scholarly journals Proteoglycan synthesis in human erythroleukaemia (HEL) cells

1992 ◽  
Vol 282 (3) ◽  
pp. 651-658 ◽  
Author(s):  
B P Schick ◽  
S Senkowski-Richardson
Keyword(s):  
Chain Length ◽  
Culture Medium ◽  
Core Protein ◽  
Dimethyl Sulphoxide ◽  
Proteoglycan Synthesis ◽  
Average Chain Length ◽  
Haematopoietic Cells ◽  
Core Proteins ◽  
Sds Page ◽  
Hel Cells

Synthesis of sulphated proteoglycans was compared in human erythroleukaemia (HEL) cells grown under control conditions and under stimulation by dimethyl sulphoxide (DMSO) and phorbol 12-myristate 13-acetate (PMA). Synthesis of [35S]sulphate-labelled proteoglycans by DMSO-treated cells was decreased by about 35% relative to controls, but synthesis of proteoglycans by PMA-treated cells increased 3-4-fold. Control and DMSO-treated cells secreted 65% of the newly synthesized proteoglycans, but PMA-treated cells secreted more than 90%. Sepharose CL-6B chromatography and SDS/PAGE suggested the presence of several proteoglycans in the cells and culture medium. The PMA-treated cells synthesized a low-Mr proteoglycan (Kav. 0.3(that was not present in controls and DMSO-treated cultures. The proteoglycans of the cells and medium from control, DMSO-treated and PMA-treated cultures could be separated into three fractions by octyl-Sepharose chromatography. The proteoglycans were resistant to trypsin but were degraded by Pronase and papain to fragments similar in size to the NaOH/NaBH4-generated glycosaminoglycans. The average chain length of the glycosaminoglycans (Kav. 0.20 on Sepharose CL-6B for controls) was decreased by DMSO (Kav. 0.25) and by PMA (Kav. 0.30-0.38). Chondroitin ABC lyase digestion of the proteoglycans from the medium of the control cultures produced two core proteins at Mr 31,000 and 36,000. The DMSO medium proteoglycans had only the 31,000-Mr core protein, and the PMA culture medium proteoglycans had core proteins of Mr 27,000, 31,000 and 36,000. Changes in synthesis of proteoglycans induced by DMSO or PMA may have relevance for the maturation of haematopoietic cells.

scholarly journals Characterization of proteoglycans isolated from associative extracts of human articular cartilage

1993 ◽  
Vol 293 (1) ◽  
pp. 165-172 ◽  
Author(s):  
V Vilím ◽  
A J Fosang
Keyword(s):  
Articular Cartilage ◽  
Molecular Mass ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Gel Chromatography ◽  
Human Articular Cartilage ◽  
Keratan Sulphate ◽  
Core Proteins ◽  
Sds Page

Approx. 10% of the total proteoglycan content of normal young human articular cartilage was extracted under associative conditions with Dulbecco's PBS. Proteoglycans isolated from the extract by Q-Sepharose chromatography were separated by gel chromatography and characterized by gradient gel SDS/PAGE and immunoblotting. Three species of small proteoglycans, two main populations of aggrecan and a population of its smaller fragments were identified. The major populations of aggrecan contained chondroitin sulphate chains, all or part of the N-terminal G1 and G2 domains and, therefore, intact keratan sulphate domains. The larger population was estimated by gradient SDS/PAGE to have a molecular mass of approx. 600 kDa or greater. The second population had an apparent molecular mass of approx. 300-600 kDa. Core proteins derived from these populations of proteoglycans separated on SDS/PAGE into several clusters of bands in the range from 120 to approx. 360 kDa. The extract further contained smaller fragments which lacked chondroitin sulphate but reacted with antibodies against keratan sulphate, and against epitopes present in the G2 domain of aggrecan. The presence of the G2 domain in a broad range of populations of decreasing size indicated extensive cleavage of the aggrecan core protein within its chondroitin sulphate domain. These findings suggest that fragmentation of aggrecan probably occurs in vivo in normal articular cartilage of young individuals. Associative extracts also contained decorin, biglycan and fibromodulin. These were resolved from aggrecan by gel chromatography and identified by immunodetection.

scholarly journals Selective inhibition of proteoglycan and hyaluronate synthesis in chondrocyte cultures by cyclofenil diphenol, a non-steroidal weak oestrogen

1984 ◽  
Vol 223 (2) ◽  
pp. 401-412 ◽  
Author(s):  
R M Mason ◽  
J D Lineham ◽  
M A Phillipson ◽  
C M Black
Keyword(s):  
Culture Medium ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Primary Cultures ◽  
Normal Subjects ◽  
Dermal Fibroblasts ◽  
Proteoglycan Synthesis ◽  
Dependent Manner ◽  
Glycoprotein Synthesis ◽  
Chondrocyte Cultures

Cyclofenil diphenol, a weak non-steroidal oestrogen, binds to albumin. In the presence of concentrations of albumin just sufficient to keep cyclofenil diphenol in solution, the compound inhibited the synthesis of [35S]proteoglycans, [3H]glycoproteins, [3H]hyaluronate and [3H]proteins in primary cultures of chondrocytes from the Swarm rat chondrosarcoma in a dose-dependent manner. When excess albumin was present, conditions were found (90 micrograms of cyclofenil diphenol and 4 mg of albumin per ml of culture medium) which completely inhibited [35S]proteoglycan and [3H]hyaluronate synthesis but had little effect on [3H]protein or [3H]glycoprotein synthesis. The time of onset of inhibition of [35S]proteoglycan synthesis by cyclofenil diphenol was very rapid (t1/2 less than 25 min) and incompatible with an action mediated through suppression of proteoglycan core protein synthesis. Cyclofenil diphenol inhibited the synthesis of [35S]chondroitin sulphate chains onto p-nitrophenyl beta-D-xyloside in the cultures. Cyclofenil diphenol had little effect on the secretion from chondrocytes of [35S]proteoglycans synthesized immediately prior to treatment. Chondrocyte cultures treated with cyclofenil diphenol recovered their biosynthetic activities almost completely within 3 h of removing the compound from the culture medium. Cyclofenil diphenol had a similar inhibitory action on the synthesis of [35S]proteoglycans in secondary cultures of human dermal fibroblasts from both normal subjects and patients with systemic sclerosis. It is proposed that cyclofenil diphenol inhibits the synthesis of [35S]proteoglycans by interfering with the formation of the glycosaminoglycan side chains of these molecules in the Golgi apparatus of cells. The action may be due to disturbance of Golgi membrane organization by the compound.

Analysis of proteoglycan synthesis by retro-ocular tissue fibroblasts under the influence of interleukin 1β and transforming growth factor-β

1994 ◽  
Vol 131 (6) ◽  
pp. 630-638 ◽  
Author(s):  
Yumi Imai ◽  
Kyomi Ibaraki ◽  
Ritsuko Odajima ◽  
Yoshimasa Shishiba
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Core Protein ◽  
Transforming Growth Factor Β ◽  
Interleukin 1Β ◽  
Chain Elongation ◽  
Ocular Tissue ◽  
Proteoglycan Synthesis ◽  
Core Proteins ◽  
Sulfate Incorporation

Imai Y, Ibaraki K, Odajima R, Shishiba Y. Analysis of proteoglycan synthesis by retro-ocular tissue fibroblasts under the influence of interleukin 1β and transforming growth factor-β Eur J Endocrinol 1994;131:630–8. ISSN 0804–4643 Retro-ocular tissue fibroblasts are supposed to be responsible for the deposition of glycosaminoglycan in Graves' ophthalmopathy. We have reported in a preliminary fashion that interleukin 1β (IL-1β) and transforming growth factor-β (TGF-β) increased the rate of [35S]sulfate incorporation into proteoglycans two to five times the control in culture of retro-ocular tissue fibroblasts. The increase in the rate of [ S]sulfate incorporation into proteoglycan will occur as a result of: (a) net increase of proteoglycan synthesis; (b) elongation of glycosaminoglycan chains; (c) increased number of glycosaminoglycan chains; (d) oversulfation of glycosaminoglycan chains; (e) increase in cell number; (f) decreased rate of degradation. We have analyzed which mechanism is important for the increase of [35S]sulfate into proteoglycans observed in human retro-ocular tissue fibroblasts under the influence of cytokines. The last two possibilities (e, f) were ruled out because during the observation period there was no consistent proliferation of the cells and no decrease in the rate of degradation of proteoglycan examined by pulse-chase experiment. Cytokines did not change the size of glycosaminoglycan chains released from proteoglycan as measured by alkaline borohydride treatment, ruling out (b). Disaccharide analysis by HPLC after chondroitin sulfate ABC digestion revealed that glycosaminoglycan mainly contains monosulfated chondroitin disaccharides and that oversulfation was not observed under the influence of IL-1β or TGF-β, ruling out (d). The capacity to synthesize glycosaminoglycan chain in the presence of an artifical acceptor of chain elongation, β-d-xylodide, was increased significantly by IL-1β but not obviously so by TGF-β. Thus, an increased number of glycosaminoglycan chains (c) is possible for IL-1β. A preliminary northern blot analysis employing probes for mRNAs for various proteoglycan core proteins showed increased expression of versican and aggrecan in the presence of IL-1β or TGF-β. This result supports the possibility of (a). In conclusion, IL-1β and TGF-β increased [35S]sulfate incorporation into proteoglycan by increasing the net increase of proteoglycan synthesis and possibly by increasing the number of glycosaminoglycan chains attached to core protein in the case of IL-1β. Y Shishiba, Division of Endocrinology, Department of Medicine, Toranomon Hospital, 2-2-2 Toranomon, Minato-ku, Tokyo, Japan

scholarly journals Independent secretion of proteoglycans and collagens in chick chondrocyte cultures during acute ascorbic acid treatment

1990 ◽  
Vol 272 (1) ◽  
pp. 193-199 ◽  
Author(s):  
M Pacifici
Keyword(s):  
Acid Treatment ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Proteoglycan Synthesis ◽  
Collagen Types ◽  
Keratan Sulphate ◽  
Sulphate Proteoglycan ◽  
Chondrocyte Cultures ◽  
Chondroitin Sulphate Proteoglycan ◽  
Core Proteins

The mechanisms regulating the secretion of proteoglycans and collagens in chondrocytes, in particular those operating at the level of the rough endoplasmic reticulum (RER), are largely unknown. To examine these mechanisms, I studied the effects of acute ascorbate treatment on the secretion of two collagen types (types II and IX) and two proteoglycan types (PG-H and PG-Lb, the major keratan sulphate/chondroitin sulphate proteoglycan and the minor chondroitin sulphate proteoglycan respectively in cartilage) in scorbutic cultures of chick vertebral chondrocytes. I found that the scorbutic chondrocytes synthesized underhydroxylated precursors of types II and IX collagen that were secreted very slowly and accumulated in the RER. When the cultures were treated acutely with ascorbate, both macromolecules underwent hydroxylation within 1-1.5 h of treatment, and began to be secreted at normal high rates starting at about 2 h. Proteoglycan synthesis and secretion, however, remained largely unaffected by ascorbate treatment. Both the half-time of newly synthesized PG-H core protein in the RER and its conversion into completed proteoglycan were unchanged during treatment. Similarly, the overall rates of synthesis and secretion of both PG-H and PG-Lb remained at control levels during treatment. The data indicate that secretion of types II and IX collagen is regulated independently of secretion of PG-H and PG-Lb. This may be mediated by the ability of the RER of the chondrocyte to discriminate between procollagens and proteoglycan core proteins.

scholarly journals Stimulation of sulphated glycosaminoglycan and decorin production in adult dermal fibroblasts by recombinant human interleukin-4

1995 ◽  
Vol 307 (3) ◽  
pp. 673-678 ◽  
Author(s):  
Y Wegrowski ◽  
V Paltot ◽  
P Gillery ◽  
B Kalis ◽  
A Randoux ◽  
...  
Keyword(s):  
Culture Medium ◽  
Core Protein ◽  
Human Fibroblasts ◽  
Inflammatory Cells ◽  
Dermal Fibroblasts ◽  
Interleukin 4 ◽  
Proteoglycan Synthesis ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Sulphated Glycosaminoglycan

Interleukin-4 (IL-4) is a pleiotropic cytokine expressed by inflammatory cells. Previous work from our laboratory has shown that it stimulates collagen synthesis in fibroblasts. Here we report the effects of recombinant human IL-4 on glycosaminoglycan (GAG) and proteoglycan synthesis in normal dermal fibroblasts from adult donors. IL-4 (10 and 100 units/ml) induced a dose-dependent increase of [3H]glucosamine and [35S]sulphate incorporation into total GAGs. The analysis of the different GAG fractions indicated the enhanced synthesis of dermatan/chondroitin sulphates. IL-4 had no effect on hyaluronan synthesis. The increase of sulphated GAG synthesis was correlated with an increase of proteoglycans in the culture medium. Decorin was identified as the major chondroitin/dermatan sulphate-containing proteoglycan in the culture medium of fibroblasts. Its synthesis was strongly stimulated by IL-4. Both the core-protein synthesis and mRNA expression were enhanced, indicating that the cytokine acted, at least in part, at the pre-translational level. These results indicate that IL-4 is able to modulate not only collagen, but also proteoglycan, production by human fibroblasts. Their implications in physiopathological processes such as wound healing or fibrosis is suggested.

scholarly journals Effects of cyclofenil diphenol, an agent which disrupts Golgi structure, on proteoglycan synthesis in chondrocytes

1992 ◽  
Vol 281 (2) ◽  
pp. 525-531 ◽  
Author(s):  
R M Mason ◽  
C A Lancaster
Keyword(s):  
Core Protein ◽  
Chondroitin Sulphate ◽  
Proteoglycan Synthesis ◽  
Specific Substrate ◽  
Inhibitory Mechanism ◽  
Similar Concentration ◽  
System A ◽  
Sds Page ◽  
Golgi Structure ◽  
Related Compounds

1. Cyclofenil diphenol (F6060), a weak non-steroidal oestrogen, was shown previously to inhibit [35S]proteoglycan synthesis [Mason, Lineham, Phillipson & Black (1984) Biochem. J. 223, 401-412] and to induce fragmentation of the Golgi apparatus into small vesicles [Lancaster, Fryer, Griffiths & Mason (1989) J. Cell Sci. 92, 271-280] in cultures of Swarm chondrosarcoma chondrocytes. Two structurally related compounds, F6204 and F6091, show a similar concentration-related effect, with complete inhibition of [35S]proteoglycan synthesis at 90 micrograms/ml. The apparent [3H]protein synthesis is only approx. 40% inhibited with [3H]lysine as precursor. Stilboestrol, clomiphene and tamoxiphen are also potent inhibitors of [35S]proteoglycan synthesis. 2. Syntheses of chondroitin 4-[35S]sulphate and chondroitin 6-[35S]sulphate, which are Golgi-mediated events, are inhibited 40-68% and 3-48% respectively by concentrations of cyclofenil between 50 and 70 micrograms/ml. [3H]Hyaluronan synthesis, which occurs by a different mechanism at the plasma membrane, is inhibited by 47-66%. These results suggest that cyclofenil may act via more than one inhibitory mechanism. Cyclofenil diphenol inhibits polymerization of chondroitin sulphate on to p-nitrophenyl beta-xyloside even when the chondrocytes are loaded with the initiator prior to treatment. 3. Cyclofenil diphenol interferes with the cellular uptake of amino acids via the system A carrier, as shown by inhibition of uptake of methylaminoisobutyric acid, a specific substrate for this system. The drug had no effect on the uptake of 2-deoxyglucose by the cells. 4. Cyclofenil diphenol (90 micrograms/ml) caused a decrease in the pool size of UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and UDP-hexoses, but this was insufficient to account for the accompanying profound inhibition of [35S]proteoglycan synthesis. Entry of [3H]glucosamine into the cell and into the UDP-N-acetylhexosamine pool did not appear to be affected. 5. Cyclofenil diphenol inhibited the substitution of 3H-labelled proteoglycan core protein with chondroitin sulphate chains. Core protein was identified in treated cultures on the basis of immunoprecipitation with an antiserum against the hyaluronate-binding region and distinguished from precipitated proteoglycan on SDS/PAGE.

Röntgenbeugungsuntersuchung an geschmolzenem Selen und Tellur sowie an Legierungen des Systems Selen—Tellur/ X-ray Diffraction Study of Molten Selenium, and Tellurium and of Molten Selenium — Tellurium-Alloys

1975 ◽  
Vol 30 (12) ◽  
pp. 1633-1639 ◽  
Author(s):  
W. Hoyer ◽  
E. Thomas ◽  
M. Wobst
Keyword(s):  
Chain Length ◽  
Short Range ◽  
Short Range Order ◽  
Coordination Shell ◽  
Range Order ◽  
Average Chain Length ◽  
X Ray Diffraction ◽  
Coordination Numbers ◽  
Nearest Neighbours ◽  
Increasing Temperature

Abstract At temperatures just above the melting point molten Selenium seems to be a mixture of long chains and eight-membered rings. With increasing temperature the number of rings and the average chain length decrease. At 460 °C the average chain length lies in the range of 10 atoms.In a slightly supercooled Tellurium-melt the number of first neighbours is two. The atoms are arranged in chains. Selenium rich Se-Te-alloy-melts are built up of mixed chains. It seems to be possible, that a smaller part of atoms forms Se6Te2 rings. At Tellurium concentrations higher than approximately 50 at.-% the chainlike structure with two next nearest neighbours changes to a disturbed Arsen-like short range order. The number of electrons in the first coordination shell, the short range order parameter introduced by Cowley and the partial coordination numbers show that Se-Te-alloys are of the "solution system" type, but in the whole concentration range for each atom there is a tendency to have "strange coordination".

scholarly journals Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length

2006 ◽  
Vol 6 (2) ◽  
pp. 328-336 ◽  
Author(s):  
Kariona A. Grabińska ◽  
Paula Magnelli ◽  
Phillips W. Robbins
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chain Length ◽  
Attachment Site ◽  
Chitin Synthase ◽  
Yeast Cells ◽  
Average Chain Length ◽  
Calcofluor White ◽  
Protein Factor

ABSTRACT Chs4p (Cal2/Csd4/Skt5) was identified as a protein factor physically interacting with Chs3p, the catalytic subunit of chitin synthase III (CSIII), and is indispensable for its enzymatic activity in vivo. Chs4p contains a putative farnesyl attachment site at the C-terminal end (CVIM motif) conserved in Chs4p of Saccharomyces cerevisiae and other fungi. Several previous reports questioned the role of Chs4p prenylation in chitin biosynthesis. In this study we reinvestigated the function of Chs4p prenylation. We provide evidence that Chs4p is farnesylated by showing that purified Chs4p is recognized by anti-farnesyl antibody and is a substrate for farnesyl transferase (FTase) in vitro and that inactivation of FTase increases the amount of unmodified Chs4p in yeast cells. We demonstrate that abolition of Chs4p prenylation causes a ∼60% decrease in CSIII activity, which is correlated with a ∼30% decrease in chitin content and with increased resistance to the chitin binding compound calcofluor white. Furthermore, we show that lack of Chs4p prenylation decreases the average chain length of the chitin polymer. Prenylation of Chs4p, however, is not a factor that mediates plasma membrane association of the protein. Our results provide evidence that the prenyl moiety attached to Chs4p is a factor modulating the activity of CSIII both in vivo and in vitro.

scholarly journals Hepatitis B viral core proteins with an N-terminal extension can assemble into core-like particles but cannot be enveloped

1999 ◽  
Vol 80 (10) ◽  
pp. 2647-2659 ◽  
Author(s):  
Eric Ka-Wai Hui ◽  
Yong Shyang Yi ◽  
Szecheng J. Lo
Keyword(s):  
Hepatitis B ◽  
Kinase Activity ◽  
Core Protein ◽  
Surface Antigens ◽  
Human Hepatoma Cells ◽  
The Core ◽  
Core Proteins ◽  
B Virus ◽  
Transfection Medium ◽  
Cscl Gradient

The structure of hepatitis B virus (HBV) nucleocapsids has been revealed in great detail by cryoelectron microscopy. How nucleocapsids interact with surface antigens to form enveloped virions remains unknown. In this study, core mutants with N-terminal additions were created to address two questions: (1) can these mutant core proteins still form nucleocapsids and (2) if so, can the mutant nucleocapsids interact with surface antigens to form virion-like particles. One plasmid encoding an extra stretch of 23 aa, including six histidine residues, fused to the N terminus of the core protein (designated HisC183) was expressed in Escherichia coli and detected by Western blot. CsCl gradient and electron microscopy analyses indicated that HisC183 could self-assemble into nucleocapsids. When HisC183 or another similar N-terminal fusion core protein (designated FlagC183) was co-expressed with a core-negative plasmid in human hepatoma cells, both mutant core proteins self-assembled into nucleocapsids. These particles also retained kinase activity. Using an endogenous polymerase assay, a fill-in HBV DNA labelled with isotope was obtained from intracellular nucleocapsids formed by mutant cores. In contrast, no such signal was detected from the transfection medium, which was consistent with PCR and Southern blot analyses. Results indicate that core mutants with N-terminal extensions can form nucleocapsids, but are blocked during the envelopment process and cannot form secreted virions. The mutant nucleocapsids generated from this work should facilitate further study on how nucleocapsids interact with surface antigens.

scholarly journals Proteomic and 3D structure analyses highlight the C/D box snoRNP assembly mechanism and its control

2014 ◽  
Vol 207 (4) ◽  
pp. 463-480 ◽  
Author(s):  
Jonathan Bizarro ◽  
Christophe Charron ◽  
Séverine Boulon ◽  
Belinda Westman ◽  
Bérengère Pradet-Balade ◽  
...  
Keyword(s):  
Isotope Labeling ◽  
Core Protein ◽  
3D Structure ◽  
Assembly Mechanism ◽  
Core Proteins ◽  
Assembly Factors ◽  
Assembly Factor

In vitro, assembly of box C/D small nucleolar ribonucleoproteins (snoRNPs) involves the sequential recruitment of core proteins to snoRNAs. In vivo, however, assembly factors are required (NUFIP, BCD1, and the HSP90–R2TP complex), and it is unknown whether a similar sequential scheme applies. In this paper, we describe systematic quantitative stable isotope labeling by amino acids in cell culture proteomic experiments and the crystal structure of the core protein Snu13p/15.5K bound to a fragment of the assembly factor Rsa1p/NUFIP. This revealed several unexpected features: (a) the existence of a protein-only pre-snoRNP complex containing five assembly factors and two core proteins, 15.5K and Nop58; (b) the characterization of ZNHIT3, which is present in the protein-only complex but gets released upon binding to C/D snoRNAs; (c) the dynamics of the R2TP complex, which appears to load/unload RuvBL AAA+ adenosine triphosphatase from pre-snoRNPs; and (d) a potential mechanism for preventing premature activation of snoRNP catalytic activity. These data provide a framework for understanding the assembly of box C/D snoRNPs.

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