Identification of glycosaminoglycans in the chondrocranium of the chick embryo before and at the onset of chondrogenesis

Development ◽  
1986 ◽  
Vol 93 (1) ◽  
pp. 29-49
Author(s):  
Charles D. Goldstein ◽  
Joseph J. Jankiewicz ◽  
Marye E. Desmond
Keyword(s):  
Chick Embryo ◽  
Cetylpyridinium Chloride ◽  
Heparan Sulphate ◽  
Relative Proportion ◽  
Fold Increase ◽  
Pulse Labelling ◽  
Cell Area ◽  
Total Head ◽  
Facial Area ◽  
Sulphated Gags

It appears that hyaluronate is associated with cell migration and the chondroitin sulphates with differentiation during morphogenesis of the chick embryo. The aim of this study was to see if such a correlation could be made for chondrocranium morphogenesis. Specifically, the purpose of this study was (1) to determine the proportion of extracellular matrix (ECM) to cell area and total head mesenchymal area during chondrocranium morphogenesis; and (2) to identify the location, types, and relative amounts of glycosaminoglycans (GAG) being synthesized in the presumptive chondrocranium at the onset of chondrogenesis and prior to this time. Morphometric analyses were made on median and parasagittal sections of heads of stage-24 and -33 embryos in order to determine relative contributions of cells and ECM to the total area of head mesenchyme at these stages. Presumptive chondrocrania (heads minus eyes) of these stage embryos were also analysed histochemically and biochemically in order to identify the GAGs present in the ECM. Sections of whole heads were stained with alcian blue at low and high pH as well as digested prior to staining with hyaluronidase (Streptomyces and testicular). Identification of GAGs was done by pulse labelling embryos with [3H]glucosamine, digesting homogenates with hyaluronidase (Streptomyces or testicular), precipitating the undigested GAGs with cetylpyridinium chloride and countingthe dissolved precipitates using scintillation spectrophotometry. The types and relative amounts of GAGs present in the presumptive chondrocranium were determined by comparing the amount of radioactivity in the precipitates of the non-digested GAG with the counts in the precipitates of the predigested GAGs. This study reports that chondrogenesis begins in the presumptive chondrocranium of the chick embryo at stage 33 and that the area of the head mesenchyme increases 60-fold between stages 24 and 33. Little change in cell density and individual cell area as well as in the relative proportion of total area allocated to cells and ECM occurs. GAGs are localized exclusively in the presumptive chondrocranium. These GAGs are restricted to the ventral half of the presumptive chondrocranium. Within this region, the GAGs are further localized to the presumptive facial area, perichordal region, ethmoid, sphenoid and periotic regions. The types of GAG being synthesized in the head mesenchyme of both stage-24 and -33 embryos are hyaluronate, the chondroitins and unidentified sulphated GAGs (dermatan, keratan, heparin and heparan sulphate). At stage 24, hyaluronate, chondroitin and the unidentified sulphated GAGs constitute about 33% each of the GAG being synthesized. At stage 33, the level of hyaluronate synthesis drops to 2%, the chondroitins to 24% and the unidentified sulphated GAGs increase to 74%. There is an 18·5-fold decrease in the percentage of hyaluronate, a 1·5-fold decrease in the amount of chondroitins and a 2·7-fold increase in the percentage of unidentified sulphated GAGs being synthesized as chondrocranium morphogenesis proceeds.

scholarly journals Properties of heparan sulphate and chondroitin sulphate from young and old human aortae

1969 ◽  
Vol 114 (1) ◽  
pp. 89-96 ◽  
Author(s):  
G. Manley ◽  
R. N. Mullinger ◽  
P. H. Lloyd
Keyword(s):  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Heparan Sulphate ◽  
Total Amino Acid ◽  
Cross Linking ◽  
Molecular Weights ◽  
Consistent Increase ◽  
Salt Gradient ◽  
Alkali Hydrolysis ◽  
Dowex 1

1. Glycosaminoglycans were liberated from old and young human ascending aortae by digestion with papain. Heparan sulphate and chondroitin sulphate were separated by the different solubilities of their complexes with cetylpyridinium chloride in solutions of sodium chloride. Final fractionation was achieved by salt-gradient column chromatography on Dowex 1 (Cl−form). 2. Heparan sulphate from old aortae showed a slight, but consistent, increase in sulphation compared with heparan sulphate from young aortae. 3. The major amino acids associated with aortic heparan sulphate and chondroitin sulphate were serine, glycine, glutamic acid and aspartic acid. Heparan sulphate and chondroitin sulphate from old aortae contained about twice as much total amino acid as heparan sulphate and chondroitin sulphate from young aortae. Alkali hydrolysis resulted in the destruction of more serine in chondroitin sulphate from old, compared with young, aortae. 4. Molecular weights of glycosaminoglycans from old and young aortae were found to be similar, and in the region of 35000. 5. It is suggested that there is an increased degree of protein–glycosaminoglycan cross-linking in old aortae.

scholarly journals Sulphated glycosaminoglycans in regenerating rat liver

1980 ◽  
Vol 188 (3) ◽  
pp. 769-773 ◽  
Author(s):  
M Edward ◽  
W F Long ◽  
H H Watson ◽  
F B Williamson
Keyword(s):  
Molecular Mechanisms ◽  
Temporal Trend ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Fold Increase ◽  
Sham Operation ◽  
Dermatan Sulphate ◽  
Weight Percentage ◽  
Biphasic Pattern ◽  
Sulphated Glycosaminoglycans

The total weight percentage glycosaminoglycan content of rat liber was found to increase by 50% in the first 30 h after partial hepatectomy. The content returned to near normal by the third day, but then increased again to a second maximum at 5-6 days, only to gradually decline to normal by the ninth day, when regeneration was nearly complete. This biphasic pattern was most marked in the chondroitin sulphate A/C component, with a 6-fold increase by the sixth day. Dermatan sulphate showed the same temporal trend, whereas heparan sulphate remained relatively unaltered. No such changes were detected in the livers of rats subjected to sham operation. The possible molecular mechanisms underlying the apparent link between cellular glycosaminoglycan content and proliferative tendency are discussed.

scholarly journals Human Serum and Urine Glycosaminoglycans in Health and in Patients with Chronic Renal Failure

1992 ◽  
Vol 29 (2) ◽  
pp. 190-195 ◽  
Author(s):  
L Bower ◽  
C Warren ◽  
G Manley
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Uronic Acid ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Heparan Sulphate ◽  
Normal Urine ◽  
Glycosaminoglycan Metabolism ◽  
Normal Controls

Quantitation of uronic acid precipitable by cetylpyridinium chloride (CPC) and electrophoretic separation of glycosaminoglycans were performed on sera from patients with chronic renal failure and compared to normal controls. Serum CPC-precipitable uronic acid (CpUA) levels in patients with renal failure were significantly higher (mean 13·7 mg/L, range 7·1–23·6 mg/L) than normal controls (mean 9·6 mg/L, range 5·1–13·9 mg/L) due to increased concentrations of low sulphated chondroitin sulphate. A positive correlation between serum CpUA and creatinine was found in renal failure patients. Urine CpUA excretion was raised in renal failure patients compared to normal controls with an increased excretion of chondroitin sulphate (Ch-S) of reduced electrophoretic mobility. Heparan sulphate (HS), a major glycosaminoglycan in normal urine, was absent from the urine of these patients. The possible origin of urine glycosaminoglycans and the role of the kidney in glycosaminoglycan metabolism are discussed.

scholarly journals Incorporation of sulphate by chick-embryo corium. Nature and products of the process in vitro

1965 ◽  
Vol 97 (2) ◽  
pp. 432-439
Author(s):  
NL Noble ◽  
RJ Boucek
Keyword(s):  
Mast Cells ◽  
Chick Embryo ◽  
Phosphate Buffer ◽  
Trichloroacetic Acid ◽  
Chondroitin Sulphate ◽  
Energy Requirement ◽  
Heparan Sulphate ◽  
Energy Of Activation ◽  
Sulphate Concentration

1. The incorporation of sulphate into the trichloroacetic acid-precipitable fraction of 9-day chick-embryo corium, incubated in Krebs-Ringer phosphate buffer, pH7, is dependent on the sulphate concentration of the medium. Uptake of sulphate is linear with time for 3.5-4hr. and is maximal at 37.5 degrees in the presence of air or oxygen. d-Glucose stimulates the incorporation of sulphate but l-glutamine has no effect. 2. Incorporation of sulphate by the chick corium is enzymic and apparently involves the synthesis of active sulphate (adenosine 3‣-phosphate 5‣-sulphatophosphate) and the transfer of sulphate from adenosine 3‣-phosphate 5‣-sulphatophosphate to acceptor glycosaminoglycuronoglycan. This proposal on the nature of the process is suggested by the similarity between the energy of activation calculated for sulphate-incorporation in the chick-corium preparation and the energy requirement reported for sulphate-activation with purified yeast enzymes. 3. The 9-day chick-embryo corium is composed principally of fibroblasts; there are no histologically demonstrable mast cells. The young fibroblast is apparently responsible for the incorporation of sulphate into glycosaminoglycuronoglycans tentatively identified as chondroitin sulphate(s), heparan sulphate and heparin-like material.

scholarly journals Isolation and characterization of the integral glycosaminoglycan constituents of human amyloid A and monoclonal light-chain amyloid fibrils

1991 ◽  
Vol 275 (1) ◽  
pp. 67-73 ◽  
Author(s):  
S R Nelson ◽  
M Lyon ◽  
J T Gallagher ◽  
E A Johnson ◽  
M B Pepys
Keyword(s):  
Light Chain ◽  
Amyloid Fibrils ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Heparan Sulphate ◽  
Cellulose Acetate Electrophoresis ◽  
Amyloid A ◽  
Isolation And Characterization ◽  
Cscl Density Gradient ◽  
A Minor

Amyloid fibrils were isolated by extraction in water from the livers and spleens of four patients who had died of monoclonal, light-chain (AL)-type, systemic amyloidosis and one with reactive systemic, amyloid A protein (AA)-type amyloidosis. Each fibril preparation contained 1-2% by weight of glycosaminoglycan (GAG) which was tightly associated with the fibrils and not just co-isolated from the tissues with them. After exhaustive digestion of the fibrils with papain and Pronase, the GAGs were specifically precipitated with cetylpyridinium chloride and were identified by cellulose acetate electrophoresis and selective susceptibility to specific glycosidases. All the preparations contained approximately equal amounts of heparan sulphate and dermatan sulphate. There was no evidence for the presence of chondroitin sulphate or other GAGs. Fine structural analysis by oligosaccharide mapping in gradient polyacrylamide gels, following partial digestion with specific glycosidases, showed very similar structures among the heparan sulphates and the dermatan sulphates, respectively. GAGs were also extracted by solubilizing amyloid fibrils in 4 M-guanidinium chloride followed by CsCl density-gradient ultracentrifugation. Although a minor proportion of the GAG material obtained in this way was apparently in the form of proteoglycan molecules, most of it was free GAG chains. The presence in amyloid fibrils of different types, in different organs and from different patients of particular GAG classes with similar structures supports the view that these molecules may be of pathogenic significance.

Cell cycle analysis of facial mesenchyme in the chick embryo

Development ◽  
1984 ◽  
Vol 81 (1) ◽  
pp. 49-59
Author(s):  
Robert Minkoff
Keyword(s):  
Cell Cycle ◽  
Chick Embryo ◽  
Generation Time ◽  
Growth Fraction ◽  
Computer Assisted ◽  
Pulse Labelling ◽  
Transit Times ◽  
Generation Times ◽  
The Difference ◽  
Slow Cycling

Cell cycle parameters were analysed in mesenchyme of the maxillary process and the roof of the stomodeum in the chick embryo from stages 19 through 28. The generation times at stages 24–26 were determined by pulse labelling of embryos with [3H]thymidine, followed by labelled mitosis counts and construction and analysis of percent-labelled mitosis curves employing computer-assisted curve-fitting techniques. The median generation time was approximately 10·6 h in the maxillary process, and 16 h in the roof of the stomodeum; corresponding values for mean generation times were approximately 12·0 and 18·2 h, respectively. Median values for transit times of G1, S, and G2 were 2·0, 5·4, and 2·5 h in the maxillary process and 5·2, 6·7, and 2·7 h in the roof of the stomodeum. The distribution of generation times of cells in the roof of the stomodeum, however, appeared to be more heterogeneous than those of cells in the maxillary process. The percentage of cells which continue to cycle rapidly (i.e. the ‘growth fraction’) was determined by repeated-labelling experiments with [3H]thymidine in chick embryos from stages 19 through 28. Cumulative labelling of mesenchymal cells in both the maxillary process and roof of the stomodeum approached 100 % at stage 19 but dropped markedly from stage 19 to 25 declining to approximately 60–70 % in the maxillary process, and to 30 % in the roof of the stomodeum. The decline in cell proliferation rates for these regions, determined in previous studies with labelling indices, appears to be a result of the removal of cells from rapidly cycling cell populations into subpopulations which are cycling more slowly and possibly into subpopulations which have become quiescent; the difference in growth rates between these regions could be attributed to the time of appearance and the size of these emerging slow cycling or quiescent subpopulations.

scholarly journals The growth of vaccinia virus in the chorio-allantois of the developing chick embryo and the production of complement-fixing antigen and haemagglutinin

1956 ◽  
Vol 54 (1) ◽  
pp. 102-113 ◽  
Author(s):  
H. B. Maitland ◽  
Barbara M. Tobin
Keyword(s):  
Chick Embryo ◽  
Vaccinia Virus ◽  
Growth Curve ◽  
Complement Fixation ◽  
Fold Increase ◽  
Growth Cycle ◽  
Soluble Antigen ◽  
Elementary Body

1. Elementary body suspensions of vaccinia virus placed on the chorio-allantoic membrane of the developing chick embryo showed immediately an enhanced infectivity which may amount to a several-fold increase in titre.2. In studying the growth curve of vaccinia virus in the chorio-allantois the distribution of virus between the liquid and the membrane had to be taken into account.3. Some virus disappeared after inoculation, but this did not necessarily indicate a non-infective phase in a growth cycle.4. Virus began to increase in 4–5 hr. and proceeded without stepwise increments, reaching a maximum in about 36 hr.5. Haemagglutinin and complement-fixing ‘soluble’ antigen were formed during the growth of virus, and the titre of each was directly related to the amount of virus. They were detectable when the titre of virus reached respectively 106.5–10.70 and 105.5–106.0 pocks per ml.6. Haemagglutinin may have been acting as a complement-fixing antigen.7. Neither haemagglutination nor complement-fixation provided evidence for the occurrence of a non-infective phase of virus as part of a growth cycle.

scholarly journals Induction of dihydrolipoamide dehydrogenase in 3T3-L1 cells during differentiation

1988 ◽  
Vol 249 (3) ◽  
pp. 897-902 ◽  
Author(s):  
D J Carothers ◽  
G Pons ◽  
M S Patel
Keyword(s):  
Gel Electrophoresis ◽  
Specific Activity ◽  
Fold Increase ◽  
Pulse Labelling ◽  
Rabbit Antibody ◽  
Dihydrolipoamide Dehydrogenase ◽  
Common Component ◽  
The Common

The activity and turnover of dihydrolipoamide dehydrogenase (E3), the common component of the three 2-oxoacid dehydrogenase complexes, were measured during the differentiation of 3T3-L1 preadipocytes into 3T3-L1 adipocytes. The specific activity of E3 increased approx. 3-4-fold in 3T3-L1 adipocytes differentiated under a regimen of insulin, dexamethasone and 3-isobutyl-1-methylxanthine for 48 h, followed by insulin alone thereafter. A rabbit antibody to pig heart E3 quantitatively precipitated the enzyme from 3T3-L1 adipocytes. By using immunoprecipitation and gel electrophoresis, a 3.3-fold increase was observed in E3 protein in 3T3-L1 adipocytes as compared with 3T3-L1 preadipocytes, on a DNA basis. Pulse-labelling experiments with L-[35S]methionine revealed a 3.5-fold increase in the rate of synthesis of E3 in 3T3-L1 adipocytes compared with that observed in 3T3-L1 preadipocytes. In contrast, the apparent half-lives of the E3 in 3T3-L1 preadipocytes (43 h) and 3T3-L1 adipocytes (33 h) were not significantly different. Therefore, the 3-4-fold increase in the specific activity of E3 in 3T3-L1 adipocytes resulted from an increased rate of synthesis of the enzyme.

Determining the metastatic potential of circulating tumor cells.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e21051-e21051
Author(s):  
Rhiana Menen ◽  
Emmett Pinney ◽  
Katarina Kolostova ◽  
Vladimir Bobek ◽  
Atsushi Suetsugu ◽  
...  
Keyword(s):  
Chick Embryo ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Fold Increase ◽  
Metastatic Potential ◽  
Cell Types ◽  
Rapid Identification ◽  
Cancer Prognosis

e21051 Background: Knowledge of the metastatic potential of circulating tumor cells would be useful for cancer prognosis and also be a rationale for targeting metastatic CTCs for therapy. Methods: Using immunomagnetic beads, CTCs were rapidly isolated from the circulation of mice orthotopically implanted with human PC-3 prostate cancer cells stably expressing green fluorescent protein (GFP). The PC-3–GFP CTCs were then expanded in culture in parallel with the parental PC-3–GFP cell line. Both cell types were then inoculated onto the chorioallentoic membrane (CAM) of chick embryos. Eight days later, embryos were harvested and the brains were processed for frozen sections. The IV-100 intravital laser scanning microscope enabled rapid identification of fluorescent metastatic foci within the chick embryonic brain. Results: Inoculation of embryos with PC-3–GFP CTCs resulted in a 3 to 10-fold increase in brain metastasis when compared to those with the parental PC-3–GFP cells (p<0.05 in all animals). Thus, PC-3–GFP CTCs have increased metastatic potential compared to their parental counterparts. Conclusions: The chick embryo represents a rapid, sensitive, imageable assay of metastatic potential for CTCs. The chick embryo assay has future clinical application for individualizing patient therapy based on the metastatic profile of their CTCs.

A Heparin Analogue that Releases Endogenous Heparan Sulphate

1979 ◽  
Author(s):  
D.P. Thomas ◽  
T.W. Barrowcliffe ◽  
E.A. Johnson ◽  
J. Stocks ◽  
J. Dawes ◽  
...  
Keyword(s):  
Lipoprotein Lipase ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Heparan Sulphate ◽  
Factor Xa ◽  
Fold Increase ◽  
Endothelial Lining ◽  
Drug Induced

Heparan sulphate (HS), a near-relative of heparin, but with much less anticoagulant activity in vitro, is bound to cell surfaces. We examined HS isolated from porcine intestinal mucosa, and found that although the material had low anticoagulant activity by APTT, it had a marked effect in anti-Factor Xa clotting assays, giving anti-Xa/APTT ratios of approximately 4:1 in terms of specific activity. In crossed immunoelectrophoresis experiments, HS bound to antithrombin III, but at higher concentrations than heparin. A heparin analogue (a polysulphated chondroitin), while virtually inactive in vitro, nevertheless when administered s.c. to man potentiated the effect of anti-Factor Xa to an extent comparable to that produced by low-dose heparin, but with an anti-Xa/APTT ratio of 4:1. The analogue also produced a marked release of lipoprotein lipase and a four-fold increase in the level of circulating PF4, as measured by radioimmunoassay. The anti-Xa and APTT effects of HS in vitro are very similar to those produced by the analogue in vivo, and it is suggested that the analogue releases not only lipoprotein lipase and PF4, but also HS. The drug-induced release of an endogenous glycosamino-glycan (probably from the endothelial lining of vessels) with anti-Xa activity may represent a fruitful approach to the prophylaxis of venous thrombosis.

Sign in / Sign up

Export Citation Format

Share Document