scholarly journals Fractionation and characterization of proteoglycans isolated from chondrocyte cell cultures

1981 ◽  
Vol 197 (2) ◽  
pp. 249-258 ◽  
Author(s):  
Sven Björnsson ◽  
Dick Heinegȧrd
Keyword(s):  
Cell Culture ◽  
Chondroitin Sulphate ◽  
Calf Serum ◽  
Void Volume ◽  
Cartilage Tissue ◽  
Foetal Calf Serum ◽  
Nasal Cartilage ◽  
Core Proteins ◽  
Minor Portion ◽  
A Minor

Chondrocyte cultures were established from foetal bovine tracheal cartilage and maintained in Ham's F12 medium with or without 10% (v/v) foetal calf serum. The proteoglycans were isolated and characterized. (1) The proteoglycans from cultures both with and without serum distributed in associative or dissociative CsCl gradients like proteoglycans from cartilage tissue. (2) The amino acid composition, protein contents and glucosamine/galactosamine ratios were grossly identical with those of the tissue derived proteoglycans. (3) Sedimentation coefficients (s0) for the monomers were 21.0S and 22.7S from cultures without and with serum respectively. The s0 values obtained for aggregates were 72.3S and 93.2S respectively. The limiting viscosity numbers [η] were 248ml/g and 298ml/g respectively. These data corresponded well to those obtained for the tissue-derived proteoglycans. (4) The sizes of the core proteins and chondroitin sulphate chains respectively were the same for both types of cell-culture proteoglycans and similar to those of the tissue proteoglycans. Both the keratan sulphate-rich region and the hyaluronic acid-binding region were identified. The latter, however, was not resistant to limit digestion with trypsin, in contrast with the fragment derived from the bovine nasal cartilage. (5) About 70% of the cell-culture proteoglycans chromatographed in the void volume on a Sepharose 2B column, whereas reduced and alkylated samples (monomers) chromatographed completely included in the column. The two link proteins present in A1 preparations of cartilage proteoglycans were also present in A1 preparations of cell-culture proteoglycans. (6) A minor portion (10%) of the 35S-labelled proteoglycans in the cultures was associated with the cells. Reduced and alkylated samples were larger compared with the monomers in the medium, and chromatographed partly (25%) excluded on the Sepharose 2B column. A larger proportion (50%) of the non-reduced samples chromatographed in the void volume of the column.

scholarly journals The degradation of cartilage proteoglycans by tissue proteinases. Proteoglycan heterogeneity and the pathway of proteolytic degradation

1977 ◽  
Vol 167 (3) ◽  
pp. 639-646 ◽  
Author(s):  
P J Roughley
Keyword(s):  
Cathepsin B ◽  
Degradation Products ◽  
Chondroitin Sulphate ◽  
Cathepsin G ◽  
Proteolytic Degradation ◽  
Single Chain ◽  
Structural Variations ◽  
Nasal Cartilage ◽  
Core Proteins ◽  
Average Size

1. CaCl2-extracted proteoglycan from bovine nasal cartilage was degraded by four tissue proteinases till no further decrease in hydroynamic size was obtained. The proteoglycan and its final degradation products were then fractionated by Sepharose 2B chromatography. 2. The average size of the degradation products was least for cathepsin B and lysosomal elastase, and greatest for cathepsin D and cathepsin G. The latter two proteinases also produced degradation products that showed the widest range of sizes. 3. The structure of the degradation products ranged from peptides containing a single glycosaminoglycan chain to those containing twelve or more chains. Of the four proteinases, only cathepsin B produced peptides that contained a single chondroitin sulphate chain. 4. The proteoglycan was very heterogeneous with respect to size and chemical composition. Its behaviour on electrophoresis suggested that at least two genetically distinct core proteins might exist. 5. Irrespective of their structural variations, all proteoglycan molecules were able to interact with hyaluronic acid. In contrast, none of the degradation products were capable of this type of interaction. 6. A pathway for the proteolytic degradation of proteoglycans is postulated in which the sites of initial cleavage may be common to the majority of proteinases, whereas the production of the final clusters is dependent on the specificity of the proteinase. Only those proteinases of broadest specificity can produce single-chain chondroitin sulphate-peptides.

Role of the cellular matrix in haemopoiesis. I. Synthesis of glycosaminoglycans by mouse bone marrow cell cultures

1983 ◽  
Vol 63 (1) ◽  
pp. 155-171
Author(s):  
J.T. Gallagher ◽  
E. Spooncer ◽  
T.M. Dexter
Keyword(s):  
Bone Marrow ◽  
Hyaluronic Acid ◽  
Chondroitin Sulphate ◽  
Close Association ◽  
Heparan Sulphate ◽  
Calf Serum ◽  
Gel Chromatography ◽  
Mouse Bone Marrow ◽  
Foetal Calf Serum ◽  
Mouse Bone Marrow Cell

Haemopoietically active mouse bone marrow cultures, incubated for 48 h with [3H]glucosamine and Na2(35)SO4, synthesized radiolabelled hyaluronic acid, heparan sulphate and chondroitin sulphate. Heparan sulphate was enriched in a trypsin extract of the adherent cells whereas hyaluronic acid and chondroitin sulphate were distributed mainly to the culture medium. Analysis of nitrous acid scission products of heparan sulphate by gel chromatography demonstrated the close association of N- and O-sulphate groups along the polysaccharide chain. Chondroitinase AC degradation established the copolymeric nature of chondroitin sulphate in which about 38% of the hexuronic acid residues were in the form of GlcUA. Studies on non-haemopoietic cultures, derived from W/Wv mice or from normal marrow cells maintained in foetal calf serum instead of horse serum, indicated that adherent stromal cells were the major source of glycosaminoglycans.

scholarly journals Ultrastructural and Biophysical Characterization of Hepatitis C Virus Particles Produced in Cell Culture

2010 ◽  
Vol 84 (21) ◽  
pp. 10999-11009 ◽  
Author(s):  
Pablo Gastaminza ◽  
Kelly A. Dryden ◽  
Bryan Boyd ◽  
Malcolm R. Wood ◽  
Mansun Law ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Structural Characteristics ◽  
Buoyant Density ◽  
Hcv Rna ◽  
Membrane Bilayer ◽  
Biophysical Characterization ◽  
Negative Stain ◽  
A Minor

ABSTRACT We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (∼40- to 75-nm diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their surfaces. Electron cryomicroscopy revealed two major particle populations of ∼60 and ∼45 nm in diameter. The ∼60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed a high infectivity-to-HCV RNA ratio. The ∼45-nm particles lacked a membrane bilayer and displayed a higher buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-low-density, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution ultrastructural information of particle populations displaying differential biophysical properties and specific infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV RNA, and viral antigens suggests that infectious particles are likely to be present in the large ∼60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.

Potassium and calcium currents activated by foetal calf serum in Balb-c 3T3 fibroblasts

1992 ◽  
Vol 1112 (2) ◽  
pp. 241-245 ◽  
Author(s):  
Davide Lovisolo ◽  
Luca Munaron ◽  
Francesco M. Baccino ◽  
Gabriella Bonelli
Keyword(s):  
Calf Serum ◽  
Calcium Currents ◽  
Foetal Calf Serum ◽  
3T3 Fibroblasts

scholarly journals Rotavirus Persistence in Cell Cultures: Selection of Resistant Cells in the Presence of Foetal Calf Serum

1983 ◽  
Vol 64 (5) ◽  
pp. 1101-1110 ◽  
Author(s):  
A. Chiarini ◽  
S. Arista ◽  
A. Giammanco ◽  
A. Sinatra
Keyword(s):  
Cell Cultures ◽  
Calf Serum ◽  
Foetal Calf Serum ◽  
Selection Of ◽  
Resistant Cells

Restriction of foetal calf serum-induced cytotoxicity by human T-cells

1981 ◽  
Vol 3 (2) ◽  
pp. 81-85 ◽  
Author(s):  
I.S. Misko ◽  
R.G. Kane ◽  
J.H. Pope
Keyword(s):  
T Cells ◽  
Calf Serum ◽  
Foetal Calf Serum ◽  
Human T Cells

Drug activity against Onchocerca gutturosa males in vitro: a model for chemotherapeutic research on onchocerciasis

1987 ◽  
Vol 61 (4) ◽  
pp. 271-281 ◽  
Author(s):  
Simon Townson ◽  
C. Connelly ◽  
A. Dobinson ◽  
R. Muller
Keyword(s):  
Culture System ◽  
Serum Proteins ◽  
Good Condition ◽  
Calf Serum ◽  
New Drugs ◽  
Foetal Calf Serum ◽  
Partial Recovery ◽  
Feeder Cells ◽  
Compound Identification

ABSTRACTAn in vitro system for chemotherapeutic research using adult male Onchocerca gutturosa has been developed as a model for O. volvulus. Using a culture system consisting of medium MEM+10% heat inactivated foetal calf serum (IFCS)+LLCMK2 (monkey kidney) feeder cells in an atmosphere of 5% CO2 in air, we examined the effects of a range of antiparasitic drugs on worm motility. Ivermectin, levamisole, furapyrimidone, Mel W, chloroquine, metrifonate, flubendazole, amoscanate and the Ciba-Geigy compounds CGP 6140, CGP 20′376 and CGI 17658 either immobilized or significantly reduced motility levels at a concentration of 5x10−5M or less within a 7-day period. Worms were affected at very low concentrations by ivermectin (effective conc. to reduce motility levels to 50% of controls, 3.14x10−8M), levamisole (7.95x10−8M), CGP 6140 (8.87x10−9M) and CGP 20′376 (2.78x10−8M). Difficulties were experienced in accurately repeating the immotile endpoint for levamisole due to an inconsistent partial recovery of motility. Over a 7-day period diethylcarbamazine had little effect on motility levels, while suramin caused a slight increase in activity compared to controls at some timepoints. Subsequent experiments demonstrated some differences in drug efficacy depending on the presence or absence of serum and feeder cells in the culture system probably because of drug avidly binding to serum proteins. However, serum and cells were found to be essential ingredients of the culture system to maintain worms in good condition, indicating that new drugs should be evaluated both in the presence and absence of serum and cells. Comparisons were made between the responses of O. gutturosa and Brugia pahangi to certain drugs and these species were found to significantly differ in their sensitivities to ivermectin and a novel compound (Wellcome), indicating that Onchocerca parasites should be used wherever possible for compound identification and development intended for the treatment of onchocerciasis. The in vitro system described here, using male O. gutturosa, provides a basis for further research and a practical alternative to O. volvulus.

‘Spontaneous’ sex reversal in organ cultures of the embryonic male gonad of the bird

Development ◽  
1974 ◽  
Vol 31 (3) ◽  
pp. 611-620
Author(s):  
Gregory F. Erickson
Keyword(s):  
Germ Cells ◽  
Primordial Germ Cells ◽  
Calf Serum ◽  
Sex Reversal ◽  
Biologically Active ◽  
Foetal Calf Serum ◽  
Developmental Sequence ◽  
Organ Cultures ◽  
Male Gonad ◽  
Ovarian Cortex

The left embryonic testis of the bird (4–8 days of incubation) was organ cultured in medium that contained 10% foetal calf serum. Under these conditions, the germinal epithelium (GE) of the 4-day gonad differentiates into an ovarian cortex and the male primordial germ cells (PGCs) complete a developmental sequence similar to normal oocytes, i.e. they divide mitotically, develop a Balbiani body, divide synchronously in groups of two, four, and eight germ cells, and some enter pre-leptotene. No medullary tissue develops in the 4-day explants. The pieces of 6- and 8-day gonad differentiate into true ovotestes in which the GE develops into a cortex and the medulla develops into seminiferous cords. The PGCs in the cortex differentiate as oocytes and those in the seminiferous cords differentiate as spermatogonia. The possibility that biologically active oestrogens are present in the growth medium is discussed.

Substitution of transferrin by FeCl3 in the development of a low foetal calf serum concentration medium for KB-26.5 hybridoma cell line

1993 ◽  
Vol 13 (2) ◽  
pp. 133-141 ◽  
Author(s):  
Bo Damgaard ◽  
Anna Sanfeliu ◽  
Jordi Joan Cair� ◽  
Carles Casas ◽  
Carles Sol� ◽  
...  
Keyword(s):  
Serum Concentration ◽  
Cell Line ◽  
Calf Serum ◽  
Foetal Calf Serum
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