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Biomedical subjects

K G Vogel

Publications and source records attributed to K G Vogel.

At least 19 recordsLinked to original sources
PubMed1994

Aggrecan in bovine tendon.

Large proteoglycans were purified by ion-exchange chromatography, gel filtration and CsCl gradient centrifugation from the compressed and tensional regions of adult bovine deep flexor tendon. Tryptic peptide maps of proteoglycan from the compressed region were very similar to maps of aggrecan from bovine articular cartilage, with evidence for the presence of all fifteen previously identified markers from the G1, G2 and G3 domains. The presence of aggrecan in these samples was confirmed by sequencing the G1 peptide YPIHTPR. The equivalent maps for large proteoglycan from tensional tendon were also consistent with the presence of aggrecan, and this was confirmed by sequencing three marker peptides from each of the G2 and G3 domains. However, G1 marker peptides were conspicuously absent from tensional samples. Northern blots for aggrecan mRNA showed high levels in cells from compressed tendon and articular cartilage. Extended exposure revealed a lower level of hybridization to RNA from tensional tendon as well. The results confirm that aggrecan, which is similar in core protein structure to articular cartilage aggrecan, is the predominant chondroitin sulfate-bearing large proteoglycan of compressed tendon. The results also indicate that aggrecan fragments lacking the G1 domain can account for the small amounts of chondroitin sulfate-bearing large proteoglycan in tensional regions of adult tendon.

Aggrecans↗
PubMed1992

The interaction of decorin core protein fragments with type I collagen.

To further define the molecular interaction between decorin and type I collagen we generated a 20 kD fragment containing the N-terminal half of the core protein by Endoproteinase Arg C digestion and a 40 kD fragment including all leucine-rich repeats in the central part of decorin core by cleavage with 2-nitro-5-thiocyanobenzoate. The fragments did not influence collagen fibril formation, even at high concentration, and radioactive fragments showed little binding to collagen fibrils. Our observations suggest that neither the N-terminal half nor the central leucine-rich repeats of the decorin core protein can, by itself, interact fully with fibrillar collagen.

Animals↗
PubMed1992

Compression loading in vitro regulates proteoglycan synthesis by tendon fibrocartilage.

The regulation of proteoglycan synthesis in a fibrocartilaginous tissue by mechanical loading was assessed in vitro. Discs of bovine tendon fibrocartilage were loaded daily with unconfined, cyclic, uniaxial compression (5 s/min, 20 min/day) and the synthesis of large and small proteoglycans was measured by incorporation of [35S]sulfate. All discs synthesized predominantly large proteoglycan when first placed in culture. After 2 weeks in culture nonloaded discs synthesized predominantly small proteoglycans whereas loaded discs continued to produce predominantly large proteoglycan. The turnover of 35S-labeled proteoglycan was not significantly altered by the compression regime. Increased synthesis of large proteoglycans was induced by a 4-day compression regime following 21 days of culture without compression. Inclusion of cytochalasin B during compression mimicked this induction. Autoradiography demonstrated that cell proliferation was minimal and confined to the disc edges whereas 35S-labeled proteoglycan synthesis occurred throughout the discs. These experiments demonstrate that mechanical compression can regulate synthesis of distinct proteoglycan types in fibrocartilage.

Animals↗
PubMed1992

The effects of transforming growth factor-beta and serum on proteoglycan synthesis by tendon fibrocartilage.

The effects of transforming growth factor-beta (TGF-beta) and serum on proteoglycan synthesis by tissue explants from the fibrocartilaginous region of adult bovine tendon and by cells in culture from this region were assessed. The most characteristic effect of added TGF-beta on both explant tissue and cells in culture was enhanced synthesis of one small proteoglycan-biglycan. Lowered serum concentration diminished incorporation of Na2 35SO4 into proteoglycans. Added TGF-beta (1 ng/ml) stimulated cell proliferation, increased overall proteoglycan synthesis, and increased the length of glycosaminoglycan chains on all secreted proteoglycans. The effect of TGF-beta on cells in culture was highly consistent whereas explants from different animals showed greater variability in the response. It was concluded that TGF-beta did not specifically promote or maintain the cartilaginous nature of this tissue because supplementing medium with TGF-beta did not significantly alter the ratio of large/small proteoglycans synthesized by tissue explants. However, the observation of enhanced biglycan synthesis by TGF-beta suggests that TGF-beta could be involved in differentiation of regions of tendon subjected to compression, because compressed tendon contains both decorin and biglycan small proteoglycans whereas tensional tendon contains primarily decorin. Excess decorin added to cell culture medium did not affect the ability of TGF-beta to enhance synthesis of biglycan.

Amino Acid Sequence↗
PubMed1990

Ultrastructure and proteoglycan composition in the developing fibrocartilaginous region of bovine tendon.

Clear distinctions in morphology and proteoglycan composition have been described in regions of adult tendon that pass under bone and are subjected to compressive as well as tensional forces. In this study, developing bovine deep flexor tendon from early fetal stages through 6 months of age was examined biochemically and by light and electron microscopy. Longitudinal collagen fibers were seen in the tensional region of tendon throughout development; whereas a well established network arrangement of collagen fibers with wide interfibrillar spaces was seen in the compressed region by 7 months of fetal age. Collagen fibril diameters of both regions increased with age with the mean diameter in tensional tissue always greater than in compressed tissue. Glycosaminoglycan hexosamine content of the tensional region remained low throughout development (approximately 0.2% of dry tissue weight), but increased in the compressed region from 0.4% of dry weight at the 7-month fetal stage, to 1.0% dry weight at 6 months. Keratan sulfate was not detectable in tensional tendon at any age as measured by inhibition ELISA, but was found in increasing quantities in the pressure bearing region of tendon from young calves. Small proteoglycans predominated in both tensional and compressed regions throughout fetal and early neonatal development, and were of both PG I (biglycan) and PG II (decorin) types. Large proteoglycans represented only a small proportion of total proteoglycans in both regions of fetal tendon. By SDS/PAGE analysis, immunoreactivity, and molecular sieve chromatography, large proteoglycans of fetal compressed tendon were similar to large proteoglycans of adult tensional tendon in that they contained only chondroitin-6-sulfate, with little if any KS, and appeared to be slightly smaller than cartilage large proteoglycans.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1989

Characteristics of the in vitro interaction of a small proteoglycan (PG II) of bovine tendon with type I collagen.

Binding of the small dermatan sulfate proteoglycan of bovine tendon (PG II type/decorin-like) to type I collagen was characterized in an in vitro fibril-forming assay, using native collagen prepared from bovine tendon by acid extraction and radiolabeled proteoglycans synthesized by bovine tendon fibroblasts in culture. Substantial binding to collagen was noted for both intact small proteoglycan and core protein from which the glycosaminoglycan chain was removed. However, binding to collagen was minimal for free glycosaminoglycan chains or large proteoglycans. Binding of the small proteoglycan was optimal at approximately physiological conditions of salt concentration and pH. Scatchard analysis showed a binding affinity constant of 3.3 x 10(7) M-1 with 0.054 proteoglycan binding sites/collagen molecule, when about 0.25-6 micrograms proteoglycan was combined with 100 micrograms collagen. Binding to preformed fibrils of native tendon collagen and to pepsin-treated bovine skin collagen was similar to binding to native tendon collagen. Binding occurred in non-ionic detergents at concentrations up to 1% and once bound, the proteoglycan was not released by washing with up to 2 M NaCl. When both PG I and PG II small proteoglycans were added to collagen, only PG II was bound. This difference is not readily explained by differences in disulfide bond position. These studies indicate a strong, specific interaction between type I collagen fibrils and the core protein of the small (PG II) proteoglycan of tendon.

Animals↗
PubMed1989

Small proteoglycan synthesis by skin fibroblasts cultured from elderly donors and patients with defined defects in types I and III collagen metabolism.

The relative synthesis of two different types of small proteoglycans with potentially distinct roles in tissue function (PGI and PGII) was investigated in human skin fibroblast cultures initiated from donors of increasing age (fetal to 92 y) and from patients with defined defects in type I and type III collagen metabolism. Because these two small proteoglycans are not distinguished by the usual methods of ion-exchange and sieve chromatography, we have separated them using gel electrophoresis and confirmed this by specific immunoprecipitation. Small proteoglycans of the PGII type were the predominant species found in the medium of all cultures from normal donors, regardless of age. Most of the mutant cell lines showed a profile of small proteoglycan synthesis like that of the normal cells (i.e., predominantly PGII) although an increased ratio of PGI/PGII was seen for two cell strains from patients with Ehlers-Danlos syndrome type IV characterized by intracellular accumulation of type III procollagen. We conclude that mutations affecting collagen primary structure and secretion appear to have little effect on the cells' synthesis and secretion of small proteoglycans. These findings fail to support an hypothesis suggesting that the metabolism of normal cellular synthetic products (proteoglycans) is altered by abnormal cellular processing of a defective product (collagen).

Adult↗
PubMed1988

Keratan sulfate is a component of proteoglycans in the compressed region of adult bovine flexor tendon.

A monoclonal antibody (ET-4-A-4) was used to identify keratan sulfate (KS) as a constituent of bovine flexor tendon. KS was present in at least 500-fold higher amounts in the fibrocartilaginous region of the tendon that is subjected to compressive forces in vivo (mean = 0.03% of tissue dry weight) than in the more proximal regions subjected only to tensional forces. The KS was associated with proteoglycans of three size categories that could be separated by Sepharose CL-4B chromatography. The largest proteoglycan (Vo) contained approximately 4% KS and was predominant in the surface region of the tendon subjected directly to compressive forces. A population of somewhat smaller molecules (Kav approximately equal to 0.3) contained less than or equal to 20% KS and proportionately less chondroitin sulfate (CS) or dermatan sulfate (DS). The KS chains of this population were not digested by keratanase. This population was dominant in tissue from the middle layer of the fibrocartilaginous region. A third population of small KS proteoglycans or fragments (Kav approximately equal to 0.6) comprised 40% of the KS found in the deepest layer of the compressed tendon region. This smaller component was independent of the small DS proteoglycans.

Animals↗
PubMed1987

Characterization and interactions of a fragment of the core protein of the small proteoglycan (PGII) from bovine tendon.

Sequence analysis showed that Staphylococcus aureus V8 protease cleaved the core protein of the small dermatan sulfate proteoglycan of bovine tendon (PGII) on the carboxy side of a glutamic acid residue located 17 amino acids from the N-terminus of the intact molecule. The remaining 40 kDa core protein fragment inhibited collagen fibrillogenesis in an in vitro assay. V8 protease readily generated this fragment in tendon tissue, but it was not released from the tissue during treatment. These results indicate that neither the 17-amino acid N-terminal peptide nor the glycosaminoglycan chain attached to this peptide is required for maintaining the interaction of this proteoglycan with a collagen matrix.

Amino Acid Sequence↗
PubMed1987

Proteoglycans of fetal bovine tendon.

The proteoglycans (PG) of bovine fetal tendon (4-8 months in utero) were extracted with 4 M guanidine HCl and partially purified by ion exchange chromatography. Proteoglycans from fetal tendon were virtually entirely small molecules (Kav approximately equal to 0.55 by Sepharose CL-4B chromatography). These small proteoglycans had dermatan sulfate glycosaminoglycan chains and a core protein (after chondroitinase ABC digestion) with Mr approximately equal to 45,000 on sodium dodecyl sulfate-polyacrylamide gels. By electrophoretic mobility, immunocross-reactivity, and V8 protease sensitivity, these proteoglycans were determined to be of both PG I and PG II types. In contrast, adult tendon contains only the PG II type of small proteoglycan. Proteoglycans synthesized by fetal tendon explant cultures were, by both chromatographic and electrophoretic mobilities, somewhat larger than those extracted from the same tissue. There was no difference in the spectrum of proteoglycans observed between those regions of fetal tendon destined to receive only tensional forces (proximal) and those regions that will be subjected as well to compressive forces (distal) in the adult. These observations indicate that the proteoglycan content and synthetic capability of all regions of fetal tendon are constant and significantly different from those of both the tensional and fibrocartilaginous regions of adult tendon.

Animals↗
PubMed1987

Proteoglycan synthesis in organ cultures from regions of bovine tendon subjected to different mechanical forces.

Synthesis of proteoglycans by morphologically and chemically distinct regions of bovine flexor tendon was investigated in explant cultures. Proximal regions of the flexor tendon which experience only tensile forces and have low contents of proteoglycans initially exhibited relatively low rates of proteoglycan synthesis but high rates of collagen synthesis. The predominant proteoglycan produced by all proximal explants was of small hydrodynamic size and appeared similar to that extracted from proximal tissue. In contrast, explants derived from the distal tendon region, which experiences frictional and compressive forces in addition to tensile forces, and has a high content of proteoglycans, showed relatively high initial rates of proteoglycan synthesis and lower rates of collagen synthesis. These distal explants produced primarily large proteoglycans on the first day in culture. Turnover of newly synthesized proteoglycans was not detectable in proximal tissue, and was low in distal tissue. Loss of unlabelled proteoglycan from proximal and distal explants was not detected during the 12 days of culture. These observations suggest that the increase in specific types of proteoglycans in regions of tendon subjected to frictional and compressive forces is the result of elevated synthesis rates in this tissue. Two alterations in proteoglycan synthesis occurred during the 12-day culture period. (1) The rate of proteoglycan synthesis by all explants increased with time in culture. (2) The proportion of small proteoglycans synthesized by distal explants increased from 32% of the total proteoglycan produced on day 1, to 80% of that produced on day 12. Explants from proximal tendon continued to produce only small proteoglycans throughout the 12 days in culture. This switch in proteoglycan phenotype, resulting in decreased synthesis of large proteoglycans by the distal tissue, may be due to a lack of compressive forces on the cultured explants.

Animals↗
PubMed1987

Site-related variations in glycosaminoglycan content and swelling properties of bovine flexor tendon.

The presence of morphologically distinct tissues within the adult bovine deep flexor tendon presented a model which we examined for correlations between proteoglycan content and tissue swelling properties. The proximal portion which experiences only tensile forces contained low levels of glycosaminoglycan (0.2% glycosaminoglycan hexosamine as percentage of dry weight) that were evenly distributed throughout its length and thickness. Collagen accounted for 80% of tissue mass, and collagen fibers formed parallel arrays running longitudinal to the direction of tensile force. In equilibrium bulk swelling tests, the properties of proximal tissue were uniform throughout the tissue and typical of collagenous tissues in which the response to equilibration in low ionic strength buffers or acid pH is dominated by the collagen network. The anterior aspect of the distal deep flexor tendon articulates with the stiff paratendinous sheath and sesamoid bones and is subjected to compressive and frictional forces in addition to longitudinal tensile forces. Along this anterior surface and extending partially into the tendon is tissue that resembles fibrocartilage with collagen fibers organized as a random network. Greatest glycosaminoglycan hexosamine contents (2-3% of dry weight) were found in the distal surface layer, and large proteoglycans were concentrated at the site receiving direct compressive and frictional loads. Equilibrium bulk swelling tests on distal tissue showed that the articulating surface layer possessed unique material properties. Distal surface layers swelled when counter ions were washed from the tissue, whereas lowered pH had little effect on distal tissue volume. Swelling properties of distal tissue correlated directly with proteoglycan content and were similar to swelling properties of articular cartilage. These results suggest that the articulating layer of distal flexor tendon is adapted for a unique set of mechanical requirements and that an elevated proteoglycan content at the site of compressive and frictional forces contributes to meeting these functional needs.

Animals↗
PubMed1987

The effect of proteoglycans on the morphology of collagen fibrils formed in vitro.

The morphology of collagen fibrils at various times during formation in vitro was quantitatively examined by negative staining and by scanning electron microscopy. The presence of a small dermatan sulfate proteoglycan from bovine tendon (5 micrograms proteoglycan/100 micrograms collagen) resulted in collagen fibrils that were significantly thinner in width at all times by both methodologies. The rate of fibril diameter increase was also retarded by the small proteoglycans, suggesting that they inhibited the lateral aggregation of forming collagen fibrils. Large proteoglycans from cartilage did not produce this effect.

Animals↗
PubMed1986

Comparisons of antibody reactivity and enzyme sensitivity between small proteoglycans from bovine tendon, bone, and cartilage.

Preparations of small proteoglycans from bovine tendon, bone, and cartilage have been compared for sensitivity to various enzymes and reactivity with different polyclonal antibodies. Chondroitinase ABC digestion of all proteoglycans generated a core protein preparation that migrated similarly in sodium dodecyl sulfate-polyacrylamide electrophoresis as a doublet band with Mr approximately equal to 45,000. The small proteoglycans of cartilage were divided into two populations based upon electrophoretic migration of the intact molecules (Rosenberg, L. C., Choi, H. U., Tank, L-H., Johnson, T. L., Pal, S., Webber, C., Reiner, A., and Poole, A. R. (1985) J. Biol. Chem. 260, 6304-6313). The core preparations of tendon, bone, and the faster-migrating (PG II) proteoglycans of cartilage all interacted in Western blot/enzyme-linked immunosorbent assay analysis with polyclonal antibody raised against either the tendon or bone proteoglycans. The slower-migrating (PG I) proteoglycans of cartilage did not react with these antibodies. Digestion of the tendon small proteoglycan with Staphylococcus aureus V8 protease released glycosaminoglycan chains from the molecule and generated a 40-kDa protein fragment that was resistant to further rapid degradation by the enzyme. This large digestion fragment was also prominent following V8 protease digestion of the faster-migrating (PG II) population of small cartilage proteoglycans, but not the small proteoglycan of bone. The N-terminal amino acid sequence of the tendon (PG II) proteoglycan was determined. These observations provide additional evidence for heterogeneity among the chemically similar small proteoglycans from different tissues.

Amino Acid Sequence↗
PubMed1986

Variability in proteoglycan synthesis among six strains of normal human adult skin fibroblasts.

Several parameters of proteoglycan synthesis (incorporation of precursor isotope, distribution in culture compartments, sensitivity to chondroitinase ABC and Sepharose CL-2B elution profile) were investigated for six strains of normal human adult skin fibroblasts in culture in order to determine the range of variability in these parameters that could be expected in a normal population. When proteoglycan accumulation was measured in all cultures the uptake of Na2 35SO4 expressed per 10(6) cells had a coefficient of variation ranging from 17% to 50% in different experiments. Variability was always less when expressed per mg cell protein. The coefficient of variation was lowest when isotopic incorporation per mg cell protein was expressed as a percent of total incorporation of that isotope. Replicate tests indicated that the variability could not be explained by the assay methodologies alone. The protocol followed was designed to determine whether any consistent distinctions for individual cell strains could be identified. No significant difference in any parameter could be detected between the six cell strains by two-way analysis of variance although differences between experiments were significant in several cases and cultured bovine tendon fibroblasts were significantly different for every parameter tested.

Adult↗
PubMed1986

Proteoglycan synthesis by fibroblast cultures initiated from regions of adult bovine tendon subjected to different mechanical forces.

Fibroblast cultures were initiated from two distinct regions of the adult bovine deep flexor tendon and synthesis of 35S-labeled proteoglycans by these cultures was investigated. The proximal/tensional region of the tendon was composed of linearly arranged dense collagen bundles, and its glycosaminoglycan hexosamine content was only 0.2% of the dry weight of the tissue. The proteoglycans of this region were predominantly small (Kav = 0.5 on Sepharose CL-4B). Cells placed into culture from this region attached to the substratum readily, and the radiolabeled proteoglycans from these cultures were 90% small proteoglycans. In a more distal region of the tendon that is subjected to compressive forces, the collagen was arranged as a network of fibrils separated from each other by a matrix that stained intensely with Alcian blue. The glycosaminoglycan content of this compressed region was up to 5-fold higher than in the proximal region, and as much as 50% of the proteoglycans were large molecules (eluted from Sepharose CL-4B in the Vo). Cells placed into culture from the distal/compressed region did not attach to the substratum as readily as those from the proximal region and were characterized by the presence of numerous cytoplasmic lipid inclusions. The [35S]proteoglycans synthesized by the distal tendon fibroblast cultures were divided into two approximately equal populations of large and small proteoglycans having elution characteristics similar to the proteoglycans extracted from this tissue. The distinct profiles of proteoglycan production were maintained by the cells in culture for several weeks, although eventually the amount of large proteoglycan synthesized by the distal tendon fibroblast cultures diminished. Both regions of tendon contained predominantly type I collagen, and collagen production was about 10% of the total protein synthesized by both cell cultures. These observations indicate that adult tendon fibroblasts in culture express stable synthesis of proteoglycan populations similar to those found in the region of tendon from which they were derived.

Animals↗
PubMed1985

Characterization of proteoglycans from adult bovine tendon.

Proteoglycans were extracted in good yield from the proximal, fibrous portion of adult bovine tendon with 4 m guanidine HCl. They comprise less than 1% of the dry weight of the tissue. Using CsCl density gradient centrifugation, gel chromatography, and ion exchange chromatography, two populations of proteoglycans were separated and purified from other tissue proteins. One was a large, chondroitin sulfate proteoglycan with high buoyant density in CsCl. This component appeared to be composed of two or three subpopulations as detected by agarose/polyacrylamide electrophoresis, although they could not be effectively separated from one another for individual characterization. As a group, the large proteoglycans eluted from Sepharose CL-2B with Kav from 0.1-0.5 and their core protein had Mr greater than 200,000 with high contents of glutamic acid, serine, and glycine. The glycosaminoglycan chains had a weight average Mr of 17,000 and more than 98% of the uronic acid was glucuronic acid. This group comprised only 12% of the total proteoglycan of the tissue. The other 88% of the proteoglycans appeared to represent one group of small molecules that eluted from Sepharose CL-2B at Kav = 0.70. They demonstrated buoyant densities in a CsCl gradient ranging from greater than or equal to 1.51 to 1.30 g/ml. Their core protein had an apparent Mr = 48,000 following removal of the glycosaminoglycan chains by digestion with chondroitinase ABC. This core protein had a particularly high content of aspartic acid/asparagine and leucine. The glycosaminoglycan chains had a weight average Mr of 37,000 and were dermatan sulfate containing 73% iduronic acid. Those molecules found at highest buoyant density appeared to have additional glycosaminoglycan chains that were shorter. Proteoglycans were also extracted from the pressure-bearing distal region of this tendon, where contents of proteoglycan per wet weight of tissue were 3-fold higher and as much as 50% of this was as large as the large proteoglycans from the proximal tissue. Preparations of large proteoglycans from both tendon regions contained molecules capable of interacting with hyaluronic acid.

Amino Acids↗