scholarly journals [3H]proline incorporation and hydroxyproline concentration in articular cartilage during the development of osteoarthritis caused by immobilization. A study in vivo with rabbits

1981 ◽  
Vol 200 (2) ◽  
pp. 435-440 ◽  
Author(s):  
T Videman ◽  
I Eronen ◽  
T Candolin
Keyword(s):  
Total Protein ◽  
Collagen Synthesis ◽  
Weight Bearing ◽  
Specific Radioactivity ◽  
Collagen Content ◽  
Synthetic Activity ◽  
Synthesis Rate ◽  
Hydroxyproline Concentration ◽  
Proline Incorporation

Proline metabolism in vivo was studied during the development of immobilization osteoarthritis in rabbits. Collagen content was measured as the hydroxyproline concentration of the tissue in question. The incorporation of [3H]proline was used as the indicator for total protein synthesis; collagen synthesis rate was estimated from measurements of the specific radioactivity of hydroxyproline. Cartilage samples from knee and hip joints were analysed after 3, 7, 11, 18, 35 and 56 days of immobilization. The total protein and collagen synthesis rates of the immobilized legs increased and reached a maximum after 11-35 days. Although they decreased thereafter, these rates remained elevated to the end of the experiment. A slight increase in the synthetic activity of the non-immobilized contralateral legs was also detected after 7--18 days of immobilization. The isotope incorporation was markedly higher in tibial marginal tissue than in weight-bearing cartilage. In spite of the increased synthesis, no clear changes were found in the collagen content of the tissues studied during the experiment.

scholarly journals Some biochemical effects of triamcinolone acetonide on rat liver and muscle

1970 ◽  
Vol 116 (3) ◽  
pp. 349-355 ◽  
Author(s):  
R. F. Peters ◽  
M. C. Richardson ◽  
Margaret Small ◽  
A. M. White
Keyword(s):  
Amino Acids ◽  
Triamcinolone Acetonide ◽  
Time Course ◽  
Muscle Protein ◽  
Specific Radioactivity ◽  
Synthetic Activity ◽  
Tissue Homogenate ◽  
Glycogen Concentration

1. The powerful anti-inflammatory glucocorticoid triamcinolone acetonide, administered to rats at 20 and 2.5mg/kg, leads to a decrease in the incorporation in vivo of [3H]uridine and [32P]orthophosphate into hind-limb skeletal muscle. 2. At the higher dose, this decrease in the rate of incorporation of precursors into RNA precedes a decrease in the incorporating ability of muscle ribosomes, which commences about 4–5h after drug administration, but is unaccompanied by any changes in the concentration of tissue ATP or free amino acids. 3. The ribosomal dysfunction extends to polyribosomes, which can only be successfully isolated from the muscle of triamcinolone-treated animals after the addition of α-amylase to the tissue homogenate to remove glycogen. 4. The specific radioactivity of muscle protein labelled in vivo with 14C-labelled amino acids does not decrease progressively after triamcinolone administration. After 2h there is an apparent stimulation of incorporation which leads to an overall discrepancy between measurements of protein-synthetic activity made in vivo and in vitro. 5. There is a significant increase in muscle-glycogen concentration between 8 and 12h after the administration of triamcinolone acetonide (20mg/kg), although a significant decrease occurs after 4h. The fall in glycogen concentration may be due to a decrease in the rate of synthesis of protein essential for glucose uptake into the tissues. 6. As judged by (a) incorporation of 14C-labelled amino acids into protein, (b) [3H]uridine and [32P]-orthophosphate incorporation into RNA, (c) the rate of induction of tryptophan pyrrolase and (d) changes in the pool sizes of taurine and tryptophan, the responses in liver followed the same time-course as those in muscle after administration of the drug.

scholarly journals Dexamethasone modulates the metabolism of type IV collagen and fibronectin in human basement-membrane-forming fibrosarcoma (HT-1080) cells

1987 ◽  
Vol 245 (1) ◽  
pp. 235-241 ◽  
Author(s):  
A Oikarinen ◽  
T Salo ◽  
L Ala-Kokko ◽  
K Tryggvason
Keyword(s):  
Total Protein ◽  
Messenger Rna ◽  
Collagen Synthesis ◽  
Glucocorticoid Receptors ◽  
Type Iv Collagen ◽  
Cell Layer ◽  
Specific Binding ◽  
Specific Radioactivity ◽  
Filtration Method ◽  
Type Iv

The effect of dexamethasone on the synthesis and degradation of type IV collagen was studied in human fibrosarcoma cells, HT-1080. A dexamethasone concentration as low as 0.1 microM markedly increased collagen synthesis in HT-1080 cells labelled with [14C]proline. The increase in type IV collagen synthesis was not specific, since total protein synthesis was also increased. Further studies indicated that part of the increase was due to an increase in the specific radioactivity of the intracellular proline pool, after dexamethasone treatment. In fact, with dexamethasone concentrations of 0.1-10 microM the relative collagen synthesis was decreased, indicating that synthesis of other protein was increased more than that of type IV collagen. This was also confirmed by measuring the relative amount of type IV collagen RNA by using recombinant plasmid cDNA specific for the human procollagen pro alpha l (IV) RNA. The results indicated that relative collagen synthesis and the relative amount of type IV collagen messenger RNA was decreased similarly, indicating that dexamethasone affected type IV collagen synthesis at the pre-translational level. The dexamethasone-induced effect on total protein and collagen synthesis was maximal after 12-24 h. Dexamethasone induced a marked accumulation of collagen into the cell layer, leading to diminished deposition of soluble collagen into the medium. Since bacterial-collagenase treatment of the cell layer drastically decreased the collagen content of the dexamethasone-treated cells, this indicates that dexamethasone caused an accumulation of collagen into the extracellular matrix of the cell layer. In contrast, the amount of fibronectin was markedly increased in the medium. Dexamethasone decreased the type IV collagen-degrading activity in HT-1080 cells. The HT-1080 cells contained glucocorticoid receptors, as demonstrated by two different methods: by a whole-cell binding assay and by using a cytosol-gel-filtration method. The number of specific binding sites was similar to that in human skin fibroblasts. In conclusion, glucocorticoids affect the metabolism of type IV collagen and fibronectin in HT-1080 cells, and, since these cells contain specific glucocorticoid receptors, the effects are apparently receptor-mediated.

Glucocorticoid-induced skeletal muscle atrophy in vitro is attenuated by mechanical stimulation

1992 ◽  
Vol 262 (6) ◽  
pp. C1471-C1477 ◽  
Author(s):  
J. A. Chromiak ◽  
H. H. Vandenburgh
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Protein Content ◽  
Total Protein ◽  
Weight Lifting ◽  
Mechanical Activity ◽  
Synthesis Rate ◽  
Protein Synthesis Rate

Glucocorticoids induce rapid atrophy of fast skeletal myofibers in vivo, and either weight lifting or endurance exercise reduces this atrophy by unknown mechanisms. We examined the effects of the synthetic glucocorticoid dexamethasone (Dex) on protein turnover in tissue-cultured avian fast skeletal myofibers and determined whether repetitive mechanical stretch altered the myofiber response to Dex. In static cultures after 3-5 days, 10(-8) M Dex decreased total protein content 42-74%, total protein synthesis rates 38-56%, mean myofiber diameter 35%, myosin heavy chain (MHC) content 86%, MHC synthesis rate 44%, and fibronectin synthesis rate 29%. Repetitive 10% stretch-relaxations of the cultured myofibers for 60 s every 5 min for 3-4 days prevented 52% of the Dex-induced decrease in protein content, 42% of the decrease in total protein synthesis rate, 77% of the decrease in MHC content, 42% of the decrease in MHC synthesis rate, and 67% of the decrease in fibronectin synthesis rate. This in vitro model system will complement in vivo studies in understanding the mechanism by which mechanical activity and glucocorticoids interact to regulate skeletal muscle growth.

scholarly journals Protein synthesis in rat lung. Measurements in vivo based on leucyl-tRNA and rapidly turning-over procollagen I

1984 ◽  
Vol 222 (1) ◽  
pp. 77-83 ◽  
Author(s):  
J Kelley ◽  
W S Stirewalt ◽  
L Chrin
Keyword(s):  
Plasma Proteins ◽  
Specific Radioactivity ◽  
Young Male ◽  
Male Rats ◽  
Synthesis Rate ◽  
Vascular Perfusion ◽  
Fractional Synthesis ◽  
Free Serum ◽  
Procollagen Iii

The relationships of the specific radioactivities of leucine in serum, leucine acylated to tRNA and leucine in procollagen I, procollagen III and total protein in lungs of unanaesthetized young male rats in vivo were assessed as a function of time during constant intravenous infusion of radiolabelled leucine. The specific radioactivity of free leucine in plasma reached a steady-state plateau value within 30 min of initiation of [3H]leucine infusion. Leucine acylated to tRNA isolated from lungs had the same specific radioactivity as free serum leucine. Leucine in procollagen I rapidly achieved a specific radioactivity equal to that of serum leucine and leucyl-tRNA, indicating that serum leucine and leucyl-tRNA isolated from total lung were in rapid equilibrium with the precursor leucine pool for procollagen I synthesis. On the basis of leucyl-tRNA or free serum leucine as the precursor, half-times of fractional conversion of procollagen I and III were calculated as 9 and 38 min respectively. The incorporation of leucine into mixed lung proteins calculated from the tracer studies was 6.8 mumol/day for the first 30 min of the infusion, after which the calculated rate increased to 15.0 mumol/day. This apparent increase correlated with the appearance of rapidly labelled plasma proteins trapped in the lungs. On the basis of short infusions lasting 30 min or less, followed by vascular perfusion of the lung, the average fractional synthesis rate of mixed pulmonary proteins in young male rats was 20%/day.

scholarly journals The effect of intermittent changes in tension on protein and collagen synthesis in isolated rabbit muscles

1981 ◽  
Vol 198 (3) ◽  
pp. 491-498 ◽  
Author(s):  
R M Palmer ◽  
P J Reeds ◽  
G E Lobley ◽  
R H Smith
Keyword(s):  
Protein Synthesis ◽  
Total Protein ◽  
Collagen Synthesis ◽  
Muscle Protein ◽  
Lactate Concentration ◽  
Constant Tension ◽  
Intact Rabbit ◽  
Mechanical Stretching ◽  
Total Muscle

Isolated intact rabbit muscles were incubated in a medium containing radioactive proline. The rates of synthesis of collagen and total muscle protein after incubation with a constant tension or intermittent mechanical stretching were compared with the rates in vivo. Muscles incubated under a constant tension synthesized protein at 22% of the rate observed in vivo; intermittent mechanical stretching resulted in an increase of 73% in the rate of protein synthesis, to 38% of that found in vivo. Collagen synthesis was affected in the same way as total protein synthesis by both types of incubation, therefore the relative rates of collagen and total protein synthesis were unchanged. ATP concentration in the isolate muscles and the uptake of glucose from the medium were increased by intermittent mechanical stretching. Incubating the muscles with a gas phase containing 5% O2 decreased the rate of protein synthesis, abolished the effect of intermittent mechanical stretching, lowered the concentration of ATP and increased the lactate concentration. The rate of protein synthesis in muscles maintained with a constant or intermittently applied tension was not affected by a previous period of incubation with the other type of stimulus.

Intestinal mucosa in diabetes: synthesis of total proteins and sucrase-isomaltase

1986 ◽  
Vol 250 (6) ◽  
pp. G788-G793 ◽  
Author(s):  
W. A. Olsen ◽  
E. Perchellet ◽  
R. L. Malinowski
Keyword(s):  
Protein Synthesis ◽  
Intestinal Mucosa ◽  
Total Protein ◽  
Specific Radioactivity ◽  
Small Intestinal Mucosa ◽  
Insulin Deficiency ◽  
Intestinal Protein ◽  
Streptozotocin Induced Diabetes ◽  
Small Intestinal

The effects of insulin deficiency on nitrogen metabolism in muscle and liver have been extensively studied with recent in vivo demonstration of impaired protein synthesis in rats with streptozotocin-induced diabetes. Despite the significant contribution of small intestinal mucosa to overall protein metabolism, the effects of insulin deficiency on intestinal protein synthesis have not been completely defined. We studied the effects of streptozotocin-induced diabetes on total protein synthesis by small intestinal mucosa and on synthesis of a single enzyme protein of the enterocyte brush-border membrane sucrase-isomaltase. We used the flooding-dose technique of McNurlan, Tomkins, and Garlick (Biochem. J. 178: 373–379, 1979) to minimize the difficulties of measuring specific radioactivity of precursor phenylalanine and determined incorporation into mucosal proteins and sucrase-isomaltase 20 min after injection of the labeled amino acid. Diabetes did not alter mucosal mass as determined by weight and content of protein and DNA during the 5 days after injection of streptozotocin. Increased rates of sucrase-isomaltase synthesis developed beginning on day 3, and those of total protein developed on day 5. Thus intestinal mucosal protein synthesis is not an insulin-sensitive process.

scholarly journals Downregulation of NOX4 Expression by Roflumilast N-Oxide Reduces Markers of Fibrosis in Lung Fibroblasts

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Daniela Vecchio ◽  
Alessandra Acquaviva ◽  
Beatrice Arezzini ◽  
Hermann Tenor ◽  
Piero A. Martorana ◽  
...  
Keyword(s):  
Collagen Synthesis ◽  
Lung Fibroblasts ◽  
Ros Generation ◽  
Phosphodiesterase 4 ◽  
Nadph Oxidase 4 ◽  
Proline Incorporation ◽  
Per Se ◽  
Oxidative Effects

The phosphodiesterase 4 inhibitor roflumilast prevents bleomycin- (BLM-) induced lung fibrosis in animal models. However, its mechanism of action remains unknown. We investigated whether roflumilast N-oxide (RNO), the active metabolite of roflumilast, can modulatein vitrothe oxidative effects of BLM on human lung fibroblasts (HLF). In addition, since BLM increases the production of F2-isoprostanes that haveper sefibrogenic activity, the effect of RNO on oxidative stress and fibrogenesis induced by the F2-isoprostane 8-epi-PGF2αwas investigated. HLF were preincubated either with the vehicle or with RNO and exposed to either BLM or 8-epi-PGF2α. Proliferation and collagen synthesis were assessed as [3H]-thymidine and [3H]-proline incorporation. Reactive oxygen species (ROS) and F2-isoprostanes were measured. NADPH oxidase 4 (NOX4) protein and mRNA were also evaluated. BLM increased both cell proliferation and collagen synthesis and enhanced ROS and F2-isoprostane production. These effects were significantly prevented by RNO. Also, RNO significantly reduced the increase in both NOX4 mRNA and protein, induced by BLM. Finally, 8-epi-PGF2α  per sestimulated HLF proliferation, collagen synthesis, and NOX4 expression and ROS generation, and RNO prevented these effects. Thus, the antifibrotic effect of RNO observedin vivomay be related to its ability to mitigate ROS generation via downregulation of NOX4.

scholarly journals Turnover of muscle protein in the fowl. Collagen content and turnover in cardiac and skeletal muscles of the adult fowl and the changes during stretch-induced growth

1978 ◽  
Vol 176 (2) ◽  
pp. 419-427 ◽  
Author(s):  
Geoffrey J. Laurent ◽  
Malcolm P. Sparrow ◽  
Peter C. Bates ◽  
David J. Millward
Keyword(s):  
Total Protein ◽  
Latissimus Dorsi ◽  
Skeletal Muscles ◽  
Collagen Synthesis ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Collagen Content ◽  
Constant Infusion ◽  
Intracellular Protein ◽  
Total Muscle

The collagen content and the rate of collagen synthesis were measured in the anterior and posterior latissimus dorsi muscles and in heart from fully grown fowl. This was done by measuring the proline/hydroxyproline ratios in the muscle and by a constant infusion of [14C]proline. These measurements were also made during the hypertrophy of the anterior muscle in response to the attachment of a weight to one wing of the fowl. In the non-growing muscles the collagen content was higher in the anterior muscle (22.8% of total protein) than in the posterior muscle (9.5% of total protein) and lowest in the heart (3.8% of total protein). In the two skeletal muscles a little over half of the collagen was accounted for by internal collagen (i.e. perimysium and endomysium). Collagen synthesis in these non-growing muscles occurred at 0.59%/day in each of the two skeletal muscles and at 0.88%/day in the cardiac muscle. During hypertrophy the collagen content of the anterior muscle increased, but not as fast as intracellular protein, so that after 58 days the concentration had fallen from 22.8 to 14.4% of total protein. This may have resulted from an incomplete production of the epimysial sheath, since the concentration of internal collagen did not fall and as a result accounted for over 80% of the total in the enlarged muscle. Collagen synthesis increased 8-fold during the first week of the hypertrophy, but never amounted to more than 4% of the total muscle protein synthesis. When the net accumulation of collagen is compared with the increased rate of synthesis it is concluded that between 30 and 70% of the newly synthesized collagen may have been degraded.

scholarly journals Lactase phlorhizin hydrolase turnover in vivo in water-fed and colostrum-fed newborn pigs

1996 ◽  
Vol 320 (3) ◽  
pp. 735-743 ◽  
Author(s):  
Mary A. DUDLEY ◽  
Douglas G. BURRIN ◽  
Andrea QUARONI ◽  
Judy ROSENBERGER ◽  
Gary COOK ◽  
...  
Keyword(s):  
Post Partum ◽  
Specific Radioactivity ◽  
Experimental Treatment ◽  
Synthesis Rate ◽  
Jejunal Mucosa ◽  
Treatment Groups ◽  
Newborn Pigs ◽  
The Absolute ◽  
Absolute Synthesis

We have estimated the synthesis rates in vivo of precursor and brush-border (BB) polypeptides of lactase phlorhizin hydrolase (LPH) in newborn pigs fed with water or colostrum for 24 h post partum. At the end of the feeding period, piglets were anaesthetized and infused intravenously for 3 h with l-[4-3H]-phenylalanine. Blood and jejunal samples were collected at timed intervals. The precursor and BB forms of LPH were isolated from jejunal mucosa by immunoprecipitation followed by SDS/PAGE, and their specific radioactivity in Phe determined. The kinetics of precursor and BB LPH labelling were analysed by using a linear compartmental model. Immunoisolated LPH protein consisted of five polypeptides [high-mannose LPH precursor (proLPHh), complex glycosylated LPH precursor (proLPHc), intermediate complex glycosylated LPH precursor (proLPHi) and two forms of BB LPH]. The fractional synthesis rate (Ks) of proLPHh and proLPHc (approx. 5%/min) were the same in the two groups but the absolute synthesis rate (in arbitrary units, min-1) of proLPHh in the colostrum-fed animals was twice that of the water-fed animals. The Ks values of proLPHi polypeptides were significantly different (water-fed, 3.89%/min; colostrum-fed, 1.6%/min), but the absolute synthesis rates did not differ. The Ks of BB LPH was not different between experimental treatment groups (on average 0.037%/min). However, the proportion of newly synthesized proLPHh processed to BB LPH was 48% lower in colostrum-fed than in water-fed animals. We conclude that in neonatal pigs, the ingestion of colostrum stimulates the synthesis of proLPHh but, at least temporarily, disrupts the processing of proLPH polypeptides to the BB enzyme.

scholarly journals Measurement of dermal collagen synthesis rate in vivo in humans

1998 ◽  
Vol 274 (4) ◽  
pp. E586-E591 ◽  
Author(s):  
Wassim A. El-Harake ◽  
Mikhail A. Furman ◽  
Brian Cook ◽  
K. Sreekumaran Nair ◽  
Jayme Kukowski ◽  
...  
Keyword(s):  
Collagen Synthesis ◽  
Current Method ◽  
Synthesis Rate ◽  
Tissue Fluid ◽  
Disease States ◽  
Human Volunteers ◽  
Human Proteins ◽  
Dermal Collagen ◽  
Fractional Synthesis

Accumulation of collagen produces organ dysfunction in many pathological conditions. We measured the fractional synthesis rate (FSR) of dermal collagen in five human volunteers from the increment of [13C]proline in detergent-soluble dermal collagen hydroxylated to hydroxyproline during a continuous infusion ofl-[1-13C]proline. In these and eight other volunteers, we measured [13C]proline enrichment in skin aminoacyl-tRNA, skin tissue fluid amino acid, and plasma. The prolyl-[13C]tRNA enrichment was one-half that in tissue fluid proline and more than threefold less than in plasma. The FSR of dermal collagen was 0.076 ± 0.063%/h (mean ± SD), similar to previously reported rates for skeletal muscle contractile proteins and substantially slower than hepatically derived circulating proteins such as albumin or fibrinogen. We conclude that the FSR of human dermal collagen resembles that of other human proteins considered to display slow turnover. The current method for its measurement may be used to determine the regulation of collagen synthesis in other organs and disease states.

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