Genetic diseases of connective tissues in animals.
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Animal Diseases↗
Biomedical subjects
R R Minor
Publications and source records attributed to R R Minor.
Defects in the processing of procollagen to collagen are demonstrable in cultured fibroblasts from patients with the Ehlers-Danlos and osteogenesis imperfecta syndromes.
This is a study of the processing of procollagen to collagen in cultures of skin and tendon fibroblasts. Processing was markedly increased by growing cells for 2-4 days postconfluence and then adding ascorbate to the medium for 2 days prior to labeling with [3H] proline. With this system, more than two-thirds of the pro-alpha chains of type I procollagen in the culture medium, and more than 90% of those in the cell layer, were rapidly processed to pC-alpha, pN-alpha, or alpha chains. Purified, exogenous procollagen was also rapidly processed in cell-free culture medium. The results showed for the first time that exogenous procollagen can be processed in conditioned cell-free medium. The system was then used to compare the processing of procollagen in the medium of normal fibroblasts, cells from one bovine and four human variants of osteogenesis imperfecta, and those from eight human variants of the Ehlers-Danlos syndrome. The cells could be divided into three groups, based on their ability to process type I procollagen: normal, consistently slow, and very slow. The cause of the decreased processing was shown to be associated with either a mutation causing a shortening of an alpha chain or decreased activity of procollagen N-proteinase in cell-free culture medium. Decreased processing of procollagen to collagen occurred with cultured fibroblasts from patients with different forms of both osteogenesis imperfecta and Ehlers-Danlos syndrome. Both of these disease syndromes are associated with abnormalities in the structure or metabolism of procollagen in fibrous connective tissues, bones, and teeth. The results show that defects in the structure, synthesis, or processing of procollagen are readily demonstrated with cultured fibroblasts.
Adolescent↗
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.
Adult↗
Biochemical composition of the connective tissue in keloids and analysis of collagen metabolism in keloid fibroblast cultures.
Keloids are histologically characterized by an abundance of the extracellular matrix of connective tissue. In the present study, we examined the connective tissue composition of keloids, and analyzed the details of collagen metabolism utilizing fibroblast cultures established from keloid tissue. Quantitative connective tissue analyses indicated that collagen was the predominant extracellular matrix component in keloids. The ratio of genetically distinct collagens type I/III was significantly increased, as compared to normal human skin. Collagen biosynthesis was measured in fibroblast cultures by the formation of radioactive hydroxyproline: 5 of 9 keloid cell cultures studied demonstrated increased procollagen production in comparison to age-, sex-, and passage-matched control skin fibroblast lines, while the remaining 4 cell lines were within the control range. Keloid fibroblast cultures which were high collagen producers also demonstrated elevated prolyl hydroxylase activity. The mechanisms of increased procollagen production in fibroblast cultures were first examined by assaying the abundance of type I procollagen-specific mRNA utilizing dot blot hybridizations with a pro alpha 2(I)-chain-specific cDNA. The type I procollagen mRNA levels were significantly increased in 4 keloid fibroblast lines, and a good correlation between the mRNA levels and the rate of procollagen production in the same cultures was noted. These observations suggest regulation of the collagen gene expression on the transcriptional level. The catabolic pathway of collagen metabolism in fibroblast cultures was examined by determining the degradation of newly synthesized procollagen polypeptides through assay of radioactive hydroxyproline in small-molecular-weight peptide fragments. In 3 keloid cell cultures, the degradation of newly synthesized collagen polypeptides was below the range of normal controls. These findings suggest that a reduced degradation of newly synthesized polypeptides might contribute to the accumulation of procollagen in some keloid fibroblast cultures. The results of this study suggest two possible mechanisms for deposition of collagen in keloid lesions in vivo: first, the growth of the lesions may result from a localized loss of control of the extracellular matrix production by fibroblasts; secondly, reduced degradation of the newly synthesized procollagen polypeptides may contribute to collagen deposition in some keloids.
Adult↗
Dermal collagen degradation and phagocytosis. Occurrence in a horse with hyperextensible fragile skin.
A 2-year-old female horse had large areas of hyperextensible, fragile skin that were interspersed with areas of normal skin. Affected skin tore easily and contained reduced amounts of dermal collagen. Collagen fibers were fragmented and disorganized, and in trichrome-stained sections, many fibers had abnormal red-stained centers. Electron microscopy showed that many collagen fibers had discrete foci of degradation in which the fibrils were fragmented, loosely packed, and widely separated by granular material. Collagen fibril fragments were present in secondary lysosomes in dermal fibroblasts, but there were no degranulated mast cells or inflammatory cells in these areas. This suggested that a noninflammatory degradation and phagocytosis of collagen had occurred in the areas of hyperextensible fragile skin in this horse. Unaffected skin had no signs of collagen degradation or phagocytosis; uniformly cylindrical collagen fibrils were densely packed into morphologically normal fibers.
Animals↗
Defects in collagen fibrillogenesis causing hyperextensible, fragile skin in dogs.
Two unrelated mixed-breed dogs were donated for studies of their fragile, hyperextensible skin. Breeding of these dogs to bitches with normal skin showed that half of their male and female offspring also had fragile, hyperextensible skin, indicating that the defect was transmitted as an autosomal dominant trait in both dogs. Electron microscopy showed distinct abnormalities in the packing of collagen into fibrils and fibers in affected skin. These packing defects in dermal collagen were identical in related dogs, but were slightly different in unrelated animals. A clinical test, the skin extensibility index, was used to quantitate the extensibility of affected and unaffected skin. This index ranged from 8% to 15% in normal dogs and from 17% to 25% in newborn pups and adult dogs with collagen packing defects. The tensile strength of dorsolateral thoracic skin of affected pups was only 5% to 10% of that of matched specimens of paired littermates. The hyperextensibility and fragility of skin were the only clinical signs, but radiographic and microradiographic studies revealed subclinical involvement of bone.
Animals↗
Cytotoxic effects of low levels of 3H-, 14C-, and 35S-labeled amino acids.
Tissue injury by radiolabeled amino acids may severely affect experimental results. In this report, 3H-, 14C-, and/or 35S-labeled proline, serine, lysine, and methionine at concentrations of 1 and 5 microCi/ml were shown to cause severe injury in organ cultures of embryonic rat lungs. This injury was evident by 6 h and was amplified by 4 days of culture. This injury was characterized with light and electron microscopy, with morphometric analysis of growth, and with quantitation of the total protein and DNA/lung. After 6 h with 5 microCi/ml of 14C- or 35S-amino-acid there were more signs of cell degeneration, and by 24 h the labeled lungs were smaller, showed more signs of cell degeneration and death, and contained 30 to 60% less new protein and DNA than control lungs. After 24 h with 5 microCi/ml of 14C- or 35S-amino-acid the total protein and DNA/lung began to decrease. This toxicity was directly proportional to the amount of intracellular decay of each isotope. With 14C- and 35S-amino-acids, lung growth slowed with approximately 100 disintegrations/cell/day (d/c/d), growth stopped with approximately 200 d/c/d, and atrophy occurred with approximately 300 d/c/d. Cell proliferation, cell differentiation, and bronchial branching continued through 4 days even though atrophy occurred with greater than 200 d/c/d. With 3H-amino-acids, growth slowed with approximately 200 d/c/d and stopped with approximately 400 d/c/d. However, no toxicity was evident with less than 60 d/c/d of 14C or 35S, or with less than 90 d/c/d of 3H. These data suggest that the amounts of intracellular decay of these weak beta-emitting isotopes should be strictly limited. Increasing amounts of tissue injury occurred with 14C or 35S at greater than 10,000 dpm/micrograms of DNA, and with 3H at greater than 20,000 dpm/micrograms of DNA.
Amino Acids↗
Hypercalcitoninism without hypercalcitoninemia.
Yearling heifers overfed protein, calcium and phosphorus with a feed recommended for high producing dairy cows developed osteopetrosis and skeletal malformations as a result of retarded bone resorption. Histologic and electron microscopic examinations showed that C cell hyperplasia was also present. The presence of C cell hyperplasia and osteopetrosis supported the diagnosis of hypercalcitoninism. Clinically unaffected heifers were studied by serum chemistry after 1 to 1.5 months on the same diet at age 6 months and after 3 months on an optimal diet to learn whether they showed evidence of hypercalcitoninism as expressed by retarded bone resorption and/or hypercalcitoninemia. The data indicated that bone resorption was retarded and that serum gastrin was elevated in the heifers without skeletal malformations while on the high calcium feed. The heifers were isocalcitoninemic. The data suggested that overfeeding calcium was sufficient to produce hypercalcitoninism but the rate of calcitonin secretion had not exceeded the rate of removal of calcitonin from blood by binding to tissue receptors and by metabolic degradation. Since calcitonin is rapidly removed from blood, hypercalcitoninism may occur without concomitant hypercalcitoninemia.
Animal Feed↗
Collagen metabolism: a comparison of diseases of collagen and diseases affecting collagen.
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Animals↗
Organ cultures of embryonic lung tissues.
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Animals↗
A light and electron microscopic study of a normal adrenal medulla and a pheochromocytoma from a horse.
The outer medullary (juxtacortical) zone of a normal equine adrenal gland had columnar chromaffin-positive cells arranged with their long axes perpendicular to fine vascular channels. The deeper medullary regions were composed of smaller irregularly round to polygonal chromaffin positive cells in small packets. Both cell types contained two types of membrane-bound cytoplasmic secretory granules. Osmiophilic granules with a homogeneous core, crenated membrane and narrow submembranous halo predominated in the columnar juxtacortical cells. The rounder, central medullary cells contained predominantly electron dense granules with a wide irregular electron lucent space between an eccentric core and the granule membrane. In contrast, irrespective of cell type or zone, cells from a pheochromocytoma contained only one type of granule similar to that described for the juxtacortical region of the normal equine adrenal medulla. The tumor cells could be classified into three subtypes based on density of granule packing but the granules were morphologically similar in all tumor cells.
Adrenal Gland Neoplasms↗
A mixed mesenchymal sarcoma in the soft palate of a dog: light and electron microscopic findings.
Soft tissue tumors containing a mixture of neoplastic fibrous tissue, cartilage, and bone have previously been classified as extraskeletal osteosarcomas in the dog. These tumors are often poorly differentiated, contain multiple neoplastic cell types, and might be more appropriately called mixed mesenchymal sarcomas. The present neoplasm caused clinical signs of stridorous respiration and dysphagia in a four and one-half year old dog. Four neoplastic cell types were demonstrated by light and electron microscopy.
Animals↗
Hereditary fragility and hyperextensibility of the skin of cats. A defect in collagen fibrillogenesis.
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Animals↗
Basement membrane procollagen is not converted to collagen in organ cultures of parietal yolk sac endoderm.
Basement membrane procollagen biosynthesis was studied in organ cultures of embryonic rat parietal yolk sac endoderm by following [14C]proline incorporation into nondialyzable proteins. After reduction with 2-mercaptoethanol the 14C-proteins synthesized were characterized by agarose gel filtration and disc electrophoresis in the presence of sodium dodecyl sulfate. The labeled procollagen was identified by its content of hydroxy[14C]proline, its sensitivity to digestion with bacterial collagenase, and its resistance to digestion with pepsin. In cultures which were continuously labeled for periods from 6 hours to 4 days, the pro-alpha chains consistently eluted as a single peak with an apparent molecular weight of 160,000. After pepsin digestion the resultant alpha chains had an apparent molecular weight between 125,000 and 140,000. This suggests that basement membrane procollagen either contains non-triple helical pepsin-resistant regions or a triple helical region which is larger than the corresponding region of interstitial procollagen. Two experiments were performed to determine whether the chains of newly synthesized basement membrane procollagen were cleaved to a smaller molecular species. In the first, the hydroxylation and secretion of procollagen were blocked with alpha, alpha'-dipyridyl, and the resulting intracellular chains of basement membrane protocollagen were found to co-elute with fully hydroxylated and secreted pro-alpha chains. In the second, cultures were labeled for 1 day and chased for 3 days with unlabeled medium. Autoradiography had shown that most of the label was chased into new basement membrane. Agarose chromotography showed that after 3-day chase the pro-alpha chains still eluted with an apparent molecular weight of 160,000. Thus, the data indicated that basement membrane procollagen was deposited in new basement membrane without undergoing a time-dependent extracellular conversion.
Animals↗
Organ cultures of the embryonic rat parietal yolk sac. I. Morphologic and autoradiographic studies of the deposition of the collagen and noncollagen glycoprotein components of basement membrane.
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Animals↗
Organ cultures of the embryonic rat parietal yolk sac. II. Synthesis, accumulation, and turnover of collagen and noncollagen basement membrane glycoproteins.
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Animals↗
The embryonic rat parietal yolk sac. The role of the parietal endoderm in the biosynthesis of basement membrane collagen and glycoprotein in vitro.
Basement membrane biosynthesis in vitro was studied in a rapidly growing embryonic tissue, the rat parietal yolk sac. This tissue consists of a thick, nonvascular basement membrane (Reichert's membrane) separating two cellular layers (parietal endoderm and trophoblast). Morphologically, Reichert's membrane appeared similar to other basement membranes. Previous analysis of the amino acid and carbohydrate composition of acellular Reichert's membrane showed it to be typical of basement membranes isolated from other tissues and species. Analysis of [14-C]proline incorporation and hydroxy [14-C]proline synthesis during the third quarter ogestation in vitro showed that basement membrane collagen synthesis in the parietal yolk sac was maximal around the 14th day of gestation. At this time, basement membrane collagen represented nearly 10% of the newly synthesized protein. The collagen synthesized in this system was characteristic of basement membrane collagen in that about 11% of the total hydroxy [14-C]proline was present as the 3-isomer. In addition, after incubation in the presence of [14-C]lysine, 83 to 94% of the hydroxy[14-C]lysine was glycosylated, with the predominant form being glucosylgalactosylhydroxy[14-C]lysine. When the parietal endoderm and trophoblast were incubated separately with [14-C]proline, it was determined that the former was solely responsible for the synthesis of basement membrane collagen since essentially all of the 4-hydroxy[14-C]proline was associated with this cell type. Autoradiographic experiments with [3-H]glucosamine also served to localize the synthesis of noncollagen basement membrane glycoprotein components to the parietal endoderm. As with the results reported for basement membrane collagen secretion in embryonic chick lens cells, there appeared to be approximately a 60-min delay between the incorporation of [14-C]proline into protein and the secretion of collagen as measured by the appearance of 4-hydroxy[14-C]proline in the culture medium. Experiments utilizing [3H]glucosamine to monitor glycoprotein synthesis did not show a delay between the incorporation of [3H]glucosamine and the secretion of nondialyzable 3-H into the medium. The results obtained using the parietal yolk sac system to study basement membrane biosynthesis were compared to those previously obtained using the kidney glomerular and embryonic chick lens systems. It was concluded that the parietal yolk sac system is superior for a number of reasons: (a) the extracellular matrix appeared to contain only basement membrane components; there was no contamination by acid mucopolysaccharides or other types of collagen; (b) only a single cell type appeared to be responsible for the synthesis of basement membrane components; and (c) a relatively large percentage of the newly synthesized protein was basement membrane collagen.
Animals↗
The ubiquitous occurrence of chondroitin sulfates in chick embryos.
The synthesis of sulfated glycosaminoglycans has been studied in a wide variety of embryonic chick tissues. All tissues studied have the capability to manufacture, but not necessarily accumulate, the chondroitin sulfates as well as other glycosaminoglycans. The relative distribution of glycosaminoglycans differs between tissues and changes with age.
Animals↗