Synthesis and degradation of collagen in the developing corium of the chick embryo.
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ABSTRACT:
SUBMITTER: Boucek RJ
PROVIDER: S-EPMC1187369 | biostudies-other | 1968 Dec
REPOSITORIES: biostudies-other
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Project description:Radioactive protein was prepared from the leg muscle of chick embryos, 11, 14, 16 and 17 days old, each injected with radioactive proline and incubated for 30, 60 or 90 min afterwards. The radioactive protein was incubated with collagenase purified by chromatography on a Sephadex G-100 column. Under this condition, only collagen is digested into products soluble in trichloroacetic acid. The relative rate of collagen synthesis was determined by comparing the amount of radioactivity released into the supernatant fraction and that in the residue, by the method of Diegelmann & Peterkofsky [(1972) Dev. Biol. 28, 443--453]. The results show that the rate of collagen synthesis remains at approx. 10% of the rate of synthesis of other non-collagenous proteins during the development of chick embryonic muscle from 11 to 17 days. This suggests that the synthesis of collagen and that of other proteins are co-ordinately regulated at these stages of development.
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Project description:Synovial joints are among the most important structures that give us complex motor abilities as humans. Degenerative joint diseases, such as arthritis, cause loss of normal joint functioning and affect over 40 million people in the USA and approximately 350 million people worldwide. Therapies based on regenerative medicine hold the promise of effectively repairing or replacing damaged joints permanently. Here, for the first time, we introduce a model for synovial joint regeneration utilizing the chick embryo. In this model, a block of tissue that contains the prospective elbow is excised, leaving a window with strips of anterior and posterior tissue intact (window excision, WE). In contrast, we also slice out the same area containing the elbow and the distal piece of the limb is pinned back onto the stump (slice excision, SE). Interestingly, when the elbow is removed via WE, regeneration of the joint takes place, whereas the elbow joint does not regenerate following SE. In order to investigate whether the regeneration response recapitulates the developmental program of forming joints, we used GDF-5 and Autotaxin (Atx) as joint tissue specific markers, and Sox-9 and Col-9 as cartilage markers for in situ hybridization on sections at different time points after WE and SE surgeries. Re-expression of GDF-5 and Atx is observed in the WE samples by 60h after surgery. In contrast, the majority of the samples that underwent SE surgery did not express GDF-5 and Atx. Also, in SE fusion of cartilage elements takes place and the joint interzone does not form. This is indicated by continuous Col-9 expression in SE limbs, whereas Col-9 is downregulated at the joint interzone in the regenerating WE samples. This order and pattern of gene expression observed in regenerates is similar to the development of a joint suggesting that regeneration recapitulates development at the molecular level. This model defines some of the conditions required for inducing joint regeneration in an otherwise nonregenerating environment. This knowledge can be useful for designing new therapeutic approaches for joint loss or for conditions affecting joint integrity in humans.
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Project description:1. The development, localization and heterogeneity of acid phosphatase and a Zn(2+)-activated acid phosphatase in cellular fractions of developing chick liver were studied. 2. Acid phosphatase is distributed abundantly in the particulate and soluble fractions. The soluble fraction is rich in Zn(2+)-activated acid phosphatase, which attains its peak activity at about 15 days of incubation. 3. The particulate acid phosphatase activity is inhibited by fluoride but not by sodium l(+)-tartrate or cysteine. On the other hand, the soluble Zn(2+)-activated acid phosphatase activity is inhibited by sodium l(+)-tartrate and cysteine but not by fluoride. 4. The pH optimum of these two enzymes is similar at about 5.6. 5. The soluble Zn(2+)-activated acid phosphatase activity appears to be thermally stabilized by the treatment with Triton X-100 or bovine serum albumin.
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Project description:The vitamin B-6 antimetabolite 4-deoxypyridoxine, when injected into 13-day chick embryos, has the effect of increasing the amount of collagen solubilized from the leg bones by buffered saline solutions, 24 h after the injection. This effect is similar to, but less marked than, that produced by the administration of the lathyrogen beta-amino-propionitrile. Since that fraction of the total collagen which is solubilized by saline represents the least-cross-linked pool, it is concluded that 4-deoxypyridoxine is a lathyrogen, decreasing the cross-linking in the developing embryo, and confirming the importance of vitamin B-6 in that process. Lysyl oxidase, the cross-linking enzyme, was measured in extracts made from the epiphysial cartilages of embryos 24 h after the injection of either 4-deoxypyridoxine or beta-aminopropionitrile. The injection of 5 mg of beta-aminopropionitrile causes the lysyl oxidase activity to fall to 61% of that of saline-injected controls; after treatment with 4-deoxypyridoxine, the activity is 74% of the control value. In the latter case, full activity is not restored to the extracts by preincubation with pyridoxal phosphate. The results are discussed in relation to the early development of the connective tissues.
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Project description:Here, we describe a highly efficient, medium-throughput strategy for cloning and in vivo screening of putative enhancers using the chick embryo. By incorporating 48 unique nanotags for use in NanoString nCounter® across three different fluorescent reporters and developing a rapid and efficient digestion/ligation type IIs restriction enzyme-based cloning protocol, we develop a multiplexed approach for rapidly identifying enhancer activity. For complete details on the use and execution of this protocol, please see Williams et al. (2019).
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Project description:The relationship between bone formation and vitamin D metabolism was investigated in the developing chick embryo. Fertilized White Leghorn eggs were incubated at 38 degrees C in an incubator for 21 days. The fresh weight and calcium content of embryonic tibiae began to increase at day 12 and attained maximal values at day 19. Bone alkaline phosphatase and citrate decarboxylation activities, both of which represent osteoblastic activity, also began to increase at days 10-12, reached maximal values at day 19 and sharply declined thereafter. Both bone enzyme activities were highly correlated with CA2+-binding activity in the chorioallantoic membrane measured by the Chelex 100 assay. When mesonephric and metanephric homogenates were incubated with 25-hydroxy[3H]cholecalciferol, a marked and concomitant increase occurred in the metanephric 1 alpha- and 24-hydroxylase activity after day 14. The production of 1 alpha, 25-dihydroxycholecalciferol attained a maximal value at day 19 and decreased thereafter, whereas that of 24,25-dihydroxycholecalciferol continued to increase until hatching. The production rate of 1 alpha, 25-dihydroxycholecalciferol by the metanephros coincided with the changes in Ca2+-binding activity in the chorioallantoic membrane and osteoblastic activity. Since both intestinal calcium absorption and bone mineral mobilization do not occur in embryonic life, these results support the idea that 1 alpha, 25-dihydroxycholecalciferol may be involved directly in bone formation or induction of a calcium-binding protein in the chorioallantoic membrane.
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Project description:BackgroundLittle is known regarding the molecular pathways that underlie the process of retinal development. The purpose of this study was to identify proteins which may be involved in development of retina. We used a proteomics-based approach to identify proteins that are up- or down-regulated during the development of the embryo chick retina.ResultsTwo-dimensional gel electrophoresis was performed with the retina of embryo chicken, which was obtained from embryos of day 7 (ED7) and of day 11 (ED11). The protein spots showing significant differences were selected for identification by MALDI mass spectrometry. Thirteen proteins were differentially expressed; seven proteins were up-regulated in embryo retina of chicken at ED 11 and six proteins were down-regulated. Significant proteins were also evaluated in embryo day 15 (ED15). Some of identified proteins were known to regulate cell proliferation, cell death, transport, metabolism, organization and extracellular matrix, and others also included novel proteins.ConclusionWe identified thirteen proteins which differentially expressed in embryonal retina of chicken at day 7, as compared to the retina of embryo of day 11. They were various regulatory proteins for cellular signaling.
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Project description:Changes in the regulation of collagen post-translational modification in transformed cells were studied in three established human sarcoma cell lines and in chick-embryo fibroblasts freshly transformed by Rous sarcoma virus. The collagens synthesized by all but one of these and by all the control human and chick-embryo cell lines were almost exclusively of types I and/or III. The relative rate of collagen synthesis and the amounts of prolyl hydroxylase activity and immunoreactive protein were markedly low in all the transformed human cell lines. The other enzymes studied, lysyl hydroxylase, hydroxylysyl galactosyltransferase and galactosylhydroxylysyl glucosyltransferase, never showed as large a decrease in activity as did prolyl hydroxylase, suggesting a more efficient regulation of the last enzyme than of the three others. The chick-embryo fibroblasts freshly transformed by Rous sarcoma virus differed from the human sarcoma cells in that prolyl hydroxylase activity was distinctly increased, whereas the decreases in immunoreactive prolyl hydroxylase protein and the three other enzyme activities were very similar to those in the simian-virus-40-transformed human fibroblasts. It seems possible that this increased prolyl hydroxylase activity is only a temporary phenomenon occurring shortly after the transformation, and may be followed by a decrease in activity later. The newly synthesized collagens of all the transformed cells that produced almost exclusively collagen types I and/or III had high extents of lysyl hydroxylation, and there was also an increase in the ratio of glycosylated to non-glycosylated hydroxylysine. The data suggest that one critical factor affecting modification is the rate of collagen synthesis, which affects the ratio of enzyme to substrate in the cell.
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