Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Characterization of the tissue-specific proteoglycans synthesized by chondrocytes from nanomelic chick embryos.

ABSTRACT: Cartilage from the avian mutant nanomelia has been reported to synthesize cartilage-specific proteoglycans, PGS(SC)-I, at 1-2% of normal values [McKeown & Goetinck (1979) Dev. Biol. 71, 203-215]. Proteoglycans were endogenously labelled with [35S]sulphate and extracted from cartilage in 4 M-guanidine hydrochloride and chromatographed on controlled-pore glass 1400. PGS(SC)-I was obtained from the void volume of these columns. Dissociative sucrose-density-gradient analysis revealed a greater than normal polydispersity in the nanomelic PGS(SC)-I. Fractions from both the controlled-pore glass 1400 void volume and sucrose gradients were tested for their ability to bind specific antibody against cartilage proteoglycan monomer. In all instances, binding of normal fractions was greater than 90%, whereas binding to nanomelic fractions ranged from 20 to 65%. Chromatography of PGS(SC)-I on controlled-pore glass 2500 resulted in 70% of the normal and 25% of the mutant proteoglycans eluting as aggregates. Chondroitin sulphate chains from mutant PGS(SC)-I appeared slightly larger than normal when chromatographed on controlled-pore glass 500. In addition, PGS(SC)-I from nanomelic cartilage is more susceptible to proteolysis in vitro than the PGS(SC)-I from normal cartilage. This evidence suggests that the small amount of cartilage-specific proteoglycan synthesized by nanomelic cartilage is not normal.

SUBMITTER: McKeown-Longo PJ

PROVIDER: S-EPMC1163655 | biostudies-other | 1982 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Further characterization of collagen galactosyltransferase from chick embryos.

Project description:Optimum extraction of collagen galactosyltransferase activity from chick embryos required relatively high concentrations of detergent and salt. The activity was inhibited by concanavalin A, and the enzyme had a high affinity for columns of this lectin coupled to agarose; these results suggest the presence of carbohydrate units in the enzyme molecule. Collagen galactosyltransferase was highly labile, and only 1% of the originally bound enzyme activity could be eluted from the concanavalin A-agarose column with a buffer containing methyl glucoside and ethylene glycol. The purification of the activity over the original supernatant of chick embryo homogenate was 250-300-fold, with the optimum reaction conditions for the purified transferase differing somewhat from those for crude enzyme preparations. The reaction was inhibited by glucose-free basement-membrane collagen, UDP and galactosylhydroxylsine, and also by Co2+ and a number of compounds resembling UDP-galactose. Hydroxylysine was also a weak inhibitor. Immobilized hydroxylysine and UDP-glucuronic acid did not bind the collagen galactosyltransferase, but the enzyme was retarded in a column of UDP-galacturonic acid linked to agarose.

| S-EPMC1184208 | biostudies-other

Characterization of newly synthesized proteoglycans from rabbit menisci in organ culture.

Project description:Rabbit menisci were incubated with Na2 35SO4 in short-term organ culture to label newly synthesized proteoglycans. The radioactive products present in both tissue and culture medium were characterized separately with respect to distribution after ultracentrifugation in CsCl isopycnic density gradients, hydrodynamic size, interaction with hyaluronic acid, and glycosaminoglycan composition (types, size and content). Analysis of proteoglycan size by gel-filtration chromatography of the most-dense CsCl fractions (A1) on Sephacryl S-500 (associative conditions) resolved three species. A peak with Kav. approx. 0.7 was present in each chromatogram, and constituted the principal component in tissue extracts. Two other peaks with Kav. values of approx. 0.2 and 0.45 were also found. When the A1 fraction from tissue was subjected to CsCl-density-gradient ultracentrifugation under dissociative conditions, 71% of the recovered radioactivity was present in the most dense (A1D1) fraction. Incubation with hyaluronic acid of either A1 or A1D1 fraction from associative extract did not alter the apparent size of the labelled product, indicating a lack of aggregate formation. Meniscal proteoglycans showed an unusual and marked tendency to adsorb irreversibly to agarose and agarose-containing gel-filtration-chromatography media. High-pressure liquid-chromatographic analyses indicated that the sulphated glycosaminoglycans consisted of chondroitin 6-sulphate (72%), chondroitin 4-sulphate (19%) and dermatan sulphate (5%). Endo-beta-galactosidase (keratanase) digestion of the material failed to detect the presence of keratan sulphate. Of the labelled glycosaminoglycans, 95% was eluted from Sephacryl S-400 as a single symmetrical peak with a Kav. of 0.5. The results of studies with tissue extracts and culture medium were similar.

| S-EPMC1144118 | biostudies-other

Partial purification and characterization of collagen galactosyltransferase from chick embryos.

Project description:Collagen galactosyltransferase was purified 50-150-fold from chick-embryo extract. The tissue homogenate was prepared in the presence of Triton X-100, since the addition of the detergent doubled the enzyme activity in the homogenate and the extract. Three species of the enzyme activity with different molecular weights were recovered on gel filtration, the mol.wts. being about 450000, 200000 and 50000. Collagen galactosyltransferase activity was strongly inhibited by p-mercuribenzoate, and stimulated by the addition of dithiothreitol to the incubation system. Studies on substrate requirements indicated that denatured citrate-soluble collagen is a more effective substrate than gelatinized insoluble collagen, as judged from their Km values. Experiments on three peptide fractions prepared from citrate-soluble collagen indicated that a fraction with an average mol.wt. of 500-600 contained peptides large enough to meet a minimun requirement for interaction with the enzyme. However, longer peptides were clearly better substrates. When native and heat-denatured citrate-soluble collagens were compared as substrates, practically no synthesis of galactosylhydroxylysine was found with native collagen. This finding suggests that the triple-helical conformation of collagen prevents the galactosylation of hydroxylysine residues.

| S-EPMC1172811 | biostudies-other

Characterization of proteoglycans synthesized by human adult glomerular mesangial cells in culture.

Project description:1. The newly synthesized proteoglycans from human adult glomerular mesangial cells labelled in vitro for 24 h with [35S]sulphate have been characterized using biochemical and immunological techniques. 2. The following proteoglycans were identified (% of total synthesized). (i) A large chondroitin sulphate proteoglycan, CSPG-I, Mr approximately 1 x 10(6) (10.6%). This proteoglycan consisted of a protein core of Mr approximately 4 x 10(5) and glycosaminoglycan chains of Mr 2.5 x 10(4), and was present in both the cell layer and the culture medium. (ii) A major small dermatan sulphate proteoglycan, DSPG-I, Mr 3.5 x 10(5) (46%), which was mainly located in the culture medium. (iii) A second minor small dermatan sulphate, DSPG-II, Mr approximately 2 x 10(5) (9.8%). This molecule was exclusively located in the culture medium. (iv) A large heparan sulphate proteoglycan, HSPG-I, Mr 8 x 10(5) (3.3%). (v) A second large heparan sulphate proteoglycan HSPG-II, Mr approximately 6 x 10(5) (23%). HSPG-I and HSPG-II were extracted from both the culture medium and the cell layer. 3. Western blot analysis of the core proteins released by chondroitin ABC lyase treatment of DSPG-I and DSPG-II identified these dermatan sulphate proteoglycans as biglycan and decorin respectively. Both DSPG-I and DSPG-II had core proteins of Mr 45,000. 4. The cell-layer-associated forms of CSPG-I, HSPG-I and HSPG-II were accessible to limited trypsin treatment, bound to octyl-Sepharose and could be inserted into liposomes, indicating a possible cell membrane location. 5. Pulse-chase experiments indicated that the cell-layer-associated [35S]proteoglycans undergo limited metabolism to inorganic [35S]sulphate, the majority of which is accounted for by the degradation of HSPG-II and to a lesser extent DSPG-I.

| S-EPMC1151194 | biostudies-other

Tissue specific regulation of the chick Sox10E1 enhancer by different Sox family members.

Project description:The transcription factor Sox10 is a key regulator of vertebrate neural crest development and serves crucial functions in the differentiation of multiple neural crest lineages. In the chick neural crest, two cis-regulatory elements have been identified that mediate Sox10 expression: Sox10E2, which initiates expression in cranial neural crest; Sox10E1 driving expression in vagal and trunk neural crest. Both also mediate Sox10 expression in the otic placode. Here, we have dissected and analyzed the Sox10E1 enhancer element to identify upstream regulatory inputs. Via mutational analysis, we found two critical Sox sites with differential impact on trunk versus otic Sox10E1 mediated reporter expression. Mutation of a combined SoxD/E motif was sufficient to completely abolish neural crest but not ear enhancer activity. However, mutation of both the SoxD/E and another SoxE site eliminated otic Sox10E1 expression. Loss-of-function experiments reveal Sox5 and Sox8 as critical inputs for trunk neural crest enhancer activity, but only Sox8 for its activity in the ear. Finally, we show by ChIP and co-immunoprecipitation that Sox5 directly binds to the SoxD/E site, and that it can interact with Sox8, further supporting their combinatorial role in activation of Sox10E1 in the trunk neural crest. The results reveal important tissue-specific inputs into Sox10 expression in the developing embryo.

| S-EPMC5810587 | biostudies-literature

Detection of isoform-specific fibroblast growth factor receptors by whole-mount in situ hybridization in early chick embryos.

Project description:We have developed "b" and "c" isoform-specific chicken fibroblast growth factor (FGF) receptor 1-3 probes for in situ hybridization. We rigorously demonstrate the specificity of these probes by using both dot blot hybridization and whole-mount in situ hybridization during neurulation and early postneurulation stages, and we compare expression patterns of each of the three isoform-specific probes to one another and to generic probes to each of the three (non-isoform-specific) FGF receptors. We show that the expression pattern of each receptor is represented by the collective expression of each of its two isoforms, with the expression of each FGF receptor being most similar to that of its "c" isoform at two of the three stages studied, and that tissue and stage differences exist in the patterns of expression of the six isoforms. We demonstrate the usefulness of these probes for defining the differential tissue expression of FGF receptor 1-3 isoforms.

| S-EPMC3092825 | biostudies-literature

A set of stage-specific gene transcripts identified in EK stage X and HH stage 3 chick embryos.

Project description:BACKGROUND: The embryonic developmental process in avian species is quite different from that in mammals. The first cleavage begins 4 h after fertilization, but the first differentiation does not occur until laying of the egg (Eyal-Giladi and Kochav (EK) stage X). After 12 to 13 h of incubation (Hamburger and Hamilton (HH) stage 3), the three germ layers form and germ cell segregation in the early chick embryo are completed. Thus, to identify genes associated with early embryonic development, we compared transcript expression patterns between undifferentiated (stage X) and differentiated (HH stage 3) embryos. RESULTS: Microarray analysis primarily showed 40 genes indicating the significant changes in expression levels between stage X and HH stage 3, and 80% of the genes (32/40) were differentially expressed with more than a twofold change. Among those, 72% (23/32) were relatively up-regulated at stage X compared to HH stage 3, while 28% (9/32) were relatively up-regulated at HH stage 3 compared to stage X. Verification and gene expression profiling of these GeneChip expression data were performed using quantitative RT-PCR for 32 genes at developmental four points; stage X (0 h), HH stage 3 (12 h), HH stage 6 (24 h), and HH stage 9 (30 h). Additionally, we further analyzed four genes with less than twofold expression increase at HH stage 3. As a result, we identified a set of stage-specific genes during the early chick embryo development; 21 genes were relatively up-regulated in the stage X embryo and 12 genes were relatively up-regulated in the HH stage 3 embryo based on both results of microarray and quantitative RT-PCR. CONCLUSION: We identified a set of genes with stage-specific expression from microarray Genechip and quantitative RT-PCR. Discovering stage-specific genes will aid in uncovering the molecular mechanisms involved the formation of the three germ layers and germ cell segregation in the early chick embryos.

| S-EPMC1894632 | biostudies-literature

Investigating right ovary degeneration in chick embryos by transcriptome sequencing.

Project description:In asymmetric chick gonads, the left and right female gonads undergo distinct programs during development, generating a functional ovary on the left side only. Despite some progress being made in recent years, the mechanisms of molecular regulation remain incompletely understood, and little genomic information is available regarding the degeneration of the right ovary in the chick embryo testis. In this study, we performed transcriptome sequencing to investigate differentially expressed genes in the left and right ovaries and gene functions at two critical time points; embryonic days 6 (E6) and 10 (E10). Using high-throughput RNA-sequencing technologies, 539 and 1046 genes were identified as being significantly differentially expressed between 6R-VS-6L and 10R-VS-10L. Gene ontology analysis of the differentially expressed genes revealed enrichment in functional pathways. Among these, candidate genes associated with degeneration of the right ovary in the chick embryo were identified. Identification of a pathway involved in ovarian degeneration provides an important resource for the further study of its molecular mechanisms and functions.

| S-EPMC5481632 | biostudies-literature

Effects of tissue compression on the hyaluronate-binding properties of newly synthesized proteoglycans in cartilage explants.

Project description:The effects of tissue compression on the hyaluronate-binding properties of newly synthesized proteoglycans in calf cartilage explants were examined. Pulse-chase experiments showed that conversion of low-affinity monomers to the high-affinity form (that is, to a form capable of forming aggregates with 1.6% hyaluronate on Sephacryl S-1000) occurred with a t1/2 of about 5.7 h in free-swelling discs at pH 7.45. Static compression during chase (in pH 7.45 medium) slowed the conversion, as did incubation in acidic medium (without compression). Both effects were dose-dependent. For example, the t1/2 for conversion was increased to about 11 h by either (1) compression from a thickness of 1.25 mm to 0.5 mm or (2) medium acidification from pH 7.45 to 6.99. Oscillatory compression of 2% amplitude at 0.001, 0.01, or 0.1 cycles/s during chase did not, however, affect the conversion. Changes in the hyaluronate-binding affinity of [35S]proteoglycans in these experiments were accompanied by no marked change in the high percentage (approximately 80%) of monomers which could form aggregates with excess hyaluronate and link protein. Since static tissue compression would result in an increased matrix proteoglycan concentration and thereby a lower intra-tissue pH [Gray, Pizzanelli, Grodzinsky & Lee (1988) J. Orthop. Res. 6, 777-792], it seems likely that matrix pH may influence proteoglycan aggregate assembly by an effect on the hyaluronate-binding affinity of proteoglycan monomer. Such a pH mechanism might have a physiological role, promoting proteoglycan deposition in regions of low proteoglycan concentration.

| S-EPMC1131369 | biostudies-other

Highly parallel assays of tissue-specific enhancers in whole Drosophila embryos.

Project description:Transcriptional enhancers are a primary mechanism by which tissue-specific gene expression is achieved. Despite the importance of these regulatory elements in development, responses to environmental stresses and disease, testing enhancer activity in animals remains tedious, with a minority of enhancers having been characterized. Here we describe 'enhancer-FACS-seq' (eFS) for highly parallel identification of active, tissue-specific enhancers in Drosophila melanogaster embryos. Analysis of enhancers identified by eFS as being active in mesodermal tissues revealed enriched DNA binding site motifs of known and putative, previously uncharacterized mesodermal transcription factors. Naive Bayes classifiers using transcription factor binding site motifs accurately predicted mesodermal enhancer activity. Application of eFS to other cell types and organisms should accelerate the cataloging of enhancers and understanding how transcriptional regulation is encoded in them.

| S-EPMC3733245 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data