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Biomedical subjects

H R Colten

Publications and source records attributed to H R Colten.

At least 73 records · Page 4Linked to original sources
PubMed1987

Structure of a human serum amyloid A gene and modulation of its expression in transfected L cells.

The structure of a human serum amyloid A (SAA) genomic clone (SAAg9) has been analyzed and the nucleotide sequence of the coding regions is compared with that of the cDNA for apoSAA1. The leader and coding sequences of exons 2 and 3 are identical to SAA1. However, there are 10 nucleotide and 7 derived amino acid substitutions in exon 4. These changes are identical to the amino acid sequence of the amyloid protein associated with familial Mediterranean fever. In particular, the amino acid substitution (Thr to Phe) at residue 69 of SAA1 may have an important role in this type of hereditary amyloidosis. The genomic clone SAAg9 has been transfected into mouse L cells, and constitutive expression of human specific mRNA and protein were observed in stable transfected clones. The expression of both SAA mRNA and protein were increased by incubation of the transfected cells with purified human interleukin-1 (IL-1), both human and mouse recombinant IL-1, and recombinant human tumor necrosis factor alpha. The induction of SAA is pretranslational and is likely to be mediated by protein factor(s) since incubation with cycloheximide diminished IL-1-dependent increase in SAA mRNA.

Amino Acid Sequence↗
PubMed1987

Macrophage membrane interleukin 1 regulates the expression of acute phase proteins in human hepatoma Hep 3B cells.

To assess the potential role of membrane interleukin 1 (mIL-1) in modulating expression of acute phase proteins, we studied the effect of fixed mouse peritoneal macrophages and isolated cell membranes on the synthesis of C3 and albumin in human hepatoma Hep 3B cells. An increase in C3 synthesis and decrease in albumin synthesis were detected after incubation of Hep 3B cells with fixed stimulated macrophages or with membrane preparations. Estimates of hepatocellular C3 and albumin mRNA indicated that the mIL-1 regulation was exerted at a pretranslational level. The changes in C3 and albumin expression induced by mIL-1 were inhibited by addition of anti-interleukin 1 (IL-1) antiserum to the macrophages, supporting the hypothesis that a form of IL-1 is present on the outer cell membrane of macrophages. Moreover, cell-surface iodination of macrophages allowed the detection of a 33-kDa IL-1 molecule, suggesting that an IL-1 species, similar to the intracellular precursor of IL-1 is present at the outer cell surface of macrophages. The expression of mIL-1 was temporally dissociated from IL-1 release, indicating that the cell-surface associated IL-1 activity is not the result of adsorbed soluble IL-1. These studies provide a basis for further investigation of the role of mIL-1 in modulating monocytes and macrophages function via cell-cell interactions.

Acute-Phase Proteins↗
PubMed1987

Complementary DNA and derived amino acid sequence of the alpha subunit of human complement protein C8: evidence for the existence of a separate alpha subunit messenger RNA.

The entire amino acid sequence of the alpha subunit (Mr 64,000) of the eighth component of complement (C8) was determined by characterizing cDNA clones isolated from a human liver cDNA library. Two clones with overlapping inserts of net length 2.44 kilobases (kb) were isolated and found to contain the entire alpha coding region [1659 base pairs (bp)]. The 5' end consists of an untranslated region and a leader sequence of 30 amino acids. This sequence contains an apparent initiation Met, signal peptide, and propeptide which ends with an arginine-rich sequence that is characteristic of proteolytic processing sites found in the pro form of protein precursors. The 3' untranslated region contains two polyadenylation signals and a poly(A) sequence. RNA blot analysis of total cellular RNA from the human hepatoma cell line HepG2 revealed a message size of approximately 2.5 kb. Features of the 5' and 3' sequences and the message size suggest that a separate mRNA codes for alpha and argues against the occurrence of a single-chain precursor form of the disulfide-linked alpha-gamma subunit found in mature C8. Analysis of the derived amino acid sequence revealed several membrane surface seeking domains and a possible transmembrane domain. These occur in a cysteine-free region of the subunit and may constitute the structural basis for alpha interaction with target membranes. Analysis of the carbohydrate composition indicates 1 or 2 asparagine-linked but no O-linked oligosaccharide chains, a result consistent with predictions from the amino acid sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Transcriptional regulation of genes encoding the acute-phase proteins CRP, SAA, and C3.

Inflammation or acute tissue injury results in a programmed change in the concentration of several plasma proteins. Among these proteins, two--C-reactive protein (CRP) and serum amyloid A protein (SAA)--increase up to 1000-fold after an acute-phase stimulus in humans and rabbits. To determine the mechanism for regulation of acute-phase gene expression, we examined changes in the rates of transcription and specific hepatic mRNA content for rabbit CRP, SAA, and some complement protein mRNA during an acute-phase response. Induction of a sterile inflammatory reaction with intramuscular injection of turpentine resulted in an increase in the hepatocellular content of CRP, SAA, C3, and factor B mRNA and the transcription of CRP, SAA, and C3 genes. These data suggest that the increase in CRP, SAA, and C3 serum concentrations observed during an acute-phase reaction is due to an increase in biosynthesis and is, at least in part, under transcriptional control.

Acute-Phase Proteins↗
PubMed1987

A dysfunctional Mo1 glycoprotein is present on a subline of the KG1 acute myelogenous leukemia cell line.

Mo1 is a glycoprotein heterodimer found on the surface of phagocytic cells. By use of monoclonal antibodies directed against various epitopes of the 155 kd alpha-chain of Mo1, two distinct functions of this glycoprotein have been identified. Mo1 serves as the receptor (CR3) for iC3b, one of the breakdown products of the third component of complement, and in addition appears to be involved in promoting cell-cell adhesion of granulocytes. In studies performed with a subline of the acute myelogenous leukemia tumor cell line KG1, we found cells from this subline (KG1m) incapable of iC3b-mediated binding despite these cells having surface Mo1. Five distinct epitopes on Mo1 identifiable on normal cells by a panel of anti-Mo1 alpha-chain monoclonal antibodies (including OKM10, thought to be identical to or close to the iC3b binding site) were equally present on KG1m by indirect immunofluorescence. The electrophoretic mobilities of both the alpha- and beta-chains of Mo1 derived from KG1m were identical to those isolated from normal granulocytes. To determine whether altered receptor mobility or decreased surface density of Mo1 was responsible for the lack of Mo1-related functions, binding to EiC3b of isolated Mo1 derived from KG1m lysate was measured. KG1m-derived Mo1 did not bind to EiC3b when compared with normal granulocyte-derived Mo1, suggesting that the lack of iC3b binding is not secondary to decreased surface receptor number or mobility. This subline of KG1 provides an excellent model for the study of the relationship between surface receptor structure and function.

Antibodies, Monoclonal↗
PubMed1987

Constitutive and IL 1-regulated murine complement gene expression is strain and tissue specific.

To study the molecular mechanisms accounting for strain- and tissue-specific variations in the production of complement proteins, complementary DNA probes were used to assess qualitative and quantitative differences in specific mRNA content of complement proteins C2, factor B, and C3 in extracts of tissues (liver, lung, spleen, kidney, and peritoneal macrophages) isolated from various mouse strains. Northern blot analysis of total hepatic RNA revealed differences in C2, factor B, and C3 mRNA levels in strains that share B10 background but differ in the H-2 region (e.g., H-2k, H-2u, H-2d, H-2f). In each instance, hepatic mRNA specific for the individual gene product corresponded in amount to the serum levels. By contrast, specific mRNA content of C2 and factor B in macrophages differed significantly from those observed in liver for each strain. Modulation of C2, factor B, and C3 expression was studied after in vivo administration of recombinant IL 1 or endotoxin to H-2k (B10.AKM) or H-2u (B10.PL) strain mice. As assessed by Northern blot analysis, neither endotoxin nor IL 1 affected liver C2-specific mRNA but increased specific C2 mRNA levels in kidney and lung. For both strains, IL 1 increased specific factor B mRNA in all tissues examined except for the H-2u strain liver factor B mRNA content, which was not affected by IL 1, whereas that of H-2k mice was increased. The lack of factor B modulation by IL 1 in the H-2u lines was specific to that gene and not a reflection of a generalized IL 1 unresponsiveness. Differences in tissue and strain specific constitutive and IL 1-regulated expression of the C3 gene were also observed in the H-2u and H-2k strains.

Animals↗
PubMed1987

DNA polymorphism of MHC III genes in inbred and wild mouse strains.

Genes encoding the second component (C2), factor B, and complement protein C4 and Slp (sex-limited protein) are members of the major histocompatibility complex class III gene cluster. In this report we describe isolation of a mouse C2 cDNA clone and its use together with factor B and C4 cDNA clones to examine the S region in a panel of 42 haplotypes in laboratory and wild mice representing 5 species and subspecies of Mus. Conservation of the C2 factor B gene duplex was evidenced by relatively limited polymorphism associated with speciation and nucleotide sequence homology between mouse and human C2 and factor B. The C4-Slp gene duplex, on the other hand, showed extensive polymorphism by DNA blot analysis. This polymorphism correlated poorly with the C2/factor B restriction fragment length polymorphism, suggesting independent evolution of these two segments of the S region. Taken together, these data will be of particular importance in studies of mouse strains with abnormal regulation of immune effector systems since the class III gene products are essential for activation of the complement cascade.

Amino Acid Sequence↗
PubMed1986

Regulation of class III major histocompatibility complex gene products by interleukin-1.

Interleukin-1 (IL-1) is a product of mononuclear phagocytes that mediates changes characteristic of the response to inflammation or tissue injury (the acute-phase response). One of two structurally and functionally homologous major histocompatibility complex (MHC) class III genes encodes a positive acute-phase protein, complement factor B. The closely linked complement C2 gene is not affected during the acute-phase response. Purified human IL-1, pH 7.0, and recombinant-generated murine IL-1, pH 5.0, increased the expression of factor B and other positive acute-phase proteins in human hepatoma cells but decreased the expression of albumin, a negative acute-phase reactant. Furthermore, in a murine fibroblast L-cell line transfected with cosmid DNA bearing the human C2 and factor B genes, IL-1 mediated a reversible dose- and time-dependent increase in factor B expression in the transfected cells. Expression of the C2 gene was not affected by IL-1. The effect of IL-1 on factor B expression involves a mechanism acting at a pre-translational level as demonstrated by an increase in specific messenger RNA content and a corresponding increase in biosynthesis and secretion of factor B. The structural basis and mechanism for selective and independent regulation of these genes provides insight into the molecular control of the inflammatory response.

Animals↗
PubMed1986

Regulation of complement protein biosynthesis in mononuclear phagocytes.

Proteins of the complement system (with the exception of the terminal components C6-9) are synthesized in mononuclear phagocytes. The extrahepatic macrophage is therefore an important local source of the complement proteins which may serve as a first-line host defence mechanism. Net synthesis and secretion of complement by these cells is a function of maturation of the mononuclear phagocytic series, the tissue from which the cells are isolated, and the state of macrophage activation. To define some of the mechanisms for regulation of complement gene expression in mononuclear phagocytes, the major histocompatibility complex class III genes and C3 have been investigated. These genes are expressed constitutively in hepatocytes and monocytes/macrophages. In the mononuclear phagocyte, interferon-gamma, at physiological concentrations, effects a dose- and time-dependent increase in factor B and C2 mRNA and a corresponding increase in factor B and C2 biosynthesis. This effect is specific inasmuch as the expression of other genes (e.g. C3) is decreased by interferon-gamma, and interferon-alpha and beta at concentrations one to two logs greater have only a minimal effect on C2 and factor B gene expression. Endotoxin acting directly on monocytes has qualitatively different effects on expression of the complement genes. These complex regulatory mechanisms are being investigated with the use of murine fibroblasts transfected with human DNA bearing the relevent complement genes.

Animals↗
PubMed1986

The human C-reactive protein gene (CRP) and serum amyloid P component gene (APCS) are located on the proximal long arm of chromosome 1.

The genes encoding two pentraxins, C-reactive protein (CRP) and serum amyloid P component (SAP), are located on the proximal long arm of human chromosome 1. Mapping of the CRP and SAP genes between the centromere and band q32 was achieved by Southern blot analysis of DNA from a panel of human X Chinese hamster somatic cell hybrids carrying defined fragments of human chromosome 1. Both genes were localized more precisely between bands q12 and q23 by in situ hybridization to human metaphase chromosomes.

Animals↗
PubMed1986

Polymorphism of the human complement C4 and steroid 21-hydroxylase genes. Restriction fragment length polymorphisms revealing structural deletions, homoduplications, and size variants.

Several autoimmune disorders as well as congenital adrenal hyperplasia (CAH) are either associated or closely linked with genetic variants of the fourth component of complement (C4A and C4B) and the enzyme steroid 21-hydroxylase (21-OH). These proteins are encoded by genes that are located downstream from the genes for complement proteins, C2 and factor B (BF) between HLA-B and -DR in the major histocompatibility complex (MHC). Previous studies of variants and null alleles were based on electrophoretic mobility of C4 protein and linkage with disease phenotypes. These data did not permit analysis of the basis for the observed null alleles and duplicated variants. We studied this region of the MHC in 126 haplotypes for a structural analysis of the four adjacent loci, C4A, 21-OHA, C4B, and 21-OHB. About half of the C4 genes typed as C4 null are deleted and several unrecognized homoduplicated C4 alleles were detected. Hence the frequencies of different C4 structural variants must be recalculated based on a direct analysis of the genes. Analysis of the C4/21-OH genes of patients with the classical (salt-wasting) form of CAH showed that some involve a deletion of the C4B and 21-OHB genes; whereas for two only the 21-OHB gene is deleted, i.e., the C4B gene is present. Together, these data provide a better understanding of the mechanisms generating and importance of deleted C4 and 21-OH null alleles in human disease.

Adrenal Hyperplasia, Congenital↗
PubMed1986

Cachectin/tumor necrosis factor regulates hepatic acute-phase gene expression.

The monokine, cachectin/tumor necrosis factor (TNF) differs from interleukin 1 (IL-1) in primary structure and in recognition by a distinct cellular receptor. It does, however, encode effector functions that are similar to those of IL-1 and characteristic of the host response to inflammation or tissue injury. Accordingly, we examined the possibility that recombinant-generated human TNF regulates hepatic acute-phase gene expression. In picomolar concentrations, TNF mediated reversible, dose- and time-dependent increases in biosynthesis of complement proteins factor B and C3, alpha 1 antichymotrypsin, and decreases in biosynthesis of albumin and transferrin in human hepatoma cell lines (Hep G2, Hep 3B). Biosynthesis of complement proteins C2 and C4, and alpha 1 proteinase inhibitor were not affected by TNF. TNF also increased factor B gene expression, but had no effect on C2 gene expression, in murine fibroblasts transfected with cosmid DNA bearing the human C2 and factor B genes. The effect of TNF on acute-phase protein expression (C3, factor B, albumin) was pre-translational as shown by changes in specific messenger RNA content.

Acute-Phase Proteins↗
PubMed1986

Regulation of complement gene expression.

The availability of molecular probes for approximately half of the 20 known complement genes now permits a detailed examination of the regulation of complement expression in liver and at extrahepatic sites in tissue macrophages. Primary cell culture, cell lines and cells transfected with DNA bearing complement genes have been used in this analysis. Pretranslational regulation of complement production has been induced by well defined cytokines such as interleukin-1 and gamma-interferon, as well as directly by endotoxin. The effect of those agents on complement genes is tissue and species specific and is developmentally regulated. These data form the basis for the elucidation of the genomic structural requirements for regulation of inflammation and, by extension, specific immune responsiveness.

Animals↗