice daily. At the time of the ponies' death, 17 weeks postoperatively, each defect had an average of 50-75% coverage with repair tissue. Exercised, medicated joints had a significantly smaller area of coverage with repair tissue than exercised, nonmedicated joints. Cartilaginous repair tissue from exercised ponies contained significantly more glycosaminoglycan and type-II collagen (r = 0.53, p < 0.05). The ratio of hydroxylysine to hydroxyproline was significantly lower and the ratio of collagen content to total protein was significantly higher in the repair tissue of medicated joints than in the repair tissue of nonmedicated joints; this is consistent with the presence of less type-II collagen in the repair tissue in medicated joints. We concluded that postoperative exercise was beneficial and that the immediate postoperative use of intra-articular polysulfated glycosaminoglycan was detrimental to the development of cartilaginous repair tissue in large osteochondral defects of equine joints.

Structure of equine type I and type II collagens.

Collagen type I was purified from equine skin and flexor tendon, and type II collagen was purified from equine articular cartilage. The proteoglycans in these tissues were extracted, using guanidine HCl; the collagens were solubilized, using pepsin digestion, then were selectively precipitated with NaCl. Gel electrophoresis indicated that the precipitates contained only type I or type II collagen. Amino acid analysis indicated that collagen constituted > 97% of the total protein in the precipitates. Hydroxylation of proline was 42.0 +/- 0.6% (mean +/- SEM) in alpha 1(I) and alpha 2(I), and was 48.1 +/- 1.3% in alpha 1(II) chains. The hydroxylation of lysine was 23.2 +/- 0.7% in alpha 1(I) and 34.1 +/- 0.9% in alpha 2(I) chains from tendon, and 49.6 +/- 4.3% in alpha 1(II) chains from cartilage. The cyanogen bromide (CB)-peptide patterns of chromatographically purified equine alpha 2(I) and alpha 1(II) chains were similar to those published previously for rat, bovine, and human alpha 2 and alpha 1 chains. However, the CB-peptide pattern of the equine alpha 1(I) chain resembled the guinea pig alpha 1(I) chain, which has no methionine between CB7 and CB6. Purified equine alpha 1(I)CB7,6 contained no methionine, methionine sulfoxide, or homoserine lactone. Mass of 42.26 kd was determined by use of mass spectrometry, and N-terminal sequence analysis established that the first 12 amino acids of this CB7,6 were identical to the sequence of human alpha 1(I)CB7.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical signs and diagnosis of osteogenesis imperfecta in three dogs.

When a young dog is evaluated for multiple fractures with minimal to no accompanying trauma, the primary differential diagnoses are metabolic disease, physical abuse, and osteogenesis imperfecta (OI). Of these, secondary hyperparathyroidism is most common, but if serum concentrations of ionized calcium, phosphorus, vitamin D, and parathormone are within reference ranges, OI must be considered. Osteogenesis imperfecta is a heritable disease characterized by brittle bones. Results of studies using cultured skin fibroblasts indicate that most cases of OI in human beings are caused by a mutation in a type-I collagen gene. Osteogenesis imperfecta was recently identified in 3 dogs. Radiographic findings included multiple fractures in various stages of healing and generalized osteopenia. Cultured fibroblasts from skin biopsy specimens were used to diagnose OI. Structural abnormalities were found in type-I collagen from each dog. This cell culture assay can be used to evaluate dogs with brittle bones.

Cross-validation of cyanogen bromide-peptide ratios to measure the proportion of type II collagen in pepsin digests of equine articular cartilage, meniscus, and cartilage repair tissue.

Collagen type I and type II were purified from equine flexor tendon and articular cartilage, respectively. Equal amounts of these collagens were cleaved with cyanogen bromide, and 11 mixtures containing increasing proportions of type II collagen were separated in seven identical sodium dodecyl sulfate-polyacrylamide gels. The density of bands was measured in wet gels and the peak areas were used to form six ratios of peptide bands that had polynomial relationships with the known proportions of type I and type II collagen in the mixtures. Calibration curves for determining the proportion of type II collagen in the mixtures were constructed using ratios and combinations of ratios of peak areas. Cross-validation was used to identify calibration curves with the smallest squared prediction error or squared average prediction error for all combinations of ratios. Ratios of peak areas of each one of the seven gels were treated, in turn, as the "unknown," and a prediction was carried out using these unknowns and the ratios from the other six gels. Two ratios had the smallest squared average prediction error and calibration curves were computed for these ratios with all seven gels. These curves were used to estimate the proportion of type II collagen in the pepsin-soluble and the pepsin-resistant fractions of articular cartilage inner and outer meniscus, and cartilage repair tissue. Cross-validation enabled selection of the cyanogen bromide-peptide ratios for calibration curves that resulted in the most accurate estimation of the proportion of type II collagen in pepsin digests of tissues.

Sequence of normal canine COL1A1 cDNA and identification of a heterozygous alpha1(I) collagen Gly208Ala mutation in a severe case of canine osteogenesis imperfecta.

The sequence of canine COL1A1 cDNA was determined from four overlapping COL1A1 RT-PCR products generated from canine fibroblast RNA. In the translated region, nucleotide identity between canine and human COL1A1 cDNA was 93.2%, although the canine sequence lacked nucleotides 204 to 215 in the region coding for the N-propeptide. Amino acid identity was 97.7%. Total RNA and type I collagen were collected from cultured skin fibroblasts of a 12-week-old male golden retriever with pathologic fractures suggestive of osteogenesis imperfecta (OI) and dentinogenesis imperfecta. Sequential, overlapping approximately 1,000-bp fragments of COL1A1 and COL1A2 cDNA were each amplified by RT-PCR using primers containing 5' T7 polymerase sites. These PCR products were transcribed with T7 RNA polymerase, hybridized into RNA duplexes, and cleaved at mismatch sites with RNase. The proband had an unique cleavage pattern for the fragment of COL1A1 mRNA spanning nucleotides 709 to 1,531. Sequence analysis identified a G to C point mutation for nucleotide 1,276, predicting a codon change from glycine (GGA) to alanine (GCA) for amino acid 208. This change disrupts the normal Gly-X-Y pattern of the collagen triple helix. Restriction enzyme digestion of the RT-PCR product was consistent with a heterozygous COL1A1 mutation. Type I collagen was labeled with 3H-proline, salt precipitated, and analyzed by SDS-PAGE. Pepsin digested alpha chains were over-hydroxylated, and procollagen processing was delayed. Thus, canine and human OI appear homologous in terms of clinical presentation, etiology, and pathogenesis.

Effect of methylprednisolone acetate on proteoglycan and collagen metabolism of articular cartilage explants.

OBJECTIVE: The effect of different doses of methylprednisolone acetate (MPA) on proteoglycan and collagen metabolism of articular cartilage from normal equine joints was tested in vitro. METHODS: Cultured explants were treated with 0, 0.0004, 0.004, 0.04, 0.4 and 4.0 mg/ml (approximately 10(-6)-10(-2) M) MPA for 72 h. Proteoglycan synthesis was measured by incorporation of sodium [35S]sulfate into proteoglycans and proteoglycan degradation was measured by release of total and radiolabeled proteoglycan into the culture media. The size of the proteoglycans was assessed with size exclusion chromatography under both associative and dissociative conditions. Total protein synthesis was measured by incorporation of [3H]proline and collagen synthesis by formation of [3H]hydroxyproline. RESULTS: Treatments of 0.0004, 0.004, and 0.04 mg/ml MPA caused a significant (p < 0.05) increase in total protein synthesis compared to the control, and doses of 0.0004 and 0.004 mg/ml MPA tended to increase (p < 0.1) collagen synthesis. In contrast, MPA doses of 0.04 mg/ml or less had no effect on proteoglycan synthesis compared to control. Proteoglycan, total protein, and collagen synthesis were severely depressed with the 0.4 and 4.0 mg/ml MPA treatments. The lowest doses of MPA had minimal effect on proteoglycan degradation, while 0.4 and 4.0 mg/ml MPA decreased degradation of total proteoglycan in a dose dependent fashion. Degradation of newly synthesized proteoglycan in the explants was significantly decreased (p < 0.05) by 4.0 mg/ml MPA. There was positive correlation (r = 0.83, p < 0.05) between the effect of MPA on proteoglycan synthesis and the release of total proteoglycan into the culture media. Chromatography under associative conditions showed that treatment with MPA (0.4 and 0.004 mg/ml) decreased the size and increased the polydispersity of aggrecan and induced synthesis of the small nonaggregating proteoglycans. Aggrecan monomers from cartilage treated with MPA included a population of smaller monomers, which resulted in greater polydispersity than those from control cartilage. CONCLUSION: As well as decreasing synthesis, loss of proteoglycan from MPA treated cartilage in vivo may be partly due to the synthesis of a population of smaller proteoglycans than those extracted from untreated cartilage, and these may not interact with hyaluronan and may not be retained permanently in the matrix. Methylprednisolone acetate may affect posttranslational modification of the core protein with the addition of smaller and possibly fewer glycosaminoglycan chains.

Local and remote matrix responses to chondrocyte-laden collagen scaffold implantation in extensive articular cartilage defects.

Chondrocyte-laden collagen scaffolds were evaluated in extensive cartilage defects in an equine model. Arthroscopic techniques were used to implant a chondrocyte-collagen culture product in 15-mm defects in the lateral trochlear ridge of the femoropatellar joint of 12 horses. Ungrafted control defects were formed in the opposite joint. Groups of six horses were terminated at 4 and 8 months after implantation and the repair sites, adjacent cartilage, and remote cartilage within each femoropatellar joint examined biochemically. Eight months following surgery the relative proportions of type II collagen in grafted and ungrafted defects, determined using the ratio of cyanogen bromide cleavage products alpha 1(II)CB10/alpha 2(I)CB3,5, were not significantly different (31.57 +/- 2.76% and 26.88 +/- 2.76%, respectively). Aggrecan content was significantly improved in grafted defects (85.61 +/- 6.51 and 74.91 +/- 10.31 micrograms/mg dry weight). Cartilage surrounding grafted defects also showed improved maintenance of cartilage glycosaminoglycan content. Thus, chondrocyte grafting in collagen scaffold vehicles improved the aggrecan content in extensive cartilage defects and surrounding normal cartilage. However, given the continued disparity between repair tissue and normal cartilage aggrecan content, and the low proportion of type II collagen in grafted defects, the utility of collagen scaffolds for chondrocyte grafting of large cartilage defects seems limited.

Sequence of canine COL1A2 cDNA: nucleotide substitutions affecting the cyanogen bromide peptide map of the alpha 2(I) chain.

The alpha2 chain of canine type I collagen was characterized with both sequence analysis of COL1A2 cDNA and chemical analysis of alpha2(I) chains. The complete sequence of canine COL1A2 cDNA was determined from reverse-transcribed and polymerase chain reaction-amplified total RNA from cultured skin fibroblasts. Pepsin-digested and cyanogen bromide-digested type I collagen peptides were analyzed with chromatography, gel electrophoresis, and mass spectrometry. Identity between the sequences of canine and human COL1A2 cDNA was 90.9%, predicting conservation of the 3 cysteine residues required for C-propeptide registration and of cleavage sites for signal peptidase, procollagen N-proteinase, vertebrate collagenase, and procollagen C-proteinase. Conservation of all 50 lysine residues was also predicted, including preservation of the 1:2 asymmetry in the X:Y distribution of the 31 lysine residues in the alpha2(I) triple helix. The human and canine sequences differed in the location of Y-position proline residues and the presence of two unique canine cyanogen bromide peptides, alpha2 CB3b and alpha2 CB3c,5. Knowledge of the conserved and unique features of canine COL1A2 will be valuable for analysis of the expression, synthesis, and structure of type I collagen as well as studies of canine osteogenesis imperfecta.

Secreted collagen induced by ascorbic acid in L5 cloned muscle cultures does not affect acetylcholine receptor expression.

Previous studies have shown that ascorbic acid increases both the total surface acetylcholine receptor (AChR) expression and the mRNA for the alpha-subunit of this receptor in myotubes of cloned L5 muscle cultures. Since ascorbic acid increases collagen synthesis in fibroblasts, we studied the effect of ascorbic acid on collagen secretion in L5 muscle cells and investigated the possibility that the effects of ascorbic acid on collagen and AChR are related. We report that L5 muscle cells secrete collagen types I, III, and V, with collagen type I being the most abundant species, and that accumulation of secreted collagens increased in the medium approximately two- to ninefold within 3 h of ascorbic acid treatment. The increase in surface AChRs, on the other hand, developed more slowly, and was detected only about 20-24 h after ascorbic acid treatment. A short (5 h) treatment with ascorbic acid is, however, sufficient to trigger an increase in AChRs 24 h later. Since ascorbic acid caused a rapid increase in collagen secretion, whereas the effect on total surface AChRs occurs more slowly, we tested the possibility that an increase in secreted collagen might be necessary for the increase in AChRs. However, when the L5 cultures were treated with bacterial collagenase, the ascorbic acid-induced increase in secreted collagen was abolished but its inductive effect on AChRs was unchanged. The increase in secreted collagen is therefore not necessary for the increase in AChRs to occur.

Changes in the types of collagen synthesized during chondrogenesis of the mouse otic capsule.

We have investigated the temporal relationship between the morphological differentiation of the mouse otic capsule and the pattern of collagen synthesis by mouse otocyst-mesenchyme complexes labeled in vitro. In 10.5- to 12-day embryos the mesenchyme surrounding the otocyst was loosely organized except for a few lateroventral condensations; explants from these embryos synthesized only small amounts of collagen. Collagen synthesis by whole explants increased by more than 50% between 12 and 13 days concomitant with metachromatic staining of the lateral periotic mesenchyme. Cartilage specific type II collagen was the predominant collagen synthesized by these explants as confirmed by SDS-PAGE, densitometry, CNBr cleavage, and V8 protease digestion. This biochemical expression of the cartilage phenotype preceded morphologic recognition of otic capsular cartilage by almost 2 days. Type II collagen synthesis continued to increase and predominate through Day 16 of gestation by which time the otic labyrinth was surrounded by mature cartilage. The minor cartilage collagen chains, 1 alpha, 2 alpha, and 3 alpha, first appeared on different days of gestation. The 1 alpha, and 3 alpha chains were synthesized by explants from 11-day embryos while the 2 alpha chain appeared during Day 13, just before overt differentiation of mature cartilage. These results suggested that the 1 alpha, 2 alpha, and 3 alpha chains may not form heterotrimers containing all three chains and that synthesis of the 2 alpha chain may be associated with stabilization of the cartilaginous matrix. Comparison of these data with the patterns of collagen production by mutant, diseased, or experimentally manipulated inner ear tissues may provide insights into the molecular basis of chondrogenic tissue interactions.

Temperature-induced post-translational over-modification of type I procollagen. Effects of over-modification of the protein on the rate of cleavage by procollagen N-proteinase and on self-assembly of collagen into fibrils.

Previous observations suggested that incubating fibroblasts at elevated temperature caused over-modification of type I procollagen by post-translational enzymes because of a delay in folding of the collagen triple helix. Here, human skin fibroblasts were incubated at 40.5 instead of 37 degrees C, and the type I procollagen secreted into the medium was isolated. Analysis of the protein indicated that there was an increase of about 5 residues of hydroxylysine/alpha chain and about 1 residue of glycosylated hydroxylysine/alpha chain. Assays with procollagen N-proteinase indicated that the N-propeptide of the over-modified collagen was cleaved at a decreased rate, apparently because the over-modification altered the conformation-dependent cleavage site for the enzyme. Assays in a system for assembly of collagen into fibrils demonstrated that the over-modified protein had a higher critical concentration for self-assembly. Also, the fibrils formed from the over-modified collagen at 31 and 29 degrees C had smaller diameters than fibrils formed from normal type I collagen. The results provide direct evidence for earlier suggestions that post-translational over-modification of a fibrillar collagen can alter the morphology of the fibrils formed. The results also indicate that some of the biological consequences of the mutations in type I procollagen causing heritable disorders must be ascribed to the effects of post-translational over-modifications that frequently occur as secondary consequences of changes in the primary structure of the protein.

Degradation of monomeric and fibrillar type III collagens by human skin collagenase. Kinetic constants using different animal substrates.

Human skin collagenase activity was examined against type III collagens, in both soluble and fibrillar form, from different animal species. In either form, human, dog, and cat type III were degraded 10- to 30-fold faster than was that from guinea pig and nearly 100-fold more readily than chick type III. These differences in susceptibility were mirrored by essentially identical differences in the rate of trypsin cleavage of the same substrates. Human, dog, and cat type III were cleaved most rapidly by trypsin, guinea pig III more slowly, and chick III was completely resistant to the serine protease. Arrhenius plots, relating enzyme activity to temperature, revealed differences in the various type III substrates consistent with their collagenase and trypsin susceptibilities. Human, dog, and cat type III collagens yielded nonlinear plots, with accompanying activation energies which decreased at temperatures above 26 degrees C; guinea pig type III displayed a plot which deviated only slightly from linearity while the plot for chick type III was completely linear. These data strongly suggest that type III collagens display substantial variability in the stability of the helix at or near the collagenase cleavage site. The susceptibility of these type III substrates as reconstituted fibrils was also examined. The relative rates of degradation of these substrates by collagenase, and by trypsin, were the same as those observed in solution. The absolute rates of degradation of collagen in fibrillar form, however, were massively lower than predicted by extrapolation from solution values. This reduction in rate is even greater for type III than for type I collagens. Thus, whereas in solution type III substrates are cleaved much faster than type I collagens, in fibrillar form these differences are less than 2-fold. These data, together with values for activation energies and deuterium isotope effects on type III fibrillar substrates, reinforce the concept that helical integrity near the collagenase cleavage site is a major specifier of the rate of collagenase activity. Furthermore, the data suggest that the exclusion of water accompanying the tight packing of monomers into fibrils presents a major energy barrier to collagenase activity, which is particularly large for type III collagen.

A mutation in the pro alpha 2(I) gene (COL1A2) for type I procollagen in Ehlers-Danlos syndrome type VII: evidence suggesting that skipping of exon 6 in RNA splicing may be a common cause of the phenotype.

Fibroblasts from a proband with Ehlers-Danlos syndrome type VII synthesized approximately equal amounts of normal and shortened pro alpha 2(I) chains of type I procollagen. Nuclease S1 probe protection experiments with mRNA demonstrated that the pro alpha 2(I) chains were shortened because of a deletion of most or all of the 54 nucleotides in exon 6, the exon that contains codons for the cleavage site for procollagen N-proteinase. Sequencing of genomic clones revealed a single-base mutation that converted the first nucleotide of intron 6 from G to A. Therefore, the mutation was a change, in the -GT-consensus splice site, that produced efficient exon skipping. Allele-specific oligonucleotide hybridizations demonstrated that the proband's mother, father, and brother did not have the mutation. Therefore, the mutation was a sporadic one. Analysis of potential 5' splice sites in the 5' end of intron 6 indicated that none had favorable values by the two commonly employed techniques for evaluating such sites. The proband is the fourth reported proband with Ehlers-Danlos syndrome VII with a single-base mutation that causes skipping of exon 6 in the splicing of RNA from either the COL1A1 gene or COL1A2 gene. No other mutations in the two type I procollagen genes have been found in the syndrome. Therefore, such mutations may be a common cause of the phenotype. The primers developed should be useful in screening for the same or similar mutations causing the disease.

Acute synovitis and intra-articular methylprednisolone acetate in ponies.

OBJECTIVE: To determine how acute synovitis, with and without intra-articular methylprednisolone acetate (MPA), affect synthesis of proteoglycan, total protein, and collagen in articular cartilage and total protein synthesis in synovial membrane. DESIGN: Synovitis was induced in 10 ponies by the injection of 0.5 ng lipopolysaccharide (LPS) into the left radiocarpal and midcarpal joints every 2 days for a total of four treatments. Synovitis was documented by clinical examination and synovial fluid analyses. Two days before euthanasia, MPA (0.1 mg/kg) was injected with the last dose of LPS into both the left and right radiocarpal and midcarpal joints of five of these ponies. Proteoglycan synthesis in articular cartilage explants from these joints was measured by incorporation of sodium [35S]sulfate. The size of the proteoglycan monomers and their aggregation with hyaluronan was assessed by size-exclusion chromatography. Protein synthesis in articular cartilage was measured by incorporation of [3H]proline and collagen synthesis by conversion of [3H]proline into [3H]hydroxyproline. Protein synthesis was measured in synovial membrane explants by incorporation of [35S]methionine. RESULTS: Ponies developed carpal effusion and mild lameness accompanied by increased total nucleated cell count and total solids in synovial fluid in response to the LPS injections. Moderate to severe synovial membrane proliferation and inflammation were observed histopathologically in joints injected with LPS but no consistent light-microscopical changes were observed in the articular cartilage from these joints. Intra-articular MPA alone was associated with decreased proteoglycan synthesis and increased protein and collagen synthesis in the cartilage explants. Total protein synthesis by synovial membrane was also increased by MPA alone. In contrast, no differences in protein or proteoglycan synthesis were observed in explants from the joints with synovitis, with or without intra-articular MPA. Treatment with MPA, LPS, and LPS/MPA did not alter proteoglycan aggregate size, but LPS-induced synovitis resulted in an increase in the second largest population of monomers. MPA increased the synthesis of small proteoglycan monomers. CONCLUSION: Based on the methods used, acute synovitis prevented changes induced by intra-articular MPA alone. Results suggested that the effect of intra-articular MPA on joint metabolism was different between inflamed and normal joints. Experimental studies must consider the effect of inflammation, as well as the potential to introduce in vitro culture artifacts when investigating the effect of intra-articular corticosteroids on chondrocyte function.

Studies of collagen in bone and dentin matrix of a Columbian mammoth (late Pleistocene) of central Utah.

A Columbian mammoth, Mammuthus columbi, was excavated at an elevation of 9000 feet in Huntington Canyon, Emery County, Utah. Radiocarbon dates on the skeleton indicated death approximately 11,200 years ago. The skeleton was removed from postglacial, Late Quaternary, lake sediments deposited as glacial runoff approximately 9500 years ago. The bones and teeth were especially well preserved in a saturated lake bed. After excavation the bones and teeth were preserved by controlled desiccation, without hardeners, over a period of 9 months. Microradiography, light and electron microscopy, medium and high angle X-ray diffraction, amino acid analysis and cyanogen bromide peptide mapping were undertaken to evaluate the packing, organization, and preservation of collagen in bone and dentin of this mammoth. Microradiography and light microscopy showed that the bone consisted of especially well preserved compact and trabecular bone, and electron microscopy of demineralized bone and tusk showed that the matrix consisted of lamellae of densely packed cylindrical collagen fibrils. Cell remnants with intact nuclei, with or without a nucleolus, as well as variable lengths of plasma membrane were occasionally present on the surface of bony trabecula. Remnants of odontoblast processes were present in some dentin tubules. High and low angle X-ray diffraction demonstrated that the demineralized matrix contained native collagen molecules and amino acid analysis showed that the composition was comparable to that of type I collagen. Cyanogen bromide peptide mapping indicated that the major peptides of type I collagen were present and had the same electrophoretic mobility as that of type I collagen of demineralized Asian elephant bone and rat tail tendon.(ABSTRACT TRUNCATED AT 250 WORDS)