Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Biosynthesis of proteoglycans in cartilage slices. Fractionation by gel chromatography and equilibrium density-gradient centrifugation.

ABSTRACT: The kinetics of incorporation of [(35)S]sulphate into slices of pig laryngeal cartilage in vitro was linear with time up to 6h. The specific radioactivities of the extracted proteoglycans (containing about 80% of the uronic acid of the cartilage) and the glycosaminoglycans remaining in the tissue after extraction were measured after various times of continuous and ;pulse-chase' radioactivity incorporation. Radioactivity was present in the isolated chondroitin sulphate after 2 min, but there was a 35min delay in its appearance in the extractable proteoglycan fraction. Fractionation of the proteoglycans by gel chromatography showed that the smallest molecules had the highest specific radioactivity, but ;pulse-chase' experiments over 5h did not demonstrate any precursor-product relationships between fractions of different size. Equilibrium density-gradient centrifugation in 4m-guanidine hydrochloride showed that among the proteoglycan fractions the specific radioactivity increased as the chondroitin sulphate content decreased, but with preparations from ;pulse-chase' experiments there was again no evidence for precursor-product relationships between the different fractions. Differences in radioactive incorporation would seem to reflect metabolic heterogeneity within the proteoglycans extracted from cartilage. This may be due either to a partial separation of different types of proteoglycans or to differences in the rates of degradation of the molecules of different size and composition as a result of the nature and specificity of the normal degrading enzymes. The results suggest that molecules of all sizes were formed at the same time.

SUBMITTER: Hardingham TE

PROVIDER: S-EPMC1178490 | biostudies-other | 1972 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

A protocol for the subcellular fractionation of Saccharomyces cerevisiae using nitrogen cavitation and density gradient centrifugation.

Project description:Most protocols for yeast subcellular fractionation involve the use of mechanical shear forces to lyse the spheroplasts produced by the enzymatic digestion of the Saccharomyces cerevisiae cell wall. These mechanical homogenization procedures often involve the manual use of devices such as the Dounce homogenizer, and so are very operator-dependent and, in consequence, lack reproducibility. Here, we report a highly reproducible method of homogenizing yeast cells based on nitrogen cavitation. This has been optimized to allow efficient release of subcellular compartments that show a high degree of integrity. The protocol remains effective and reproducible across a range of sample volumes and buffer environments. The subsequent separation method, which employs both sucrose and iodixanol density gradients, has been developed to resolve the major membrane-bound compartments of S. cerevisiae. We present an integrated protocol that is fast, facile, robust and efficient and that will enable 'omics' studies of the subcellular compartments of S. cerevisiae and other yeasts.

| S-EPMC4282465 | biostudies-literature

Characterization of gastric mucoproteins isolated by equilibrium density-gradient centrifugation in caesium chloride.

Project description:1. The mucoprotein from pig gastric mucus has been purified by equilibrium centrifugation in a CsCl gradient. 2. This procedure removes the non-covalently bound protein, which is closely associated with the mucoprotein and not easily removed from it by gel filtration. 3. The purified mucoprotein is separable by gel filtration into a high-molecular-weight mucoprotein A (mol.wt. 2.3x10(6)) and a low-molecular-weight mucoprotein B/C (mol.wt. 1.15x10(6)). 4. These two mucoproteins have the same chemical analysis namely fucose 11.3%, galactose 26%, glucosamine 19.5%, galactosamine 8.3% and protein 13.6%. 5. Mucoprotein A contains 3.1% ester sulphate. 6. These mucoproteins are isolated without enzymic digestion and have a higher protein content than the blood-group-substance mucoproteins from proteolytic digestion of gastric mucus. Detailed amino acid analysis shows that the extra protein in the non-enzymically digested material is composed of amino acids other than serine and threonine. 7. Mucoproteins A and B/C contain respectively 130 and 9 half-cystine residues per molecule of which about 78 and 6 residues are involved in disulphide linkages. 8. Cleavage of these disulphide linkages by mercaptoethanol splits both mucoproteins into four equally sized subunits of mol.wt. 5.2x10(5) for mucoprotein A and 2.8x10(4) for mucoprotein B/C. 9. The sole N-terminal amino acid of mucoprotein A is aspartic acid, whereas mucoprotein B/C has several different N-terminal amino acid residues.

| S-EPMC1168167 | biostudies-other

Separation of Stabilized MOPS Gold Nanostars by Density Gradient Centrifugation.

Project description:This article describes the stabilization and postsynthetic separation of gold nanostars (AuNS) synthesized with a morpholine-based Good's buffer, 3-(N-morpholino)propanesulfonic acid. Resuspension of AuNS in ultrapure water improved the shape stability of the particles over 30 days. We demonstrated the sorting of nanostars via rate-zonal centrifugation through a linear sucrose gradient based on branch length and number. We determined that one round of centrifugation was sufficient for separation. Also, we improved the structural homogeneity and stability of the nanoparticles through the optimization of the storage conditions and established a robust method to sort AuNS based on size and shape.

| S-EPMC6641882 | biostudies-literature

Separation of Bacteria, Protozoa and Carbon Nanotubes by Density Gradient Centrifugation.

Project description:Sustainable production and use of carbon nanotube (CNT)-enabled materials require efficient assessment of CNT environmental hazards, including the potential for CNT bioaccumulation and biomagnification in environmental receptors. Microbes, as abundant organisms responsible for nutrient cycling in soil and water, are important ecological receptors for studying the effects of CNTs. Quantification of CNT association with microbial cells requires efficient separation of CNT-associated cells from individually dispersed CNTs and CNT agglomerates. Here, we designed, optimized, and demonstrated procedures for separating bacteria (Pseudomonas aeruginosa) from unbound multiwall carbon nanotubes (MWCNTs) and MWCNT agglomerates using sucrose density gradient centrifugation. We demonstrate separation of protozoa (Tetrahymena thermophila) from MWCNTs, bacterial agglomerates, and protozoan fecal pellets by centrifugation in an iodixanol solution. The presence of MWCNTs in the density gradients after centrifugation was determined by quantification of 14C-labeled MWCNTs; the recovery of microbes from the density gradient media was confirmed by optical microscopy. Protozoan intracellular contents of MWCNTs and of bacteria were also unaffected by the designed separation process. The optimized methods contribute to improved efficiency and accuracy in quantifying MWCNT association with bacteria and MWCNT accumulation in protozoan cells, thus supporting improved assessment of CNT bioaccumulation.

| S-EPMC5132190 | biostudies-literature

Purification of functional mouse skeletal muscle mitochondria using percoll density gradient centrifugation.

Project description:ObjectiveOur goal was to isolate purified mitochondria from mouse skeletal muscle using a Percoll density gradient and to assess bioenergetic function and purity via Seahorse Extracellular Flux (XF) Analyses and mass spectrometry.ResultsMitochondria isolated from murine quadriceps femoris skeletal muscle using a Percoll density gradient method allowed for minimally contaminated preparations with time from tissue harvest to mitochondrial isolation and quantification in about 3-4 h. Percoll purification from 100 to 200 mg fresh tissue yielded ~ 200-400 ug protein. Mitochondrial bioenergetics evaluated using the Seahorse XFe96 analyzer, a high-throughput respirometry platform, showed optimum mitochondrial input at 500 ng with respiratory control ratio ranging from 3.9 to 7.1 using various substrates demonstrating a high degree of functionality. Furthermore, proteomic analysis of Percoll-enriched mitochondria isolated from skeletal muscle using this method showed significant enrichment of mitochondrial proteins indicating high sample purity. This study established a methodology that ensures sufficient high quality mitochondria for downstream analyses such as mitochondrial bioenergetics and proteomics.

| S-EPMC10544150 | biostudies-literature

Comparative proteomic analysis of spermatozoa isolated by swim-up or density gradient centrifugation.

Project description:Reports about the morphologic and functional characteristics of spermatozoa prepared by density gradient centrifugation (DC) or swim-up (SU) have produced discordant results. We have performed a proteomic comparison of cells prepared by DC and SU providing a molecular insight into the differences between these two methods of sperm cell isolation.Protein maps were obtained by 2-dimensional (2-D) separations consisting of isoelectrofocusing (IEF) from pI 3 to 11 followed by SDS-polyacrylamide gel electrophoresis. 2-D gels were stained with Sypro Ruby. Map images of DC and SU spermatozoa were compared using dedicated software. Intensities of a given spot were considered different between DC and SU when their group mean differed by >1.5-fold (p<0.05, Anova).No differences were observed for 853 spots, indicating a 98.7% similarity between DC and SU. Five spots were DC>SU and 1 was SU>DC. Proteins present in 3 of the differential spots could be identified. One DC>SU spot contained lactate dehydrogenase C and gamma-glutamylhydrolase, a second DC>SU spot contained fumarate hydratase and glyceraldehyde-3-phosphate dehydrogenase-2, and a SU>DC spot contained pyruvate kinase M1/M2.The differences in protein levels found on comparison of DC with SU spermatozoa indicate possible dissimilarities in their glycolytic metabolism and DNA methylation and suggest that DC cells may have a better capacitation potential.

| S-EPMC4410587 | biostudies-literature

Electrostatic and non-electrostatic contributions of proteoglycans to the compressive equilibrium modulus of bovine articular cartilage.

Project description:This study presents direct experimental evidence for assessing the electrostatic and non-electrostatic contributions of proteoglycans to the compressive equilibrium modulus of bovine articular cartilage. Immature and mature bovine cartilage samples were tested in unconfined compression and their depth-dependent equilibrium compressive modulus was determined using strain measurements with digital image correlation analysis. The electrostatic contribution was assessed by testing samples in isotonic and hypertonic saline; the combined contribution was assessed by testing untreated and proteoglycan-depleted samples. Though it is well recognized that proteoglycans contribute significantly to the compressive stiffness of cartilage, results demonstrate that the combined electrostatic and non-electrostatic contributions may add up to more than 98% of the modulus, a magnitude not previously appreciated. Of this contribution, about two thirds arises from electrostatic effects. The compressive modulus of the proteoglycan-depleted cartilage matrix may be as low as 3kPa, representing less than 2% of the normal tissue modulus; experimental evidence also confirms that the collagen matrix in digested cartilage may buckle under compressive strains, resulting in crimping patterns. Thus, it is reasonable to model the collagen as a fibrillar matrix that can sustain only tension. This study also demonstrates that residual stresses in cartilage do not arise exclusively from proteoglycans, since cartilage remains curled relative to its in situ geometry even after proteoglycan depletion. These increased insights on the structure-function relationships of cartilage can lead to improved constitutive models and a better understanding of the response of cartilage to physiological loading conditions.

| S-EPMC2900255 | biostudies-literature

New device for sperm preparation involving migration-gravity sedimentation without centrifugation compared with density-gradient centrifugation for normozoospermic intrauterine insemination.

Project description:ObjectiveTo investigate the efficacy of a new device for sperm preparation involving migration-gravity sedimentation without centrifugation (MIGLIS), compared with density-gradient centrifugation (DGC) for normozoospermic intrauterine insemination (IUI).DesignRetrospective cohort study.SettingNot applicable.PatientsA total of 10,318 cases of IUI (3,015 MIGLIS and 7,303 DGC) between October 2013 and September 2019.InterventionsNone.Main outcome measuresSperm analysis, subsequent pregnancy outcomes, and complications.ResultsMIGLIS was associated with a lower sperm recovery rate and fewer injected sperm compared with DGC. However, the overall pregnancy rates following MIGLIS and DGC were similar (MIGLIS 8.8%, DGC 9.3%). In a subanalysis according to age, the pregnancy rate was higher for MIGLIS among women 40-41 years of age (8.6% vs. 5.9%). Peritonitis was the only recorded complication, with similar frequencies in the MIGLIS and DGC groups (MIGLIS two cases, DGC four cases). No cases became severe, and all improved after antibiotic treatment. There were no cases of uterine cramping or pain symptoms.ConclusionsMIGLIS is a new sperm preparation method that does not require centrifugation. Its use was associated with pregnancy rates similar to those with DGC and a higher pregnancy rate in older women. MIGLIS is a novel sperm preparation method for selecting spermatozoa with high motility and good fertilization ability in patients undergoing IUI, in vitro fertilization, and intracytoplasmic sperm injection.

| S-EPMC8244315 | biostudies-literature

Fractionation of heparin-derived oligosaccharides by gradient polyacrylamide-gel electrophoresis.

Project description:Heparin-derived oligosaccharides, prepared by using flavobacterial heparinase, having a high degree of heterogeneity (sequence variability) were resolved into sharp well-defined bands by using polyacrylamide gel electrophoresis (PAGE). The use of a stacking gel and a high-density-pore-gradient resolving gel was primarily responsible for the success of this separation. Low-Mr standards of known structure and having a degree of polymerization (dp) 2-6 were used to establish that the separation on gradient PAGE was primarily dependent on molecular size. High-Mr oligosaccharides (dp 8-20) were prepared using strong-anion-exchange h.p.l.c. and were used to help characterize the gradient PAGE separation. Kinetic profiles were obtained for the depolymerization of heparin and heparan sulphate with heparinase and heparitinase respectively. The utility of this approach in sequencing oligosaccharides derived from glycosaminoglycans is discussed.

| S-EPMC1148026 | biostudies-other

Effects of Semen Processing on Sperm Function: Differences between Swim-Up and Density Gradient Centrifugation.

Project description:PurposeAndrology research has evolved notoriously in the latest years, particularly since male factor contribution to couple infertility has been undoubtedly demonstrated. However, sperm function investigations results are sometimes contradictory, probably as a result of the use of different sperm processing techniques. In this work, we underwent a systematic functional comparison of human sperm samples simultaneously processed by swim-up and density gradient centrifugation, which are the preferred sperm processing methods used in basic and clinical laboratories.Materials and methodsTo compare functional characteristics of sperm isolated by swim-up and density gradient centrifugation followed by incubation at different times under capacitating conditions.ResultsSemen samples processed in parallel by these two procedures resulted in sperm preparations with significant differences in redox state, spontaneous intracellular calcium oscillations, hyperactivation, protein tyrosine phosphorylation, and acrosome reaction responsivity to calcium ionophore. Such differences showed time-dependent specific patterns for spontaneous intracellular calcium oscillations, hyperactivation and protein tyrosine phosphorylation. Sperm retrieved by density gradient centrifugation showed more hyperactivation and tyrosine phosphorylation than swim-up sperm, suggesting a higher degree of capacitation.ConclusionsOur results account for functional differences observed in spermatozoa processed with these two methods and therefore may contribute to a better interpretation of outcomes obtained in different laboratories as well as to improve experimental designs aimed to study sperm physiology and fertility potential.

| S-EPMC8443982 | 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