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G M Fuller

Publications and source records attributed to G M Fuller.

At least 19 recordsLinked to original sources
PubMed1992

Dynamics of interleukin-6 internalization and degradation in rat hepatocytes.

We have investigated the dissociation, internalization, and degradation of 125I-interleukin-6 (125I-IL-6) by primary rat hepatocytes. Temperature shift experiments following saturation binding of 125I-IL-6 to cell surface receptors in hepatocytes showed a rapid loss of surface-bound 125I-IL-6 (t1/2 = 15 min), concomitant with a rapid rise in internalized radiolabeled ligand. After reaching a maximum by 30 min at 37 degrees C, the level of internalized 125I-IL-6 decreased with time and appeared in the culture media in a non-trichloroacetic acid-precipitable (degraded) state. The addition of the lysosomotropic agent chloroquine inhibited this receptor-mediated degradation of IL-6 without affecting ligand internalization. Polyacrylamide gel electrophoresis analysis of internalized 125I-IL-6 confirms these results. Additionally, we show that the IL-6.IL-6 receptor complex is stable, and dissociation of these two molecular species occurs at a pH below 5.0. In contrast to published results, data presented in this study clearly indicate that IL-6 is rapidly internalized and degraded within hepatocytes by a receptor-mediated mechanism.

Animals↗
PubMed1992

Identification of a human cDNA with high homology to yeast omnipotent suppressor 45.

Omnipotent suppression is a well-established phenomenon in yeast and bacteria in which nonsense mutations are misread. Wild-type (wt) suppressors are presumed to be involved in ensuring the fidelity of translation. We report a human homolog to wt yeast omnipotent suppressor 45 which shares 63% identity at the nucleotide level in the area of open reading frame (ORF) and 73% similarity at the amino acid (aa) level. The aa sequence of the human protein was deduced from a 2.3-kb cDNA (TB3-1) isolated from an adenocarcinoma T84 cell line cDNA library. The cDNA contains an ORF of 1284 bp which encodes a 47.8-kDa protein. Two transcripts for the clone were identified (2.6 and 4.0 kb) in a variety of human cell types. The strong structural similarity to yeast omnipotent suppressor 45, and its widespread expression suggest that this cDNA may play a role in the accurate recognition of nonsense codons in mammalian cells.

Amino Acid Sequence↗
PubMed1992

Chromosomal localization of the IL-6 receptor signal transducing subunit, gp130 (IL6ST).

Signal transduction in eukaryotic cells is a complex process mediated, normally, by the interaction of soluble extrinsic protein factors and their cognate receptors. One example of this phenomena is the inflammatory cytokine interleukin-6 and the IL-6 receptor. However, the IL-6 receptor, once its ligand is bound, associates with another membrane glycoprotein, gp130, to potentiate the cytokine response. To further understand the basis of this interaction, and its possible implications in cellular transforming events, the corresponding gene(s) must be studied. Here we find that the human gp130 gene product is homologous to two distinct chromosomal loci on chromosomes 5 and 17. Furthermore, the presence of two distinct gp130 gene sequences is restricted to primates and is not found in other vertebrates.

Antigens, CD↗
PubMed1992

Chromosomal localization of a human cDNA containing a DIDS binding domain and demonstrating high homology to yeast omnipotent suppressor 45.

We recently have identified a full-length cDNA (TB3-1) from a human adenocarcinoma cell line T84 cDNA library that encodes a 47.8-kDa protein. TB3-1 shares identity with the putative yeast translation termination factor omnipotent suppressor 45. Using human-mouse somatic cell panel analysis, a family of sequences with high homology to the TB3-1 cDNA clone were localized to human chromosomes 5, 6, 7, and X. Southern analysis of a panel of mammalian and chicken genomic DNA demonstrates that TB3-1 is well conserved in higher vertebrates.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Molecular cloning and characterization of the rat liver IL-6 signal transducing molecule, gp130.

Interleukin-6 (IL-6) is a multifunctional cytokine that exerts its effects on different target cells by interacting with a specific receptor. This interaction leads to the association and activation of a second membrane glycoprotein, gp130, which is the IL-6 signal transducing molecule. The nucleotide sequence of gp130 from a human B-cell line has been reported. We report here the cloning and sequence analysis of the gp130 molecule derived from rat liver. Comparison of gp130 molecules from the different species and cell types reveals 78% overall amino acid homology and 94% identity in the growth factor signaling domain. Two gp130 mRNA species, a moderately abundant species of 7.5 kb and a lesser one of 9.0 kb, were present in rat hepatocytes. Ribonuclease protection analyses demonstrated the presence of gp130 mRNA in four different nontransformed cell types: hepatocytes, astrocytes, fibroblasts, and endothelial cells. The sequences between both gp130s in the different cell types are quite similar, supporting the prediction that the different responses initiated by IL-6 on different target cells are modulated by cell-specific proteins distal to the activated gp130 molecule.

Amino Acid Sequence↗
PubMed1992

Differential regulation of interleukin-6 receptor and gp130 gene expression in rat hepatocytes.

Interleukin-6 (IL-6) relays an important signal to hepatocytes during the early stages of an acute inflammatory response, causing an alteration in the expression of several major defense proteins. Additional regulation of this signal could occur either by altering the number of IL-6 receptors (IL-6-R) or of the signal transducing protein, gp130. We employed ribonuclease protection assays to measure the expression of IL-6-R and gp130 mRNA in primary rat hepatocytes in response to IL-6, interleukin-1, dexamethasone, and combinations thereof. Dexamethasone increases receptor mRNA levels 2.7-fold above controls but has no detectable effect on that of gp130. Such treatment increased surface expression of IL-6-R from 600 receptors per cell to greater than 6000, without a change in Kd (2.5-4.6 x 10(-10) M). In contrast to the stimulatory effect of the steroid signal, the inflammatory cytokines, individually and together, down-modulated both the mRNA and the cell surface expression of IL-6-R. These findings demonstrate for the first time that a sensitive control system exists between inflammatory mediators and IL-6-R.

Animals↗
PubMed1991

Isolation and characterization of biologically active murine interleukin-6 produced in Escherichia coli.

Interleukin-6 (IL-6) is a multi-functional cytokine produced and secreted by several different cell types, including those of the immune system. A cDNA coding for the mature murine IL-6 (mIL-6), which extends from amino acid (aa) 25 through 211, was cloned into a prokaryotic vector and then expressed in Escherichia coli. The recombinant mIL-6 (remIL-6) was isolated from bacterial inclusion bodies by solubilization in 4 M guanidine hydrochloride followed by gel-filtration chromatography. The protein was refolded to an active conformation by dialysis against 25 mM Na. acetate pH 5.5. A final step of purification and concentration on a cation exchange resin yielded pure and biologically active remIL-6. The purified preparation had the expected aa composition, as confirmed by aa analysis and pI of 7.0-7.1. The biological activity of the recombinant protein was measured in two systems; a proliferation assay employing 7TD1 cells, and a fibrinogen biosynthesis assay employing primary rat hepatocytes. Both assay systems demonstrated that the remIL-6 was active in the range of 10(8) units/mg, which is similar to that estimated for native cytokine. Antibodies raised in rabbits against remIL-6 neutralized the biological activity of both recombinant and native IL-6.

Animals↗
PubMed1991

Identification and characterization of a C-terminally extended form of recombinant murine IL-6.

Murine interleukin-6 (mIL-6) was expressed in Escherichia coli in the insoluble fraction of cell lysates. Approximately equal amounts of two polypeptide species, reactive with anti-IL-6 antibodies, were produced. The two forms of mIL-6 were isolated and found to have identical N-terminal sequences initiated by Met-Phe-Pro-Thr-Ser-Gln-. Peptide mapping after endoproteinase glu-C digestion led to isolation and characterization of the C-terminal peptides from each of the two forms and allowed the source of the heterogeneity to be identified as a C-terminal addition of three amino acids, Gln-Lys-Leu, to authentic mIL-6. Inspection of the nucleotide sequence of the plasmid containing the mIL-6 gene and expression of the plasmid in other strains suggested that the addition of three amino acids was caused by a readthrough of the termination codon arising from an unexpected suppressor mutation in the original host strain. Although the C-terminus of IL-6 is critical for the activity of this cytokine, the IL-6 variant with extended C-terminus was fully active in two separate bioassays. This suggests that the additional amino acids do not disrupt the structure or function of this important region of the molecule.

Amino Acid Sequence↗
PubMed1991

Transcription and translation are required for fibrinogen mRNA degradation in hepatocytes.

Fibrinogen synthesis increases significantly during the early stages of an inflammatory reaction. In this study, we analysed quantitatively the fate of each fibrinogen transcript in primary rat hepatocytes during and following stimulation with interleukin-6 (IL-6). Northern blot hybridization analysis demonstrated a coordinated increase in the levels of fibrinogen mRNAs within 30 min following addition of IL-6. The half-life for each fibrinogen mRNA species was determined to be 8 h, and the decline in the level of all three fibrinogen transcripts occurred in a tightly coordinated fashion. When inhibitors of transcription (actinomycin-D) or translation (cycloheximide) were added following a maximal induction of fibrinogen mRNA expression by IL-6, the decay of mRNA was significantly diminished. Furthermore, the addition of cycloheximide (CHX) to hepatocytes increased fibrinogen mRNA levels, but only if the cells had been stimulated with IL-6. These data suggest that lability of the fibrinogen mRNAs may be due, in part, to the presence of a specific short-lived protein(s) that enhances their degradation. Constant exposure to IL-6 was required for the continual increase in expression of the fibrinogen mRNAs. Taken together, these results provide evidence that the turnover of fibrinogen mRNAs is stringently coordinated, and involves specific regulatory molecules yet to be characterized.

Animals↗
PubMed1991

The putative role of fibrin fragments in the biosynthesis of fibrinogen by hepatoma cells.

The current study reinvestigated whether pure plasminolytic fragments D and E from fibrin directly increase fibrinogen synthesized by the hepatocyte. Fibrinogen protein levels and fibrinogen mRNA levels in an interleukin-6 (IL-6) responsive rat hepatoma cell line (FAZA) were measured quantitatively by [35S]-methionine pulse-chase experiments and Northern hybridizations, respectively. The results demonstrate that neither fragments D nor E, alone or in combination, in the presence or absence of dexamethasone, had any influence on the production of fibrinogen.

Animals↗
PubMed1990

The role of carbohydrate in the function of human plasminogen: comparison of the protein obtained from molecular cloning and expression in Escherichia coli and COS cells.

A cDNA library was constructed in the phage lambda gt11 from human liver mRNA enriched for plasminogen mRNA by chromatography on Sepharose 4B. A full-length cDNA clone of human plasminogen was isolated. The 2.7 kb cDNA encoded the entire plasminogen molecule, a signal peptide sequence and two start codons with a 5'-untranslated region of about 80 base pairs. In the 3'-non coding region of 280 base pairs a consensus signal AATAAA was found at a distance of 46 base pairs upstream of the poly(A) tail. The plasminogen cDNA was subcloned in the eukaryotic expression vector p91023 (B), and human plasminogen was expressed in monkey kidney (COS m6) cells and in Escherichia coli. The recombinant molecule obtained from COS cells has physicochemical and biological properties similar to native human plasminogen I, indicating that it has folded in a manner similar to plasminogen synthesized by liver. By contrast, plasminogen expressed in E. coli could not be activated and showed biological properties which are very different from glycosylated forms of plasminogen. However, the non-glycosylated plasminogen was bound by lysine-Sepharose and reacted with a conformation dependent monoclonal antibody to kringles 1 to 3. These data suggest that the protein has properly folded kringle domains. Our studies suggest that the carbohydrate domains may play an important role in the function of the plasminogen molecule.

Animals↗
PubMed1990

Induction and regulation of interleukin-6 gene expression in rat astrocytes.

Cells that produce interleukin-6 (IL-6) require the presence of signaling molecules since this cytokine is not normally constitutively expressed. It is now established that astrocytes produce IL-6; however, the precise inducing molecules and the kinetics of their action have not yet been clearly identified. In the current study, we show that either interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha) exert a strong inducing signal for IL-6 in primary rat astrocytes. When the two cytokines are added together the response is synergistic, suggesting that each cytokine may induce IL-6 gene expression by different pathways. Interferon-gamma (IFN-gamma) does not affect IL-6 expression although if it is added in conjunction with IL-1 beta, an augmented induction of IL-6 occurs. In addition to the cytokines, bacterial lipopolysaccharide (LPS) and the calcium ionophore, A23187, induce IL-6 expression. IL-6 expression can be blocked by the glucocorticoid analogue, dexamethasone. IL-6 induction by LPS/Ca2+ ionophore is more sensitive to the suppressive effects of dexamethasone than is IL-6 induction by TNF-alpha/IL-1 beta. Cycloheximide (CHX), an inhibitor of protein synthesis, markedly increased levels of IL-6 mRNA in both unstimulated and stimulated astrocytes, indicating that ongoing protein synthesis is not required for astrocyte IL-6 gene expression. We propose that astrocyte-produced IL-6 may have a role in augmenting intracerebral immune responses in neurological diseases such as multiple sclerosis (MS), AIDS dementia complex (ADC), and viral infections. These diseases are characterized by infiltration of lymphoid and mononuclear cells into the central nervous system (CNS), and intrathecal production of immunoglobulins. IL-6 may act to promote terminal differentiation of B cells in the CNS, leading to immunoglobulin synthesis.

Animals↗
PubMed1988

Fibrinogen.

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Animals↗
PubMed1987

Regulation of angiotensinogen gene expression in a human hepatoma cell line.

Angiotensinogen is the precursor of biologically active peptide angiotensin II and its synthesis is increased in the liver during acute inflammation. We have used radiolabeled human angiotensinogen cDNA to study the effect of hepatocyte stimulating factor (HSF), a protein synthesized in differentiating monocytes which increases the synthesis of various hepatic proteins during inflammation, on angiotensinogen mRNA levels in human hepatoma cells (HepG2). Our results indicate that angiotensinogen mRNA is present in human hepatoma (HepG2) cells and its levels are decreased when treated with hepatocyte stimulating factor. Although dexamethasone elevated angiotensinogen mRNA levels, HSF reduced this increase. These results suggest that a factor other than HSF may be involved in elevating the angiotensinogen mRNA levels in the liver during inflammation.

Angiotensinogen↗
PubMed1987

Effects of hyaluronic acid and other glycosaminoglycans on fibrin polymer formation.

Previously, we reported that the glycosaminoglycan (GAG) hyaluronic acid (HA) specifically bound to the plasma protein fibrinogen [LeBoeuf, R. D., Raja, R. R., Fuller, G. M., & Weigel, P. H. (1986) J. Biol. Chem. 261, 12586]. The binding of other macromolecules to fibrinogen could influence the conversion of fibrinogen to fibrin. Therefore, we tested whether HA and other GAGs could alter the kinetics of fibrin polymer formation and the physical structure of the resulting gel. In this study, we present data showing that the GAGs HA and chondroitin sulfate (CS) affect fibrin formation in three specific ways: (i) they decreased the clotting time of fibrinogen 3-10-fold; (ii) both GAGs increase significantly the rate of fibrin polymer formation; and (iii) fibrin gels containing HA or CS had a final A450 that was greater than controls, indicating that these two glycosaminoglycans influence either the final size of fibrin fibrils or the extent of the lateral association between fibrils. These results demonstrate that the interactions of HA and CS with forming fibrin polymers can alter both the kinetics of formation and may produce structural changes in fibrin gels.

Animals↗