The behavior of (14C)-D-penicillamine in collagen metabolism.

The most important effect of penicillamine on collagen metabolism is the reduction of collagen crosslinking. However, even after long time application of penicillamine, collagen is crosslinked to a certain degree. After intravenous injection of a trace dose of (14C) labelled D-penicillamine it can be determined that this substance is rapidly bound to neutral salt soluble, acetic acid soluble and urea soluble collagen fractions and to a lesser extent to insoluble collagen as well. The amount of penicillamine which binds to any of the collagen fractions depends on the turnover rate. When different tissues are compared, penicillamine seems to have the greatest affinity to tissues with a high collagen turnover. Further studies of neutral salt soluble collagen by CM-cellulose chromatography revealed a stable linkage of penicillamine to collagen alpha chains.     

Collagen Synthesis by Lathyrogen-Treated 3T6 Fibroblasts

The lathyrogen, beta-aminopropionitrile (BAPN), in ascorbate-free medium increased the synthesis of both collagenous and non-collagenous proteins in 3T6 fibroblasts. Cell layer collagen was underhydroxylated and more easily extracted with neutral salt in treated cultures. The ratios of proline: hydroxyproline in sequentially extracted collagen in the cell layer of both BAPN and control cultures showed virtually no difference in the acid soluble and insoluble fractions; however, the BAPN salt-soluble fraction was definitely underhydroxylated when compared to control cultures. The data suggest that the salt-soluble macromolecule represents, in addition to a cross-linking defect, an underhydroxylated intracellular collagen precursor.     

The effects of colchicine and its derivates on the collagen biosynthesis in vitro

Recent evidence shows that colchicine decreases collagen production and favourably influences some fibroproductive processes in experimental animal and clinical practice. In our study we investigated the influence of two colchicine derivates — demecolcine and desacetylcolchiceine — on the synthesis of collagen and non-collagen proteins in new born rat skin after the incubation of tissues slices with¹⁴C-proline for 2 h. Collagen was separated in fractions soluble in either 0.45M NaCl or 2% semicarbazide that in an insoluble residue. Demecolcine (10^–3–5·10^–5 M), desacetylcolchiceine (10^–2–5·10^–4 M), as well as colchicine (10^–4–10^–6), inhibited the synthesis of soluble collagen, depending on the drug concentration in the incubation media. Higher concentrations of tested agents also decreased the formation of insoluble collagen. Significant inhibition of non-collagen protein synthesis was ascertained only in the presence of desacetylcolchiceine.     

Salt-soluble collagen and elastin in the human aorta and pulmonary artery

Collagen and elastin, the major structural components of blood vessels, have a very low turnover. In disease, this rate may be increased and an elevation of the tissue concentration of the soluble degradation fragments might be anticipated. In this preliminary study the concentration of extractable collagen and elastin in the aorta and pulmonary artery of eight human subjects postmortem was determined. The proportion of pulmonary artery collagen and elastin that was soluble was generally either equal to or greater than that in the abdominal aorta. The fraction of collagen that was salt extractable was larger than the soluble elastin fraction.     

Isolation and partial characterization of collagen fibrils, fibers and fiber-bundles from insoluble calf dermis

Collagen fibrils, fibers and fiber bundles have been isolated from insoluble calf skin chips by disaggregation with a neutral salt buffer followed by differential centrifugation. The ratio of type I collagen/type III collagen was relatively constant, regardless of the size of the fibrous materials. On the other hand, the ratios of glycosaminoglycans/collagen and non-collagenous glycoproteins/collagen were significantly higher in the thinner fiber fractions. However, the ratio of hyaluronic acid to dermatan sulfate was unchanged. The role of the non-collanenous constituents in collagen fibrillogenesis in vivo is discussed.     

Biosynthesis of proteins of the blood serum, liver, and aorta in experimental atherosclerosis

Summary Labelled glycine-1-C¹⁴ and methionine-1-C¹⁴ were used to study the biosynthesis of protein fractions of the blood serum, proteins of the subcellular fractions of the liver and fractions of aorta proteins in rabbits with experimental cholesterol atherosclerosis. It has been found that in atherosclerosis there is an unequal decrease in the biosynthesis of all protein fractions of the blood serum. The decrease in the biosynthesis of liver proteins in experimental atherosclerosis is due to a decrease in protein synthesis in nuclear and microsome fractions of the liver cell. In experimental atherosclerosis there is a decrease in the synthesis of collagen and no change in the biosynthesis of soluble proteins and elastin of the aorta.(Presented by Active Member of the Academy of Medical Sciences of the USSR A. L. Myasnikov). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 62, No. 10, pp. 51–53, October, 1966.     

Relationship Between the Dose of Ascorbic Acid and its Structural Analogs and Proline Hydroxylation in Various Biological Systemst

The effect of ascorbic acid on the rate of in vitro collagen formation was examined in several guinea pig tissues having different rates of collagen metabolism and in chick embryo skin after various conditions of ascorbic acid supplementation.

It was found that in embryonic skin, carrageenan granuloma and liver the extent of C¹⁴-hydroxyproline forniation was markedly influenced by the amount of ascorbic acid present in the tissue, which, in turn, reflected the dose of ascorbic acid administered in vitro. The increase of collagen synthesis above the normal rate was directly proportional to the increase in concentration of ascorbic acid in the tissue. This was observed not only in tissues from animals dependent on an exogenous supply of ascorbic acid but also in tissues from organisms capable of ascorbic acid synthesis. However, the rate of collagen synthesis was not affected by ascorbic acid in organs which normally exhibit a low metabolic turnover of collagen (lung, muscle); in these collagen synthesis was found to be independent of the amount of available ascorbic acid. The presence of additional ascorbic acid in the incubation medium had a stirnulatory effect on collagen synthesis in chick embryo skin and carrageenan granuloma, but this immediate effect was less pronounced than that of the endogenous ascorbic acid, elevated by in viao supplementation. It is suggested that the stimulatory effect of ascorbic acid on collagen synthesis may be dependent on the availability of the precursor form of proline hydroxylase.

Comparison of the in vitro and in vivo effect of some ascorbic acid analogs upon proline hydroxylation in carrageenan granuloma from scorbutic guinea pigs indicates that although some of the derivatives were effective in vitro, their efficiency in vivo was not comparable to the effect of L-ascorbic acid.

Using the measure of radioactivity of ultrafilterable hydroxproline as an index of collagen degradation, it was found that ascorbic acid deficiency did not accelerate degradation of collagen which had been hydroxylated.     

Distribution of Ingested 14C-Cholesterol in the Macromolecular Fractions of Rat Connective Tissues

The distribution of ¹⁴C-cholesterol was studied in the connective tissues of rats fed on a normal diet and on a high-cholesterol diet. The tissues studied are the skin, subcutaneously implanted polyvinyl sponge and aorta. Radioactivity and cholesterol content were determined in the chloroformmethanol extracts of these tissues as well as in macromolecular fractions obtained by their 'chemical dissection'.

Semilog plots of radioactivity determined in successive chloroform-methanol extracts gave an indication of the speed of extraction of cholesterol from the tissues studied. This speed was about equal for skin and sponge tissues and significantly higher for aorta; it was not influenced by the regime or by the sex of the animals. The first two chloroform-methanol extracts contained over 95 % of the total radioactivity. The remaining radioactivity showed non-random distribution between the macromolecular fractions of the tissues. A significant fraction of radioactivity remained associated with the polymeric fibrous stroma (several times extracted with 1 M CaC1₂) containing mainly collagen, structural glycoproteins and elastin. The fraction of radioactivity associated with elastin was significantly increased in the high cholesterol diet groups.

The specific activity of tissue-cholesterol varied between large limits and was significantly different from the blood serum cholesterol. Especially high specific activities were found in the residual cholesterol fraction associated with elastin. These results support the assumption of several separate, slowly equilibrating tissue pools of cholesterol and suggest that the cholesterol associated with the macromolecular components of the intercellular matrix (elastin, collagen, structural glycoproteins) may represent such separate pools.     

The Effects of Acetic Acid on Collagen Cross-Links

Native, acid-soluble and acid-insoluble collagen from the tail tendons of rats and calves have been reduced by ³H-sodium borohydride. The reduced cross-link derivatives were separated by ion exchange chromatography. The destruction of the cross-links under the conditions of hydrolysis was measured. By applying the appropriate correction factors, the approximate levels of the respective cross-link structures in the variously treated tissues were assessed. We conclude that treatment of collagen by 0.05 M acetic acid for 1 hour or less imposes a profound rearrangement of intermolecular bonds in both the soluble and insoluble preparations. The nature of the intermolecular bonding in native collagen therefore cannot be meaningfully assessed if the preparation is previously exposed to acid.     

Protein turnover,amino acid requirements and recommendations for athletes and active populations

Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers (¹³C-lysine, ¹⁵N-glycine, ²H₅-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg⁻¹·day⁻¹ compared to 0.8 g·kg⁻¹·day⁻¹ in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.     

Cell Death and Proliferation and Its Relation to Collagen Degradation in Uterine Involution of Rat

Collagen concentration, procollagenase localization, and their association with cell proliferation and apoptosis during postpartum involution, were investigated biochemically and histochemically in postpartum day 1, 3, 5, and 7 rat uterine tissues. In control animals, uterine wet weight, soluble protein, and collagen decreased rapidly during days 1 to 3 postpartum, and the DNA concentration in the uterine horn rapidly decreased, as noted by others. Simultaneously, both apoptosis and cell proliferation were observed in these tissues. These processes were highest in smooth muscle cells on day 3 postpartum. Procollagenase was found in the cell cytoplasm through days 1 to 3 postpartum, was highest on the third day postpartum, and appeared to gradually diminish by day 5 postpartum. Disorganization of collagen fibers was observed, under polarized microscopy by a strong birefringence of collagen fibers of the circular smooth muscle cell layers. However, this disorganization of the uterine collagen diminished progressively from day 3 to day 7. Treatment with estradiol or a combination of estradiol and progesterone suppressed cellular turnover and attenuated the changes in DNA, total amino acids, and collagen on day 3 postpartum. In this study, cellular turnover and biochemical and morphological changes appeared to be closely associated. Gonadal steroid hormones appear to influence these changes and retard uterine involution. This study suggests that a dynamic turnover of the cellular population takes place during uterine involution. It is possible that other factors, in addition to steroid hormones, contribute to uterine involution. It is to be postulated that these factors either are themselves decreased or, alternatively, may increase the inhibition of other unknown factors by an indirect mechanism.     

Collagen and mechanical strength in various organs of rats treated with D-penicillamine or amino-acetonitrile.

After oral treatment with D-penicillamine (D-Pc) or with aminoacetonitrile (AAn) for 10 days, mechanical and chemical parameters were studied simultaneously in various organs of Sprague Dawley rats. Tensile strength of skin strips and of tail tendons, breaking strength of femur bones and tensile strength of granuloma tissue (induced by implanted glass rods) were measured and calculated. In the same tissue the soluble collagen fractions and the insoluble collagen were determined. Total collagen and the ratio insoluble vs. soluble collagen were calculated. Tensile strength of skin, tendon and granuloma tissue were greatly reduced by D-Pc treatment but only minimally influenced by AAN treatment. On the other hand only AAN significantly reduced the breaking strength of bone. All these changes were closely correlated with the content of insoluble collagen in the respective tissues. The correlation coefficients to total collagen were similar but lower. The correlation coefficients between strength and the ratio insoluble vs. soluble collagen were generally still lower. Earlier findings in aged and corticoid treated rats, proving that insoluble collagen content determines mechanical strength of connnective and supporting tissue thus could be confirmed.     

Sodium pump quantity and turnover in rabbit descending colon at different rates of sodium absorption

³H-Ouabain binding to isolated epithelia and basolateral membrane vesicles of Na⁺-transporting epithelial cells of rabbit descending colon was determined to quantify the number of operative Na⁺-pump sites at different rates of transcellular Na⁺ transport which was varied over a wide range by chronic dietary Na⁺ restriction or Na⁺ loading. Both in intact epithelia and in basolateral membrane vesicles the maximal number of specific ouabain binding sites was higher in preparations from animals transporting Na⁺ at high rates than in preparations from animals transporting Na⁺ at low rates. The affinity of ouabain to its binding site and the association and dissociation rate constants were not dependent on the rate of Na⁺ transport. In intact epithelia the Na⁺ turnover rate per pump unit was twice as high in tissues with high Na⁺ transport than in tissues with low Na⁺ transport. In basolateral membrane vesicles the Na⁺ turnover rate was considerably higher than in intact epithelia and there was no difference in turnover rate between vesicle preparations obtained from tissues transporting Na⁺ at high or low rates. Hence, factors within the intact cell appear to control the turnover rate of the Na⁺-pump.     

Collagen Alteration in Medial Collateral Ligament Healing in a Rabbit Model

The purpose of this study was to evaluate changes over time in surgically ruptured medial collateral ligament (MCL) with respect to collagen synthesis, collagen degradation and collagen type. Our method involved prelabeling collagen in growing rabbits using ³H-proline followed by surgical injury to the MCL. Animals were sacrificed at intervals up to 40 weeks post-injury. At each time point total hydroxyproline, changes in collagen mass, and collagen synthesis and degradation were determined and related to the unruptured control MCL. A separate semiquantitative determination of collagen type was also performed on the midsubstance of each healing and control ligament. Results showed an increase in total collagen mass and a decrease in collagen concentration in all healing ligaments. Concommitantly, an increase in the collagen turnover rate was observed. Relative turnover was greatest at 3 to 6 weeks after injury and returned toward the normal rate by 40 weeks. Type I collagen was partially replaced by type III, probably as a result of increased synthesis during scar formation. The mechanism for collagen remodeling (replacement of mass and concentration in an organized fashion) in untreated rabbit MCL scar appears functional by virtue of its chronicity but, due to a shift in collagen type, it may also be qualitatively inadequate.     

Collagen and Mechanical Strength in Various Organs of Rats Treated With D-Penicillamine or Amino-Acetonttrile

After oral treatment with D-penicillamine (D-Pc) or with aminoacetonitrile (AAN) for 10 days, mechanical and chemical parameters were studied simultaneously in various organs of Sprague Dawley rats. Tensile strength of skin strips and of tail tendons, breaking strength of femur bones and tensile strength of granuloma tissue (induced by implanted glass rods) were measured and calculated. In the same tissue the soluble collagen fractions and the insoluble collagen were determined. Total collagen and the ratio insoluble vs. soluble collagen were calculated. Tensile strength of skin, tendon and granuloma tissue were greatly reduced by D-Pc treatment but only minimally influenced by AAN treatment. On the other hand only AAN significantly reduced the breaking strength of bone. All these changes were closely correlated with the content of insoluble collagen in the respective tissues. The correlation coefficients to total collagen were similar but lower. The correlation coefficients between strength and the ratio insoluble vs. soluble collagen were generally still lower. Earlier findings in aged and corticoid treated rats, proving that insoluble collagen content determines mechanical strength of connective and supporting tissue thus could be confirmed.     

Synthesis of lung collagen in hamsters with elastase-induced emphysema

Lung collagen was studied in hamsters which had been intratracheally instilled with elastase. Total and insoluble collagens in lungs among the elastase-instilled were higher than those of the age-matched controls and continued to be elevated even 4 months after the elastase-instillation. A transient increase in salt-soluble collagen during the period 6 to 20 days after the elastase-instillation was also found among the experimentals. Studies of incorporation of l-[¹⁴C]proline into lung collagen showed higher specific radioactivities in both hydroxyproline and collagen fractions as well as a many-fold increase in total radioactivities in collagen fractions of lungs among the elastase-instilled hamsters. The results indicated that collagen metabolism in hamster lungs was activated after the elastase-injury which led to an increase of collagen in lung.     

The structural basis of cell-mediated immunological reactions of collagen: Reactivity of separated α-chains of calf and rat collagen in cutaneous delayed hypersensitivity reactions

Denatured calf and rat skin collagen and α1- and α2-chains from these collagens were tested for their capacity to sensitize guinea-pigs for the cutaneous delayed hypersensitivity reaction and to elicit this reaction in sensitized animals. Alpha-chains from neutral salt extracted and from urea extracted collagen were also compared. All preparations were fully active as sensitizers as well as elicitors of the skin reaction. In spite of extensive cross-reactions, significant differences between α1- and α2-chains of a given species as well as between corresponding chains of different species could be detected. Differences between chains from neutral salt extracted collagen and from urea extracted collagen were also revealed.     

Collagen-bound collagenase

The presence of collagenase bound to collagen extracted and purified from several animal and human sources by a standard procedure has been confirmed by different methods. Polyacrylamide (10%) gel electrophoresis at pH 8.1 of intact or "spontaneously"degraded neutral salt soluble collagen results in the separation of two components: the upper one says at the origin and represents collagen or collagen ragments, whereas the lower protein component contains no collagen, often preserves specific collagenolytic activity, and migrates as a single band in SDS/polyacrylamide electrophoresis. With lower polyacrylamide gel concentration the electrophoretic separation of the two components is less clear. Removal of the lower protein component from collagen solutions by two different methods (TCA-ethanol purification cycles and pepsin digestion) results in concomitant loss of their "spontaneous" instability. Eluates of the lower protein component stimulate the heterologous production of a monospecific antibody capable of inhibiting the collagenolytic activity of homologous crude collagenase preparations. It is suggested that collagen-bound collagenase is not an artifact of the extraction procedure but rather a physiological reality, probably corresponding in the living animal to the enzyme closely associated with extracellular collagen fibers, revealed by immunohistochemical methods.     

Dermal collagen implants

The feasibility of using preparations of cell-free, fibrous dermal collagen, prepared fay trypsin-treatment of skin, for the repair of soft body tissues has been examined both as subcutaneous implants and as a replacement for dermis in skin wounds in rats. Increased collagen stability, and suppression or reduction of tissue antigenrcity in collagen heterografts, was achieved by crosslinking with weak solutions of aldehydes while still allowing implant recellularization and revascularization. Tritium-labelled collagen turnover studies have shown that maintenance of collagen mass in implants crosslinked with glutaraldehyde occurs primarily by inhibition of loss of original implant collagen. Some of the in vitro growth characteristics of human fibroblasts on animal collagen preparations are also described.     

Polarized Light Microscopic Analysis of Bone Formation After Inhibition of Cyclooxygenase 1 and 2

Potassium diclofenac is a potent nonsteroidal anti‐inflammatory drug (NSAID) and COX isoforms (COX‐1 and COX‐2) inhibitor. Quantitative analysis of birefringence with polarized light microscopy is a useful method to investigate the macromolecular orientation and organization of collagen fibers in connective tissues. The aim of this research was to analyze the collagen structure and maturation in bone formed after potassium diclofenac administration, during first molar orthodontic movement. Sixty Wistar rats were divided in two equal groups (N = 30): control (C) and potassium diclofenac (PD). The animals in Group C received 0.9% saline solution and the PD group received potassium diclofenac Cataflam^® (5 mg/kg). Animals were sacrificed 3, 7, or 14 days after a NiTi unilateral closed‐coil spring was stretched between the upper right first molar and the incisors. The first molar area was fixed, decalcified, and histologically processed using picrosirius pigment. The collagen birefringence of bone turnover was analyzed by phase retardation. Two‐way ANOVA and Tukey's test showed that optical retardation was influenced by time and treatment. There was increase in the collagen organization over time. On the third day, the C group showed better collagen organization than the PD group. Potassium diclofenac interfered in collagen maturation, reducing fibril organization in the initial phase of orthodontic movement. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.