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Biomedical subjects

T J Koob

Publications and source records attributed to T J Koob.

At least 19 recordsLinked to original sources

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

Proteoglycan synthesis in normotensive and spontaneously hypertensive rat arteries in vitro.

Proteoglycans (PGs) were analyzed and compared in the media of the thoracic aorta, abdominal aorta, left carotid artery and superior mesenteric artery of age-matched Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Two ages were examined; 10 week old, during the development of hypertension and 28 week old, when hypertension is well established in the SHR. Large chondroitin sulfate PG, large heparan sulfate PG and biglycan (PGI) and decorin (PGII) small PGs were identified. Biglycan was the predominant small PG found in all arteries. Newly synthesized PGs were labelled in vitro with 35SO4 for quantitation. The synthesis of large and small PGs was similar in the media of the thoracic aorta, abdominal aorta, left carotid artery, and superior mesenteric artery. The large to small ratio value, a measure of the artery PG composition, was also similar among the four arteries but was highest in the mesenteric artery. In both WKY and SHR arteries there was significantly decreased PG synthesis in the 28-week old compared to 10-week old animals. This was especially true for large PG. Hypertensive changes in PG synthesis were seen mainly in the carotid artery. In this artery, synthesis of both large and small PG was increased in the SHR, at both ages. The ratio of large to small PG was not significantly different between SHR and WKY arteries. We conclude that 28-week old WKY and SHR rat arteries synthesize less large and small PG than 10-week old arteries. The most prominent change seen in hypertensive rats is an increase in PG synthesis in the carotid artery.

Animals

Effects of chondroitinase-ABC on proteoglycans and swelling properties of fibrocartilage in bovine flexor tendon.

Fibrocartilaginous regions of bovine deep flexor tendon were treated with chondroitinase-ABC and trypsin in order to extract proteoglycans from the extracellular matrix and thereby investigate the contribution of proteoglycan and collagen organization to tissue material properties. Chondroitinase-ABC digestion of tendon specimens for 24 h resulted in extraction of 60% of tissue glycosaminoglycan and leaching of the degraded large proteoglycan from the tissue residue. The totally degraded core protein of the small dermatan sulfate proteoglycan remained with the tissue residue, indicating that it is specifically associated with the tissue residue and that this association is not dependent on the glycosaminoglycan chains. Treatment of residues with trypsin after chondroitinase-ABC digestion depleted the specimens of proteoglycan. Bulk swelling tests on enzyme-extracted specimens showed that the distinct swelling properties of the fibrocartilaginous regions of the distal flexor tendon could be partially accounted for by elevated levels of proteoglycan. Swelling tests also showed that the distinct collagen organization of this region contributes significantly to the tissue's material properties. These results suggest that the fibrocartilaginous organization and composition of the articulating layer of distal tendon are adapted for mechanical requirements unique to this site, which receives compressive and frictional loads in addition to tensile loads.

Animals

Collagen and proteoglycan in a sea urchin ligament with mutable mechanical properties.

The "problematic ligament" of sea urchins is a connective tissue which crosses the ball-and-socket joint between spine and body wall. The problem of this ligament is that it is composed of parallel collagen fibrils, yet normally undergoes rapid and dramatic alterations in mechanical properties and in length. Previous work has suggested that the collagen fibrils of the ligament are able to slide past one another during length changes but are inhibited from sliding when the ligament is in "catch". In this model of the ligament both the collagen fibrils and the interfibrillar matrix are mechanically important. We have found that the collagen fibrils of the spine ligament of the pencil urchin Eucidaris tribuloides are discontinuous and end by tapering within the body of the ligament. Intact fibrils that have been isolated from the ligament vary by more than an order of magnitude in length and in radius but have a constant length/radius (aspect) ratio of about 5,300. This is the first determination of the aspect ratio of collagen fibrils from any source. The constant aspect ratio of the fibrils is consistent with their functioning as the discontinuous fiber phase in a fiber-reinforced composite material, while the high value of the aspect ratio indicates that the nonfibrillar matrix, which must act to transfer stress between fibrils, can produce a stiff and strong ligament even if it is several orders of magnitude weaker and more compliant than the fibrils. Moreover, the tensile properties of the ligament may be determined by the properties of the matrix. A prominent component of the interfibrillar matrix is a proteoglycan which associates with specific bands at the surface of the collagen fibrils through noncovalent binding of its core protein. The glycosaminoglycan moiety of this proteoglycan is partly comprised of chondroitin sulfate/dermatan sulfate polymers. These results are consistent with the "sliding fibril" hypothesis and suggest that the proteoglycan may be an important component of the stress-transfer matrix.

Animals

Quantitation of hyaluronic acid in tissues by ion-pair reverse-phase high-performance liquid chromatography of oligosaccharide cleavage products.

A method for quantifying hyaluronic acid in biological tissues and fluids is described. The assay uses ion-pair HPLC to resolve and quantify the oligosaccharide end products of Streptomyces hyaluronidase digestion. Tissue samples were solubilized by papain, and the nondiffusate after dialysis was exhaustively digested with Streptomyces hyaluronidase. The resulting tetrasaccharide and hexasaccharide cleavage products were resolved by reverse-phase high-performance liquid chromatography in the presence of the ion-pairing agent, tetrabutylammonium phosphate. The saccharides were detected and quantified by their absorbance at 232 nm due to the alpha, beta-unsaturated carboxyl group generated by the eliminase reaction. In control experiments 93 +/- 3% of a hyaluronic acid standard so treated was reproducibly recovered as its tetra- and hexasaccharide cleavage products. As little as 0.5 microgram of the oligosaccharides could be quantified with no interference from a vast excess of chondroitin sulfate or other tissue components. The assay was applied to various types of human, bovine, and rabbit cartilage and to samples of other tissues including nucleus pulposus, annulus fibrosus, skin, aorta, cervix, cockscomb, synovial fluid, and vitreous humor. Results on human articular cartilage showed a linear increase in the content of hyaluronate from 0.1 to 0.5% of tissue dry weight between birth and 80 years of age.

Adolescent

Collagen cross-linking: distribution of hydroxypyridinium cross-links among invertebrate phyla and tissues.

1. Using a specific HPLC assay, a wide variety of marine invertebrate connective tissues was screened for the 3-hydroxypyridinium amino acids that are prominent intermolecular cross-linking residues in the collagens of many vertebrate connective tissues. 2. One or both of the two structural forms that exist, hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP), was found in organisms from the following phyla: coelenterata, Annelida, Echinodermata, Mollusca and Arthropoda. 3. Neither amino acid was found in tissues from representative species of Porifera and Chordata. 4. Of special note was an unusually high ratio of LP to HP in Limulus polyphemus gill cartilage.

Animals

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

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

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

Streaming potentials: a sensitive index of enzymatic degradation in articular cartilage.

Under physiological conditions, the extracellular matrix of articular cartilage contains a high fixed-charge density, associated with its ionized proteoglycan (PG) molecules. Compression of the highly charged cartilage matrix within the physiologic range leads to the production of electrical streaming potentials. We observed significant changes in the potential response due to chemical modifications of the matrix, such as extraction of PG and glycosaminoglycan (GAG) moieties using chondroitinase-ABC adn trypsin. The streaming potential was a sensitive index of the degradative loss of these matrix constituents and of the kinetics of the enzymatic degradative process.

Animals

Plasma estradiol, testosterone, and progesterone levels during the ovulatory cycle of the skate (Raja erinacea).

Amounts of estradiol, testosterone, and progesterone in plasma were measured during the reproductive cycle of female Raja erinacea. Estradiol titers correlated directly with follicle size in females undergoing ovarian recrudescence, while highest concentrations were found in females with preovulatory follicles. These data indicate that as follicles grow, their steroidogenic capacity increases. In mature, nonspawning females, titers of estradiol and testosterone varied markedly. Progesterone was not detected in peripheral plasma of skates that did not produce eggs during the observation period. In females producing eggs, estradiol and testosterone predominated during the follicular phase of each spawning cycle. While estradiol and testosterone were elevated, progesterone was not detectable in the peripheral circulation. As ovulation and formation of capsules approached, plasma estradiol and testosterone declined to near baseline levels. Circulating progesterone rose sharply two days before encapsulation of ovulated eggs and remained elevated for only two days. On the day of encapsulation, concentrations of plasma progesterone had fallen to nearly baseline levels. Progesterone titers remained low throughout egg retention and oviposition. These measurements demonstrate that progesterone titers are elevated at specific times during the reproductive cycle of the skate and clearly suggest that progesterone is critically involved in events occurring at ovulation, encapsulation, and possibly oviposition.

Animals

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

Quantitation of hydroxypyridinium crosslinks in collagen by high-performance liquid chromatography.

An HPLC method for quantifying the 3-hydroxypyridinium crosslinks of collagen is described. It can be applied to crude hydrolysates of all types of connective tissue. Mineralized tissues can be hydrolyzed directly and analyzed without interference from the mineral ions. The hydroxylysyl (HP) and lysyl (LP) forms of hydroxypyridinium residue were resolved on a reverse-phase C18 column using a gradient of acetonitrile in water and 0.01 M n-heptafluorobutyric acid as an ion-pairing agent. The crosslinking amino acids were accurately quantified down to 2 PM (1 ng) injected, by detecting their natural fluorescence with a spectrofluorometer. Tissues in which hydroxypyridinium crosslinks were plentiful included all forms of cartilage, bone, dentin, ligament, tendon, fascia, intervertebral disc, lung, gut, cervix, aorta, and vitreous humor. Among normal tissues, LP, the minor form of the crosslink, was present in significant amounts relative to HP only in bone and dentin. Both crosslinks were essentially absent from skin, cornea, rat tail tendon, and basement membranes.

Amino Acids

External incubation alters the composition of squamate eggshells.

Eggshells of the skink, Eumeces fasciatus, and of the rough green snake, Opheodrys vernalis, contain 28-40% of total shell mass as calcium at oviposition. After incubation, both calcium concentration and content were reduced 17-41%. Protein accounted for 33-65% of shell mass in Eumeces and Opheodrys eggshells at oviposition. Reductions in protein concentration and content from 22-86% following incubation were observed. Comparing amino acid compositions of eggshells collected following oviposition with those collected from the same clutch after hatching reveals similarities among all eggshells except those of Eumeces from Michigan which lack desmosine and isodesmosine. Concentrations of desmosines in eggshells from Missouri and Michigan Eumeces appeared to decrease following incubation by 49.7% and 12.2%, respectively. Although neither the mechanisms involved in apparent losses of calcium and protein, nor the fate of the mobilized materials are known, possible pathways for mobilization of eggshell constituents are outlined and their pertinence to the physiology of squamate eggs and the evolution of viviparity in reptiles is suggested.

Amino Acids

Effects of relaxin and insulin on reproductive tract size and early fetal loss in Squalus acanthias.

These experiments demonstrate that both porcine relaxin and bovine insulin can increase the cervical cross-sectional area in Squalus acanthias. When given after estradiol priming, the magnitude of this response is greater. This effect is limited to Stage C females in which pregnancy is over 75% complete. The overall result is premature loss of developing fetuses. The effect of relaxin or insulin on cervical cross-sectional area and fetal loss is unrelated to an effect on blood sugar. Cervical weights are affected little by hormone treatment. Relaxin and insulin do not increase the cross-sectional area of the anterior uterine constriction. These results suggest the involvement of a relaxin-like molecule during normal parturition in the spiny dogfish.

Animals

Early changes in material properties of rabbit articular cartilage after meniscectomy.

We have correlated early material and biochemical changes in articular cartilage in a surgical model for cartilage degeneration. Medial meniscectomy was performed on the left knee of 17 adult, female New Zealand white rabbits. The equilibrium Young's modulus of cartilage was assessed by an indentation test in situ at defined sites on the medial and lateral tibial plateaus of the operated and control knees; the cartilage was then excised and analyzed biochemically. Focal changes were consistently observed in the medial surface of the operated knee. The equilibrium modulus and the glycosaminoglycan content fell rapidly, reaching a minimum by 2 weeks after surgery; the lateral tibial surface was essentially unaffected. Six months after surgery, the glycosaminoglycan content had returned to normal and the modulus to near normal. Independent measurements on cored plugs from the medial surface 2 weeks after surgery revealed a significant decrease in both the dynamic stiffness and the streaming potential in the operated knee compared with the control. The findings suggest that normal ambulatory loads in vivo will deform the affected medial cartilage much more than normal. It remains to be seen if altered mechanical stresses are solely responsible for initiating and sustaining matrix remodeling by the chondrocytes.

Animals

Hormonal interactions in mammalian collagenase regulation. Comparative studies in human skin and rat uterus.

The production of collagenase by human skin explants in culture is prevented by 10(-8) M dexamethasone, 5 . 10(-4) M dibutyryl cyclic AMP, or 2.5 . 10(-3) M theophylline. Decreases in collagenase activity are paralleled by reductions in the degradation of explant collagen during the culture period. Progesterone, which effectively inhibits collagenase production in rat uterine explant cultures, has no effect on human skin explants. The inhibition by cyclic AMP is nucleotide specific. When partially inhibitory concentrations of dexamethasone and dibutyryl cyclic AMP, or dexamethasone and theophylline, are added to culture medium together, the resultant inhibition is that predicted by additivity. Synergistic inhibition, as observed in rat uterus between progesterone and dibutyryl cyclic AMP, fails to occur. Dexamethasone inhibits the production of collagenase by cultured explants of rat uterus, with complete inhibition occurring at 10(-7) M steroid. Synergism between glucocorticoids and dibutyryl cyclic AMP or between dexamethasone and progesterone could not be demonstrated in the uterine culture system. These results suggest the existence of three regulatory systems for the control of collagenase production in mammalian tissues, and that cooperativity between systems may occur on a tissue-specific basis.

Animals