scholarly journals Structure and properties of an under-sulfated heparan sulfate proteoglycan synthesized by a rat hepatoma cell line.

1984 ◽  
Vol 98 (3) ◽  
pp. 946-953 ◽  
Author(s):  
J Robinson ◽  
M Viti ◽  
M Höök
Keyword(s):  
Heparan Sulfate ◽  
Rat Liver ◽  
Culture Medium ◽  
Hepatoma Cell ◽  
Hepatoma Cells ◽  
Heparan Sulfate Proteoglycan ◽  
Heparan Sulfate Proteoglycans ◽  
Hepatoma Cell Line ◽  
Sulfate Proteoglycan ◽  
Rat Hepatoma Cell Line

A rat hepatoma cell line was shown to synthesize heparan sulfate and chondroitin sulfate proteoglycans. Unlike cultured hepatocytes, the hepatoma cells did not deposit these proteoglycans into an extracellular matrix, and most of the newly synthesized heparan sulfate proteoglycans were secreted into the culture medium. Heparan sulfate proteoglycans were also found associated with the cell surface. These proteoglycans could be solubilized by mild trypsin or detergent treatment of the cells but could not be displaced from the cells by incubation with heparin. The detergent-solubilized heparan sulfate proteoglycan had a hydrophobic segment that enabled it to bind to octyl-Sepharose. This segment could conceivably anchor the molecule in the lipid interior of the plasma membrane. The size of the hepatoma heparan sulfate proteoglycans was similar to that of proteoglycans isolated from rat liver microsomes or from primary cultures of rat hepatocytes. Ion-exchange chromatography on DEAE-Sephacel indicated that the hepatoma heparan sulfate proteoglycans had a lower average charge density than the rat liver heparan sulfate proteoglycans. The lower charge density of the hepatoma heparan sulfate can be largely attributed to a reduced number of N-sulfated glucosamine units in the polysaccharide chain compared with that of rat liver heparan sulfate. Hepatoma heparan sulfate proteoglycans purified from the culture medium had a considerably lower affinity for fibronectin-Sepharose compared with that of rat liver heparan sulfate proteoglycans. Furthermore, the hepatoma proteoglycan did not bind to the neoplastic cells, whereas heparan sulfate from normal rat liver bound to the hepatoma cells in a time-dependent reaction. The possible consequences of the reduced sulfation of the heparan sulfate proteoglycan produced by the hepatoma cells are discussed in terms of the postulated roles of heparan sulfate in the regulation of cell growth and extracellular matrix formation.

scholarly journals Molecular cloning of the major cell surface heparan sulfate proteoglycan from rat liver.

1992 ◽  
Vol 267 (6) ◽  
pp. 3894-3900
Author(s):  
A Pierce ◽  
M Lyon ◽  
I.N. Hampson ◽  
G.J. Cowling ◽  
J.T. Gallagher
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Molecular Cloning ◽  
Rat Liver ◽  
Heparan Sulfate Proteoglycan ◽  
Sulfate Proteoglycan

scholarly journals Characterization of a novel heparan sulfate proteoglycan found in the extracellular matrix of liver sinusoids and basement membranes.

1991 ◽  
Vol 113 (5) ◽  
pp. 1231-1241 ◽  
Author(s):  
C J Soroka ◽  
M G Farquhar
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Heparan Sulfate ◽  
Rat Liver ◽  
Core Protein ◽  
Heparan Sulfate Proteoglycan ◽  
Basement Membranes ◽  
Sulfate Proteoglycan ◽  
Sinusoidal Cell ◽  
Space Of Disse

A novel heparan sulfate proteoglycan (HSPG) present in the extracellular matrix of rat liver has been partially characterized. Proteoglycans were purified from a high salt extract of total microsomes from rat liver and found to consist predominantly (approximately 90%) of HSPG. A polyclonal antiserum raised against this fraction specifically recognized HSPG by immunoprecipitation and immunoblotting. The intact, fully glycosylated HSPG migrated as a broad smear (150-300 kD) by SDS-PAGE, but after deglycosylation with trifluoromethanesulfonic acid only a single approximately 40-kD band was seen. By immunocytochemistry this HSPG was localized in the perisinusoidal space of Disse associated with irregular clumps of basement membrane-like extracellular matrix material, some of which was closely associated with the hepatocyte sinusoidal cell surface. It was also localized in biosynthetic compartments (rough ER and Golgi cisternae) of hepatocytes, suggesting that this HSPG is synthesized and deposited in the space of Disse by the hepatocyte. The anti-liver HSPG IgG also stained basement membranes of hepatic blood vessels and bile ducts as well as those of kidney and several other organs (heart, pancreas, and intestine). An antibody that recognizes the basement membrane HSPG found in the rat glomerular basement membrane did not precipitate the 150-300-kD rat liver HSPG. We conclude that the liver sinusoidal space of Disse contains a novel population of HSPG that differs in its overall size, its distribution and in the size of its core protein from other HSPG (i.e., membrane-intercalated HSPG) previously described in rat liver. It also differs in its core protein size from HSPG purified from other extracellular matrix sources. This population of HSPG appears to be a member of the basement membrane HSPG family.

scholarly journals Expression of the liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA in FAO-1 cells

1993 ◽  
Vol 293 (1) ◽  
pp. 173-179 ◽  
Author(s):  
C Espinet ◽  
A M Vargas ◽  
M R el-Maghrabi ◽  
A J Lange ◽  
S J Pilkis
Keyword(s):  
Skeletal Muscle ◽  
Cell Line ◽  
Rat Liver ◽  
Hormonal Regulation ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Bifunctional Enzyme ◽  
Dependent Manner ◽  
Rat Hepatoma Cell Line ◽  
Rat Hepatoma

The hormonal regulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression was studied in the rat hepatoma cell line FAO-1. Both 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities were detected in FAO-1 cells, at 68% of the levels found in rat liver. Northern blot analysis showed that FAO-1 cells, like rat liver, contained a predominant species of bifunctional enzyme mRNA, which is 2.2 kb in size. A sensitive RNAase protection assay revealed the presence in FAO-1 cells of an additional mRNA species, which is generated when transcription is initiated from the skeletal muscle promoter of the rat liver/skeletal muscle gene. The liver/skeletal muscle mRNA ratio in FAO-1 cells was 10:1, which is similar to that observed in rat liver. In contrast, in another rat hepatoma cell line, FTO-2B, only the skeletal muscle mRNA was detected. Insulin and dexamethasone induced the liver bifunctional enzyme mRNA in FAO-1 cells by 2-4-fold and 10-20-fold respectively in a concentration- and time-dependent manner, and their effects were antagonized by cyclic AMP. Transcription of the gene in FAO-1 cells, measured by nuclear run-on assays, was also enhanced by dexamethasone and insulin. It is concluded that the FAO-1 cell line is similar to liver with respect to both the preferential use of the liver promoter of the gene and its regulation by hormones, and is therefore an excellent model for the study of the hepatic expression of this gene.

scholarly journals Identification of a plasma membrane glutamine transporter from the rat hepatoma cell line H4-IIE-C3

2002 ◽  
Vol 368 (1) ◽  
pp. 371-375 ◽  
Author(s):  
Matthew POLLARD ◽  
David MEREDITH ◽  
John D. McGIVAN
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Rat Liver ◽  
Hepatoma Cell ◽  
Single Amino Acid ◽  
Hepatoma Cell Line ◽  
Normal Rat ◽  
Rat Hepatoma Cell Line ◽  
Rat Hepatoma ◽  
Glutamine Uptake

Glutamine is taken up into the rat hepatoma cell line H4-IIE-C3 by a Na+-dependent transport system which is specific for glutamine, alanine, serine, cysteine and asparagine and does not tolerate substitution of Na+ by Li+. Glutamine transport was relatively weakly inhibited by a 50-fold excess of leucine and was not inhibited by phenylalanine or N-methyl aminoisobutyrate. These general properties are characteristic of the recently identified ASCT/B0 family of transporters. Using a reverse transcriptase PCR-based homology cloning approach, we have characterized a cDNA for a novel member of this transporter family (H4-ASCT2) from H4-IIE-C3 cells. The cDNA encodes a 551-amino acid protein which exhibits similarities of between 75 and 85% with ASCT/B0 transporters previously cloned from other sources. When expressed in Xenopus oocytes, this transporter catalyses Na+-dependent glutamine uptake with characteristics very similar to those of glutamine uptake into the H4-IIE-C3 cells. This newly characterized transporter possesses a number of amino acid sequence differences from ASCT2 clones recently isolated from rat astroglial cells and from normal rat liver. In particular, the loop region between transmembrane helices 3 and 4 from H4-ASCT2 shares less than 60% sequence similarity with ASCT2 from rat liver; furthermore, there are some 25 single amino acid substitutions elsewhere in the H4-ASCT2 sequence compared with that from rat liver. Thus enhanced glutamine uptake in rat hepatoma cells is mediated by the expression of a novel ASCT/B0 transporter isoform rather than by increased expression of the ASCT2 mRNA found in normal rat liver.

scholarly journals Membrane-Associated Heparan Sulfate Proteoglycan Is a Receptor for Adeno-Associated Virus Type 2 Virions

1998 ◽  
Vol 72 (2) ◽  
pp. 1438-1445 ◽  
Author(s):  
Candace Summerford ◽  
Richard Jude Samulski
Keyword(s):  
Gene Therapy ◽  
Heparan Sulfate ◽  
Cell Lines ◽  
Heparan Sulfate Proteoglycan ◽  
Heparan Sulfate Proteoglycans ◽  
Target Cells ◽  
Sulfate Proteoglycan ◽  
Viral Receptor ◽  
Adeno Associated Virus

ABSTRACT The human parvovirus adeno-associated virus (AAV) infects a broad range of cell types, including human, nonhuman primate, canine, murine, and avian. Although little is known about the initial events of virus infection, AAV is currently being developed as a vector for human gene therapy. Using defined mutant CHO cell lines and standard biochemical assays, we demonstrate that heparan sulfate proteoglycans mediate both AAV attachment to and infection of target cells. Competition experiments using heparin, a soluble receptor analog, demonstrated dose-dependent inhibition of AAV attachment and infection. Enzymatic removal of heparan but not chondroitin sulfate moieties from the cell surface greatly reduced AAV attachment and infectivity. Finally, mutant cell lines that do not produce heparan sulfate proteoglycans were significantly impaired for both AAV binding and infection. This is the first report that proteoglycan has a role in cellular attachment of a parvovirus. Together, these results demonstrate that membrane-associated heparan sulfate proteoglycan serves as the viral receptor for AAV type 2, and provide an explanation for the broad host range of AAV. Identification of heparan sulfate proteoglycan as a viral receptor should facilitate development of new reagents for virus purification and provide critical information on the use of AAV as a gene therapy vector.

scholarly journals Association between heparan sulfate proteoglycan excretion and proteinuria after renal transplantation.

1996 ◽  
Vol 7 (12) ◽  
pp. 2670-2676
Author(s):  
I Stefanidis ◽  
B Heintz ◽  
G Stöcker ◽  
C Mrowka ◽  
H G Sieberth ◽  
...  
Keyword(s):  
Renal Transplantation ◽  
Basement Membrane ◽  
Heparan Sulfate ◽  
Glomerular Basement Membrane ◽  
Excretion Rate ◽  
Heparan Sulfate Proteoglycan ◽  
Heparan Sulfate Proteoglycans ◽  
Second Phase ◽  
Sulfate Proteoglycan ◽  
Glomerular Proteinuria

The aim of the study presented here was to investigate whether, in patients showing immediate graft function after renal transplantation, cold-ischemia and reperfusion lead to damage of the glomerular basement membrane and consequently to a loss of heparan sulfate proteoglycans. Loss of these heparan sulfate proteoglycans is a major cause of proteinuria. Time-dependent changes in urinary excretion rates of heparan sulfate proteoglycans but also of total protein, albumin, low- and high-molecular-weight proteins were analyzed quantitatively and by polyacrylamid-gel-electrophoresis in eight patients. Immediately after renal transplantation, severe proteinuria with an excretion rate of up to 251 +/- 108 mg/min was apparent and rapidly declined within 24 h to 4.11 +/- 2.80 mg/min. The gel-electrophoretic pattern showed a nonselective glomerular and tubular proteinuria. The excretion rate of heparan sulfate proteoglycan was increased in this initial reperfusion phase (up to 7 h), most probably because of ischemia- and reperfusion-induced damage of the glomerular basement membrane. The initial nonselective glomerular proteinuria disappeared within 48 h, changing to a mild selective glomerular proteinuria. In this second phase (7 to 48 h), lower levels of heparan sulfate proteoglycan excretion were observed (0.54 +/- 0.54 microgram/min versus 1.66 +/- 1.93 micrograms/min, P < 0.05). However, during the repair process of the glomerular basement membrane, heparan sulfate proteoglycan is synthesized de novo, leading to an increasing heparan sulfate proteoglycan content of the glomerular basement membrane. This second phase is paralleled by the change from a nonselective to a selective glomerular proteinuria. In the third phase, when the heparan sulfate proteoglycan content of the glomerular basement membrane normalizes, glomerular proteinuria was abolished in most of the patients.

scholarly journals Comparison of the multiple deoxyribonucleic acid-dependent ribonucleic acid polymerase forms of whole rat liver and a minimal-deviation rat hepatoma cell line

1972 ◽  
Vol 126 (3) ◽  
pp. 675-681 ◽  
Author(s):  
C. J. Chesterton ◽  
S M Humphrey ◽  
P H W Butterworth
Keyword(s):  
Cell Line ◽  
Rat Liver ◽  
Animal Cell ◽  
Hepatoma Cell ◽  
Rna Polymerases ◽  
Hepatoma Cell Line ◽  
Enzyme Preparations ◽  
Minimal Deviation ◽  
Rat Hepatoma Cell Line ◽  
Rat Hepatoma

To investigate the possibility that the pattern of multiple DNA-dependent RNA polymerases of an animal cell exerts a controlling influence on its nature, the activities of these enzymes were compared in differentiated rat liver and in a rapidly growing minimal-deviation rat hepatoma cell line by using established techniques of enzyme extraction, separation and determination. Relative to the DNA content of the tissues, RNA polymerase activities of forms AI, AII and B were approx. ninefold, twofold and twofold higher respectively in the cell line than in the liver. Tests indicated that these results could not be explained by differences in extraction efficiency or by the presence of unbound inhibitors or stimulators of polymerase activity in the final enzyme preparations. New forms of the enzyme were not detected in either tissue. The significance of these findings with respect to the possible role of multiple RNA polymerases in the control of cellular activities is discussed.

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