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G Fey

Publications and source records attributed to G Fey.

At least 19 recordsLinked to original sources
PubMed1993

Regulation of IL-6 and the hepatic IL-6 receptor in acute inflammation in vivo.

The serum levels of interleukin 6 (IL-6) and the expression (mRNA) of the 80 kDa IL-6 receptor (IL-6R) were examined in three different models of acute inflammation. Rats were treated with either Freund's complete adjuvant (FA) via intraperitoneal injection, LPS via intravenous injection, or turpentine via subcutaneous injection. Using bio- and specific immunoassays, rat serum levels of IL-6, corticosterone, and acute phase proteins were quantified. LPS treatment induced the quickest and greatest serum IL-6 response (> 100 ng/ml within 3 h). In comparison, sera from turpentine and FA-treated rats contained much lower levels of IL-6 activity (< 10 ng/ml). Serum corticosterone levels increased by 3 h after injection in all three models, and equivalent raised serum levels of acute phase proteins were detected within 12-24 h. The expression of IL-6 receptor mRNA in hepatocytes increased markedly as early as 3 h after treatment and message levels began to decline by 6-12 h in all three models. To analyze the individual effects of raised corticosterone and IL-6 on the expression of hepatic IL-6R mRNA, rats were injected with either dexamethasone (Dex) or purified recombinant rat IL-6 (rIL-6) via intraperitoneal injection. Rats injected with rIL-6 showed highly induced IL-6R mRNA levels as early as 1 h after injection, and Dex-injected rats showed a significant but less dramatic rise in IL-6R message levels. Dex- or rIL-6-injected rats demonstrated a distinct profile of acute phase protein response different from that seen in the three experimental models. Regulation of IL-6R gene expression in the liver in vivo depends on a complex interaction between the hepatocyte and a combination of cytokines and other hormones.

Acute Disease↗
PubMed1992

Tumor necrosis factor alpha negatively regulates hepatitis B virus gene expression in transgenic mice.

It is well known that several inflammatory cytokines can modulate hepatocellular gene expression in a complex physiological process known as the hepatic acute-phase response. Since hepatitis B virus (HBV) characteristically induces a vigorous lymphomononuclear inflammatory response in the liver during acute and chronic hepatitis, it is possible that hepatocellular HBV gene expression may also be modulated by one or more of the cytokines produced by these cells. Using bacterial lipopolysaccharide (LPS) as a surrogate inducer of inflammatory cytokines in vivo, we have tested this hypothesis in a transgenic mouse model system. In experiments with two independent transgenic mouse lineages that express the HBV envelope region under the control of either HBV or cellular promoters, we observed a 50 to 80% reduction in the hepatic steady-state content of a 2.1-kb HBV mRNA following administration of a single intraperitoneal dose of LPS. The regulatory influence of several inflammatory cytokines known to be induced by LPS was also examined in this system. The negative regulatory effect of LPS was consistently reproduced by the administration of a single nontoxic dose of tumor necrosis factor alpha, and it was occasionally observed following the administration of high doses of alpha interferon and interleukin-6, while no effect was detectable in response to high-dose interleukin-1 alpha or to gamma interferon. These observations suggest that tumor necrosis factor alpha and perhaps other cytokines may activate a heretofore unsuspected intracellular pathway that negatively regulates HBV gene expression. The intracellular mechanism(s) responsible for this effect and its pathophysiologic relevance remain to be elucidated.

Acute-Phase Reaction↗
PubMed1989

Heterogeneous lobular distribution of hepatocytes expressing acute-phase genes during the acute inflammatory reaction.

Functional heterogeneity in the lobule with regard to plasma protein synthesis is still debated. Therefore, we have localized in liver sections from normal rats and from rats with turpentine-induced AIR the mRNA and protein products of three genes with different alterations in their hepatic expression during an AIR: alpha 2M and alpha 1PI, two positively reacting acute-phase genes, and alpha 1I3, a negative acute-phase reactant. In normal liver, all hepatocytes expressed alpha 2M and alpha 1I3 mRNA, but a preferential expression of alpha 2M and alpha 1I3 mRNA and protein in the PP and ML zones was observed. During an AIR, the level of alpha 2M mRNA increased fourfold in the cytoplasm of PP and ML hepatocytes, while the level of cytoplasmic alpha 1I3 mRNA was decreased about fourfold in the same zones, with parallel variations in the expression of the corresponding proteins. In contrast, no significant modulation of the RNA and protein concentrations of both genes was detected in PV areas. alpha 1PI mRNA was expressed at the same levels in the three lobular zones in normal liver, but staining for the alpha 1PI protein was more intense in the PV zones. During the acute-phase response alpha 1PI mRNA levels were increased twofold in all three lobular zones, and alpha 1PI staining became homogeneous within the lobule. These results demonstrate that the location of a hepatocyte with the liver lobule can influence the expression of the three genes under study both at pre- and post-translational levels, in basal conditions, as well as during modulation of their expression during the inflammatory reaction.

Acute Disease↗
PubMed1989

Complete intron/exon organization of DNA encoding the alpha' chain of human C3.

The third component of human complement (C3), a central molecule in both the classical and alternative pathways of complement, is comprised of two polypeptides, termed the alpha and beta chains. Activation of C3 cleaves the alpha chain into two fragments, C3a, an inflammatory peptide, and the alpha' chain which remains covalently linked to the beta chain. Proteolytic fragments derived from the alpha' chain during activation and regulation of complement play a significant role in host defense and regulation of the immune response. Two cosmid clones covering the alpha' chain region were used to characterize the structure of this portion of the C3 gene. The alpha' chain is encoded by 24 exons, which range in size from 52 to 213 base pairs (bp) with an average size of 115 bp. The splice donor sequence at the beginning of intron 12 has a rare sequence variant of GC instead of the usual GT sequence. Ten introns have been completely sequenced and were surprisingly short, ranging in size from 85 to 242 bp with an average of 140 bp. Other introns range in size from 250 bp to over 4 kilobases in length. The gene size for this portion of C3 is estimated to be 23-24 kilobases. Comparison of exon structure with protein domains and with peptide mapping studies demonstrates that several binding sites on C3 are encoded by single exons. These data support the hypothesis that individual exons can code for functional protein domains.

Base Sequence↗
PubMed1989

The gene for human complement component C9 mapped to chromosome 5 by polymerase chain reaction.

The gene for human complement component C9 has been mapped to chromosome 5. This was achieved by using a novel application of the polymerase chain reaction to amplify specifically the human C9 gene on a background of rodent DNA in somatic cell hybrids. The assignment to chromosome 5 was confirmed by in situ hybridization to human metaphase chromosomes, giving a regional localization of 5p13.

Animals↗
PubMed1985

Nucleotide sequence of cDNA encoding human alpha 2-macroglobulin and assignment of the chromosomal locus.

Six alpha 2-macroglobulin (alpha 2M) cDNA clones were isolated from a human liver cDNA library by using synthetic oligonucleotides as hybridization probes. One of these, p alpha 2M1, carries a 4.6 kilobase-pair insert, which was sequenced. The insert contains the coding sequences for the mature alpha 2M polypeptide (1451 amino acids) and for a 23-amino acid signal peptide at the NH2 terminus of the precursor pro-alpha 2M. At the 3' end of the insert a poly(A) addition signal A-A-T-A-A-A and part of the poly(A) tail of the messenger RNA were found. The protein sequence deduced from the nucleotide sequence agrees with the published alpha 2M amino acid sequence for all except three residues. The alpha 2M locus was assigned to human chromosome 12 by Southern blot analysis with DNA from a panel of mouse/human somatic cell hybrids, using alpha 2M cDNA as a hybridization probe.

Animals↗
PubMed1983

Structure and expression of the C3 gene.

To map the multiple interactive sites on the C3 polypeptide, it is advantageous to combine the approaches of protein chemistry, nucleic acid technology, and molecular biology. This review summarizes the currently known molecular properties of mouse liver C3 mRNA, cloned C3 cDNA, and genomic DNA. Original data communicated have specified the amino acid sequence of the 215 amino-terminal residues of mature mouse C3 beta. Southern blot analysis of liver DNA indicated that the mouse genome contains only one type of C3 gene, that murine and human C3 sequences strongly cross-hybridize, and that the human C3 gene is not somatically rearranged. Included are descriptions of the first human C3 genomic DNA clones, their preparation, and their use to map the human C3 gene to chromosome 19 in linkage with the myotonic dystrophy (DM) locus. After a brief survey of reports describing inherited human C3 deficiencies, we discuss a Dutch family and their three members with total homozygous C3 deficiency who were the subjects of a recent publication. The restricted synthesis of C3 in major and minor producer tissues is discussed and it is proposed that the C3 gene provides a good model system for studying the molecular basis of tissue-specific gene expression. Data are presented documenting the production of C3 in two established mouse macrophage-like cell lines and two rat hepatoma cell lines in tissue cultures. A short account covers the extensive literature on regulation of C3 serum concentrations in acute and chronic inflammation and the very incomplete picture that presently depicts hormonal regulation of C3 synthesis. The final experiment reported demonstrates that nucleic acid hybridization with cloned cDNA probes is a sensitive assay for quantitative determinations of C3 mRNA. With the help of cloned cDNA and genomic DNA, researchers can address questions concerning the functional topography of the C3 polypeptide, the gene's structure, and the molecular nature of inherited C3 deficiencies in humans.

Animals↗
PubMed1983

Linkage analysis of myotonic dystrophy and sequences on chromosome 19 using a cloned complement 3 gene probe.

Variations in DNA sequence generate polymorphisms which can be followed through families. A cloned gene specific probe for human complement 3 (C3) was hybridised to DNA samples digested with restriction endonucleases. The C3 probe detects several restriction fragment length polymorphisms (RFLPs) that occur frequently in the general population. These DNA alleles can be readily used in linkage analyses of loci on chromosome 19, since most families studied are informative. The inheritance of one such polymorphism was followed through myotonic dystrophy families. The segregation data for both the C3 protein polymorphism and the C3 RFLP support the linkage of myotonic dystrophy (DM) and C3.

Chromosome Mapping↗
PubMed1983

Exclusion of the C3 gene from the 19q133 to 19qter region by Southern analysis of human-rodent somatic cell hybrids, employing a cloned genomic C3 gene fragment.

By Southern analysis of human-rodent-somatic cell hybrids, the C3 gene could be excluded from the distal portion of the long arm of chromosome 19. In this study, a cloned genomic DNA fragment of the human C3 gene was employed as hybridisation probe. Since C3 is linked with the myotonic dystrophy (DM) and the Lewis (Le) locus, this exclusion may be relevant too for the regional assignment of these and other genes of the large chromosome 19 linkage group.

Animals↗
PubMed1982

Assignment of the structural gene for the third component of human complement to chromosome 19.

The third component of complement (C3) is synthesized and secreted by cultured human primary fibroblasts. A monoclonal antibody having specificity for an antigenic determinant carried by human but not mouse C3 was used to study the continued expression of human C3 in three panels of independently derived human-mouse somatic cell hybrids. Expression of the human product was shown to segregate with human chromosome 19 and with no other chromosome or group of chromosomes. A unique-sequence human genomic C3 DNA clone was isolated and used as a probe in DNA hybridization experiments with DNA prepared from appropriate human-mouse somatic cell hybrids to confirm assignment of the human C3 gene to chromosome 19.

Chromosome Mapping↗
PubMed1982

Isolation and analysis of genomic DNA clones encoding the third component of mouse complement.

A gene library was constructed with DNA from strain A mice by using the phage lambda vector lambda 1059. By screening with cloned cDNA for the third component of mouse complement, C3, four different C3 genomic clones were isolated from this library. Two of the recombinant phages carry insertions of 14 and 18 kilobase pairs, respectively, which together cover one complete copy of the C3 gene and several hundred nucleotides of its 5' and 3' flanking sequences. The distance from the 5' end of the gene, which includes the hexanucleotide T-A-T-A-A-A and a translation initiation codon, to its 3' end as defined by the poly(A) attachment site is 24 kilobase pairs. From the genomic DNA sequence, a signal peptide of 24 amino acid residues is predicted at the NH2 terminus of the initial translation product. The signal peptide and the next two amino acids are encoded by the first exon of this gene.

Animals↗
PubMed1982

Characterization of the mRNA and cloned cDNA specifying the third component of mouse complement.

Eighteen cDNA clones containing inserts specific for the third component of complement (C3) have been derived from high molecular weight mouse liver mRNA. The inserts span 4,600 nucleotides of the C3 coding sequence, including the 3' end of C3 mRNA. The length of C3 mRNA was determined to be 5,100 +/- 200 nucleotides, including a poly(A)-containing tail of mean length 170 nucleotides. From cDNA sequence analysis of the 5'-proximal region of C3 mRNA, the NH2-terminal amino acid sequence of the mature C3 beta chain was predicted to be Ile-Pro-Met-Tyr-Ser-Ile-Ile-Thr-Pro-Asn-Val-Leu-Arg-Leu-Glu. This sequence is in good agreement with the reported amino acid sequences of human and guinea pig C3 beta chains. These data position the C3 beta subunit to the NH2-terminal portion of the precursor C3 molecule (pro-C3) and establish the order of subunits in pro-C3 to be NH2-beta-alpha-COOH. In addition, the cDNA sequence indicates that an NH2-terminal extension peptide precedes the beta chain in pro-C3. The amino acid sequence of the mouse C3a fragment and its flanking regions was determined. The data indicate the presence of four arginine residues located between the COOH terminus of the C3 beta and the NH2 terminus of the C3 alpha subunits in pro-C3. The coding sequences of the amino acids that constitute the internal thioester domain in C3 were determined. Unexpectedly, the glutamyl residue that has been shown to participate in the thioester bond in native C3 was found to be encoded as a glutamine.

Animals↗
PubMed1981

Biosynthesis of complement components.

Many of the serum complement components are synthesized primarily in the liver but extrahepatic synthesis in mononuclear phagocytes has been noted. This local production of complement, particularly at sites of inflammation, may be of importance in host defenses and immunopathological reactions. Recent studies of complement biosynthesis in vitro have revealed some of the genetic and microenvironmental factors that affect the rates of complement synthesis, posttranslational modification, and secretion of these proteins. The third, fourth, and fifth complement components are synthesized as single chain, procomplement proteins that require proteolytic cleavage to generate the native C4 protein is mediated by a plasmin-like enzyme. These studies, plus the most recent developments in analysis of the complement genes at a molecular level, will provide more complete understanding the mechanisms of regulation of complement production.

Animals↗
PubMed1981

Mouse complement components C3 and C4. Characterization of their messenger RNA and molecular cloning of complementary DNA for C3.

Mouse liver mRNA species which direct the cell-free synthesis of pro-C3 and pro-C4 polypeptides with an apparent molecular weight of 175,000 and 190,000, respectively, were shown to sediment faster than 28 S. By electrophoresis in a denaturing gel the mRNA for C3 was determined to contain approximately 7,500 ribonucleotides. cDNA was synthesized from size-fractionated mouse liver mRNA and cloned in the plasmid pBR 322. Among the cDNA clones recovered three were identified as being complementary to portions of the mRNA for C3.

Animals↗