Total Amount Of Collagen Research Articles

INFLUENCE OF EXTERNAL POTASSIUM ON THE SYNTHESIS AND DEPOSITION OF MATRIX COMPONENTS BY CHONDROCYTES IN VITRO

The effect of a high external potassium concentration on the synthesis and deposition of matrix components by chondrocytes in cell culture was determined. There is a twofold increase in the amount of chondroitin 4- and 6-sulfate accumulated by chondrocytes grown in medium containing a high potassium concentration. There is also a comparable increase in the production of other sulfated glycosaminoglycans (GAG) including heparan sulfate and uncharacterized glycoprotein components. The twofold greater accumulation of GAG in the high potassium medium is primarily the result of a decrease in their rate of degradation. In spite of this increased accumulation of GAG, the cells in high potassium fail to elaborate appreciable quantities of visible matrix, although they do retain the typical chondrocytic polygonal morphology. Although most of the products are secreted into the culture medium in the high potassium environment, the cell layer retains the same amount of glycosaminoglycan as the control cultures. The inability of chondrocytes grown in high potassium to elaborate the typical hyaline cartilage matrix is not a consequence of an impairment in collagen synthesis, since there is no difference in the total amount of collagen synthesized by high potassium or control cultures. There is, however, a slight increase in the proportion of collagen that is secreted into the medium by chondrocytes in high potassium. Synthesis of the predominant cartilage matrix molecules is not sufficient in itself to ensure that these molecules will be assembled into a hyaline matrix.

Lack of effect of chronic lithium chloride on bone composition and metabolism.

Lithium chloride (LiCl, 1.43 ±changes in the total amount of soluble collagen, 0.13 mEq/k/day) was administered in distilled the rate of collagen resorption under standardized water to 13 adult female rats for 40 days; 6 rats conditions in vitro, or the rate of collagen forma-served as controls. LiCl had no effect on theserumtion. Serum lithium rose to 0.42 ± 0.10 mEq/1 total calcium and inorganic phosphorus or on and bone lithium content to 29/μg/g wet weight. metaphyseal bone composition or collagen metabo-The data fail to demonstrate any effect of lithium lism. There were no significant changes in the on female rat bone composition or metabolism percentage bone wet weight of mineral salts,ni doses which are many times the usual human organic matrix, or fat and water; there were no prophylactic dose of 0.2–0.4 meq/k/day and ad-changes in the content per unit wet weight of ministered under the experimental conditions calcium, hydroxyproline, or DNA. There were nodescribed.(Endocrinology 94: 915, 1974)

Rebound of rat carrageenin granuloma following cessation of anti-inflammatory steroid therapy

Glucocorticoid therapy induced rapid involution of chronic granulomatous inflammation in rats by subcutaneous injection of carrageenin. Hydrocortisone acetate injected into the granuloma pouch at doses higher than 3 mg/kg/day for 3 days caused maximum involution. After withdrawal of the corticoid therapy, rebound of the granulomatous inflammation took place resulting in rapid recovery of the wet weight and total content of tissue DNA and non-collagen proteins. A dose of 3 mg/kg/day was optimal for observing this rebound phenomenon. In order to investigate metabolic aspects of the rebound phenomenon minced granuloma was incubated in vitro with [ 3H] thymidine or [ 3H] proline. The rate of incorporation of the labeled precursor into non-collagen protein was elevated near to the normal level by 24 hr after the interruption of the corticoid treatment. A second step in the course of the recovery was a rapid increase in the incorporation of labeled thymidine into DNA which was attained by 48 hr after the last injection of the corticoid. The rate of recovery of the total amount of non-collagen protein, however, was rather slow compared with that of DNA which reached the control level 3 days after the withdrawal of the corticoid therapy. The total non-collagen protein of the granuloma reached almost complete recovery 1 day later. These results suggest that the synthesis of some fractions of the granuloma proteins which involve proteins essential for DNA synthesis was activated before the reactivation of the synthesis of DNA and some other proteins. Recovery of collagen synthesis was not complete until 4 days after the cessation of the corticoid treatment. Consequently, the total amount of collagen was still lower than that of the control on the last day of the experiment.

The Synthesis of Collagen and Glycosaminoglycans by Dedifferentiated Chondroblasts in Culture

Marked changes occur in the morphology of chick chondroblasts grown for 5 days in F-10 medium containing either 5-bromo-2′-deoxyuridine or embryo extract. The cells lose the characteristic polygonal morphology and assume a flattened ‘fibroblastic’ appearance. To determine whether the morphological changes reflect a biochemical transformation toward frank fibroblasts, changes in collagen and glycosaminoglycan synthesis were examined in these ‘dedifferentiated’ cells. Growth in either medium did not significantly affect the total amount of collagen synthesized. However, the subunit composition of the collagen chains was different. Freshly isolated cartilage trunks or control chondroblast cultures synthesized only α1 subunits (suggesting exclusive synthesis of α1(ll)3-type collagen), whereas dedifferentiated cultures synthesized both α1 and, in addition, some α2 subunits (suggesting synthesis of fibroblasttype α1(l)2α2-type collagen). Incorporation of labelled glucosamine in F-10 medium showed that the major glycosaminoglycan synthesized by either cartilage trunks or chondroblast monolayers is chondroitin sulphate; little, if any, hyaluronic acid could be detected. With growth in embryo extract (EE) glucosamine was incorporated equally into chondroitin sulphate and hyaluronic acid, whereas in BUdR, chondroitin sulphate synthesis was completely inhibited. Distinct biochemical differences were therefore found for both collagen and glycosaminoglycan synthesis during growth in either BUdR or EE. Such changes were not identical but both demonstrate changes in synthetic programme tending to approach that of frank fibroblasts.

Effect of long-term physical stress on collagen growth in the lung, heart, and femur of young and adult rats.

Male Wistar rats, 1.8 and 7.5 months old, were adapted to exercise on the treadmill for a period of 7 weeks. The animals were trained 3 h daily at the treadmill speed of 24 m/min. Wet and dry weights and collagenous hydroxyl-proline content in the lung, heart, and femur were analyzed. Physical stress did not affect any of the parameters studied in the lung. Cardiomegaly developed only in the young rats exposed to training. There was significantly higher total amount and density of collagen in the hyper-trophic heart. In contrast to the reactivity of the heart to physical stress, the amount and density of collagen in the femora was significantly increased only in the adult rats. There was no effect on the content of calcium in the bone. The calcium-collagen ratio was, therefore, significantly lower in the trained rats. It is concluded that collagen in every organ reacts differently to physical stress in relation to age.

Connective tissue reactions to lambda-carrageenan in the rat: Lambda-Carrageenan-Collagen Interaction

The development of the λ-carrageenan granuloma in the rat has been quantitatively examined. The wet weight, weight after freeze-drying and ether extraction, and the amount of DNA per granuloma reached a maximum twice: the first one within three days, the second maximum at the sixth day. They probably represent the maximal nonproliferative and proliferative response to λ-carrageenan, respectively. Through labeling of the collagen of young rats with 3H-proline it was shown that after 12 days about 45% of the acid insoluble collagen, preexisting in the granuloma at the third day, was no longer present in the gelatin fraction, while simultaneously new acid insoluble collagen was formed. The total amount of collagen increased from 3 up to 30 days. This finding contrasts with the rapid decrease in the amount of collagen starting at about the 15th day in guinea pig granulomata provoked by commercial carrageenan preparations. In vitro, native acid soluble collagen from rat skin was precipitated with λ-carrageenan. From this precipitate the collagen was only partially extractable by gelatinization. Its implications for the figures on collagen extracted from the granuloma were discussed.

Collagen synthesis in the secondary palate of the developing rat

The presence of hydroxyproline in whole rat foetuses and isolated palates was used to calculate collagen synthesis from the 13th day of gestation to the first neonatal day. In the palate the amount of collagen per unit dry weight increased significantly during the gestational period just prior to and during palatal closure, while a much greater rate of increase was noted after closure during the period of bone formation. In the whole foetus, the amount of collagen per unit dry weight was similar to the values observed in the palate during the period prior to and during closure, while a marked difference was noted at later gestational stages. The total amount of collagen in the palate and foetus increased in an exponential manner during the time of palatal closure.

Effect of estradiol-17β on collagen biosynthesis, degradation and re-utilization in vivo

Estradiol-17β was administered to adult, female guinea pigs in which collagen had been labelled during rapid growth by the chronic administration of 14C-proline. In skin, treatment reduced collagen biosynthesis and increased collagen degradation for de-novo biosynthesis elsewhere. In the granuloma produced by γ-carrageenan, estradiol-17β produced no change in the total amount of newly-synthesized collagen, in the amount of 14C-label derived from the degradation of prelabelled collagen or in the amount derived by the re-utilization of acid-soluble collagen. In the uterus and metaphyseal bone, estradiol-17β increased both collagenous and non-collagenous de-novo protein biosynthesis. The proline for this was derived, at least in part, from the degradation of prelabelled collagen, probably from skin.

Degradation and turnover of collagen in the mouse skin and the effect of whole body x-irradiation

1. 1. Male mice were given L-[ 3H]proline intraperitoneally at 4 weeks of age. The total radioactivity of hydroxyproline in the whole body skin increased with time up to the 6th week after the injection, and then gradually decreased to 19.8% of the peak value in the following 50 weeks. The half-life of the labeled skin collagen was calculated as 80 days in the mice at 18–30 weeks of age, 130 days at 30–45 weeks of age and 200 days at 45–60 weeks of age, respectively. 2. 2. The degredation of skin collagen in adult mice was accelerated by whole body exposure to a total of 900 or 1200 R X-ray. Exposure to 300 R 4 times resulted in a 30% increase in the degradation of labeled collagen in the 30–60-week-old mice. 3. 3. The total amount of collagen in the whole body skin was also significantly decreased by a total of 900 or 1200 R irradiation. 4. 4. The turnover rate of 1 M NaCl-soluble collagen was higher than that of insoluble collagen and this tendency was amplified by X-irradiation. 5. 5. The decrease in the solubility of skin collagen with ageing was accelerated by X-irradiation.

PHYSICAL AND CHEMICAL PROPERTIES OF EPIMYSIAL ACID‐SOLUBLE COLLAGEN FROM MEATS OF VARYING TENDERNESS

SUMMARY: Components of the molecular structure of epimysial acid‐soluble collagen (ASC) from meats of varying tenderness were studied by several methods. The ASC was studied since it contains an appreciable amount of intramolecular cross‐links but is still soluble. Though the amount of total collagen in epimysial tissue was found to have no correlation with meat tenderness, the molecular studies indicated some correlation of the type of epimysial ASC with meat tenderness. Sucrose density‐gradient ultracentrifugation analyses of denatured epimysial ASC suggested that this type of collagen contains a lesser degree of cross‐links when obtained from tender meat samples. Viscosity measurements were found to be correlated to tenderness of meat in a manner similar to the results obtained by ultracentrifugation, in that the intrinsic viscosity of epimysial ASC from tender meat was lower than that from tough meat, indicating differences among the relative sizes of the collagen molecules from the different samples. Partial amino acid analyses of the epimysial ASC samples via gas chromatography showed no differences in the levels of aspartic, serine or hydroxyproline, amino acids which may be involved in the ester type of cross‐links. Results of chemical estimation of the ester type of cross‐links indicated that the proportions of esters in the various ASC samples were similar. The amount of lysine was found to be significantly higher (P < 0.05) in epimysial ASC from tough meat compared to tender meat, suggesting an increased potential of the aldehydic‐type of cross‐link, which is formed via an aldol condensation of two aldehydes derived biosynthetically from lysine. This was strengthened by the results obtained in the chemical estimation of the aldehydic‐type of cross‐link, in that the epimysial ASC of tough meat contained significantly more aldehyde than did that of tender meat (P<0.05).

Influence of hydrocortisone acetate on the chemical composition of experimental granulomas.

SummaryGranulomas were induced by the bilateral implantation of weighed sterile cotton sponges in the dorsum of male Sherman rats. Hydrocortisone acetate (300 μg) impregnation significantly inhibited granuloma growth for approximately 5 days. In addition, steroid treatment significantly reduced the total amount and concentration of collagen, total protein, DNA and RNA. Water content was increased by about 4% in the steroid treated granulomas. Hydrocortisone acetate (300 μg) impregnation resulted in significant reduction of both total C14-L-proline incorporation and specific activity (cpm/μg protein), measured at 12 and 24 hours after implantation.

Quantitative variations in total and soluble collagen in testes of prepubertal and adult rats.

Total and soluble collagen (neutral and citrosoluble fractions) from albuginea and glandular tissue of prepubertal and adult rat testes were determined with the following results: 1) In the albuginea total collagen increased significantly with age; soluble collagen, however, decreased progressively with age; 2) in glandular tissue, comprising seminiferous tubules and intertubular structures, no significant variation was observed in the content of total collagen;soluble collagen, however, showed an increase at a prepubertal age (around 20 days), followed by a decrease in adulthood; 3) a progressive increase with age was observed in the absolute amount of total collagen in the albuginea and in the glandular tissue. The significance of the variations observed in the amount of collagen in both structures is discussed in relation to gonadal maturation, mechanical forces and permeability of the tubular wall. (Endocrinology 80: 1163, 1967)

Changes of collagen content in skin, femur and uterus of 17-beta-estradiol benzoate-treated rats.

The collagen concentration and the total amount of collagen per tissue were determined in the uterus, skin and femurs of intact female rats and in the skin and femurs of intact male rats which had received specific quantities of 17β-estradiol benzoate for 14 days (1.8, 3.6 and 14 μg/100 g body wt daily and 50 and 100 μg/ 100 g body wt every 2nd day) and in similar untreated male and female animals. In the estradiol benzoate-treated rats, the collagen from the 3 tissues showed contrasting changes. Although the mg of collagen/uterus of the rats treated with 1.8, 3.6 and 14 μg of estradiol benzoate/100 g body wt daily were increased significantly (p <0.01, 0.01, 0.05), the per cent collagen in the dry fat-free uterus was decreased in each group of steroid-treated animals (decrease significant at p<0.05 for 1.8 μg/100 g body wt daily and p <0.01 for other dosages). A significant decrease (p <0.05, 0.01, 0.05, 0.05) in g of collagen/total skin was observed with female rats at estradiol benzoate dosages of 1.8 ...

The Solubility of Intramuscular Collagen in Meat Animals of Various Ages

SUMMARYThe percentage of total intramuscular collagen solubilized by heating in 1/4‐strength Ringer's solution, muscles from 98 cattle, 15 sheep, and 9 pigs at 77°C for 1 hour decreased as chronological age increased. Total intramuscular collagen was high in very young (8‐9 weeks) bo vines and in two of the three very old (7‐15 years) bovines studied. In the age range of 16 weeks to d1/2 years, variation in total bovine intramuscular collagen was wide. There was no indication of a systematic increase as age increased. The results provide strong presumptive evidence of an increase with age in the number or strength of the cross links of intramuscular collagen and furnish an acceptable explanation as to why total amounts of intramuscular collagen are inadequate to explain toughness associated with increasing chronological age.

CONNECTIVE TISSUE 13. EFFECT OF ESTRADIOL BENZOATE UPON COLLAGEN SYNTHESIS BY SPONGE BIOPSY CONNECTIVE TISSUE.

Summary1. The effect of estradiol benzoate upon collagen synthesis in sponge biopsy connective tissue has been studied in normal and ovariectomized rats. 2. A dose of 300 μg, twice weekly for 4 weeks, produced a decrease in sponge weight, collagen concentration and total amount of collagen. However, the radioisotopic data demonstrated a stimulating effect of estrogen on collagen synthesis, as there was an increase in the specific activity of collagen, hydroxylysine and lysine and the total counts per minute recovered per incubation. These results lead to the conclusion that estradiol stimulates the metabolism of collagen. 3. The stimulatory effect of estradiol on collagen synthesis in sponge biopsy connective tissue is more readily demonstrated in the ovariectomized than in the intact rat.

The chemical composition of the human vitreous body as related to age and myopia

Chemical and viscometric studies were carried out on the vitreous bodies of 42 human post-mortem eyes. The age range in this series, which included normal and myopic eyes, was from newborn to 85 years old. In all cases, the vitreous body was divided into two portions, viz. cortical layer and central portion, for the various determinations. The findings of previous investigators that the concentrations of proteins and hyaluronic acid (calculated from the hexosamine content) are higher in the cortical layer than in the central region were confirmed. Very little collagen could be detected in the cortical layer, almost all of it being in the central portion. In confirmation of previous investigations, the viscosity numbers of the central region were found to be higher than those of the cortical layer in normal eyes. The concentrations of both collagen and hyaluronic acid were very low in the infant group (0–2 years old), but they appeared to reach adult levels by the age of 13. In contrast, the protein levels at birth were nearly equal to those of adults. Although the total amounts of protein, hyaluronic acid and collagen in the central portion were nearly the same in myopic and nonmyopic eyes, the concentrations of all three were considerably lower in the myopic group. In 5 of the 7 myopic eyes studied, the viscosity number of the cortical layer was higher than that of the central portion.

Seasonal Variation in the Collagen Content of Pacific Herring Tissues

The total amount of collagen and its relative solubility was determined in 3-year-old herring of the same population caught at two periods, June and January, representing two extremes of the sexual cycle. Sexually mature herring, caught in January, contained 40% more collagen than sexually immature fish caught in June. When fractionated into neutral salt-soluble, acid-soluble and insoluble collagen, the proportions of the three fractions remained much the same at both seasons of the year. When various tissue fractions of the fish were examined at the two seasons it was found that not only did the concentration of collagen change in the tissues with maturation but the contribution which some of the tissues made to the total body weight also changed. The skin + scales fraction contributed most to the increase in collagen in February herring. This was due partly to a higher concentration of collagen in these tissues at this season and partly to the higher proportion of skin + scales at this time of year. The significance of these findings in relation to the biochemistry of sexual maturation is considered.

Studies on the formation of collagen. II. The influence of growth rate on neutral salt extracts of guinea pig dermis.

The total amount of neutral salt-extractible collagen in the skin of growing, suckling guinea pigs amounted to about 10 per cent of the total collagen of the dennis. This is roughly equivalent to a 1 to 2 day increment in dermal collagen incident to growth. Fourteen days of static weight maintained by limited caloric intake reduced the neutral salt-extractible collagen to very low levels. Following this period, 5 to 7 days of steady weight gain induced by ad lib. feeding was required to produce significant increases in this collagen fraction. Return to control levels occurred within 12 days of continuous growth. The amount of collagen extracted from the dermis of young guinea pigs with cold neutral salt solutions varied directly with growth rate (weight gain) and was greatly diminished after short periods of restricted caloric intake. Two days of fasting diminished the total extracted collagen by one-half. Three consecutive extractions with citrate buffer pH, 3.5, of the residues remaining after exhaustive saline extraction removed 40 per cent more collagen from the skins of actively growing animals than from those of animals fasted for 2 days. However, subsequent extraction of residues with dilute acetic acid equalized the total amount of collagen extracted at acid pH from the two groups. The viscosity of cold neutral extracts was unrelated to the concentrations of non-collagenous proteins and carbohydrates but varied directly with the collagen content.