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H R Colten

Publications and source records attributed to H R Colten.

At least 19 recordsLinked to original sources
PubMed1992

Type I human complement C2 deficiency. A 28-base pair gene deletion causes skipping of exon 6 during RNA splicing.

Two variants of a genetic deficiency of complement protein C2 (C2D) have been previously identified. No C2 protein translation is detected in type I deficiency, while type II deficiency is characterized by a selective block in C2 secretion. Type I C2 deficiency was described in a family in which the C2 null allele (C2Q0) is associated with the major histocompatibility haplotype/complotype HLA-A25,B18,C2Q0,BfS,C4A4, C4B2,Drw2; this extended haplotype occurs in over 90% of C2-deficient individuals (common complotype/haplotype). To determine the molecular basis of type I C2 deficiency, the C2 gene and cDNA were characterized from a homozygous type I C2-deficient individual with the common associated haplotype/complotype. We found a 28-base pair deletion in the type I C2Q0 gene, beginning 9 base pairs upstream of the 3'-end of exon 6, that generates a C2 transcript with a complete deletion of exon 6 (134 base pair) and a premature termination codon. In studies of eight kindred, the 28-base pair deletion was observed in all C2Q0 alleles associated with the common type I deficient complotype/haplotype; this deletion was not present in normal C2 nor in type II C2-deficient genes. These data demonstrate that: 1) type I human complement C2 deficiency is caused by a 28-base pair genomic deletion that causes skipping of exon 6 during RNA splicing, resulting in generation of a premature termination codon, 2) the 28-base pair deletion in the type I C2Q0 gene is strongly associated with the HLA haplotype/complotype A25,B18,C2Q0,BfS,C4A4,C4B2,Drw2, suggesting that all C2-deficient individuals with this haplotype/complotype will harbor the 28-base pair C2 gene deletion, and 3) type II C2 deficiency is caused by a different, as yet uncharacterized, molecular genetic defect.

Amino Acid Sequence↗
PubMed1992

Cis- and trans-acting elements required for constitutive and cytokine-regulated expression of the mouse complement C3 gene.

The third component of complement (C3) is an important mediator of inflammation. Murine and human genomic cosmid clones were isolated, characterized and sequenced 5' to the complement C3 gene transcriptional initiation sites to determine cis elements that participate in constitutive and regulated C3 gene expression. The murine and human 5' flanking regions are 51% identical overall, with positions -36 to -1 and -146 to -68 showing 80% identity. Four TATA boxes were identified upstream of the murine transcriptional initiation site, but deletion and transfection analysis using reporter gene constructs in HepG2 cells indicated that only the TATA element at position -30, together with sequences -395 to -111, are essential for constitutive expression of murine C3 in hepatocytes. Deletion analysis also suggested that sequences between -1457 and -800 contain regulatory elements that are involved in suppressing basal expression. Sequences between -90 to -41 confer both enhancer activity and interleukin-1/-6 (IL-1/IL-6)-responsiveness. Mutation analyses showed that both sequences between -88 and -83 and -77 to -72 are essential for enhancer activity and responsiveness to IL-1, but only sequences between -88 and -83 are necessary for IL-6-responsiveness. A gel-retardation assay showed that several nucleoproteins, perhaps of the C/EBP family, from HepG2 cells bound to sequences between -88 to -83. Collectively, these results localize cis-acting elements involved in constitutive and IL-1/IL-6-regulated murine C3 gene expression and provide evidence for specific transacting factors.

Animals↗
PubMed1992

cis and trans elements differ among mouse strains with high and low extrahepatic complement factor B gene expression.

Factor B (Bf), an enzyme of the alternative pathway of complement activation, is one of four major histocompatibility complex (MHC) class III genes. To ascertain the genetic mechanism for tissue-specific constitutive and regulated expression of Bf, we sequenced the regulatory regions 5' of the gene from mice of different H-2 MHC haplotypes and assessed trans-acting factors, specific DNA binding nucleoproteins, in liver and kidney. Striking tissue-specific differences in constitutive expression of Bf were demonstrated in mice of H-2f or H-2z haplotypes when compared with H-2d or H-2u (kidney and intestinal Bf in H-2d or H-2u much greater than H-2f or H-2z). These differences correlated with a point nucleotide substitution 3 bp downstream of the upstream Bf initiation site that affects interaction with a DNA binding protein. This and additional cis differences localize the sequence substitutions responsible for previously identified restriction fragment length polymorphisms among inbred mouse strains and also reveal two previously unrecognized polymorphisms generated by SmaI and HinfI digestion. Evidence for differences in trans was found in a comparison of DNA binding nucleoproteins from kidney, but not liver, of B10.PL when compared with B10.M. These data, together with the high degree of sequence homology between human and mouse Bf 5' flanking regions, should prompt a search for polymorphic restriction sites and cis binding elements in the Bf promoter that could serve as markers of human MHC-associated renal pathology and variants in local MHC class III gene expression.

Animals↗
PubMed1992

Complement deficiencies.

The complement system consists of about two dozen plasma and cell membrane proteins which function as cofactors in defense against pathogenic microbes and in the generation of many immunopathogenic disorders. The complexity of this system and its role in other biological functions has been appreciated within the last two decades. Recognition of genetic deficiencies of the complement proteins and their phenotypic expression has provided additional insights into the physiological role of the complement system. Complementary DNA (cDNA) clones for most of the complement components are now available, and the gene structures for many have been elucidated. Application of molecular biological methods to studies of the complement system and its deficiencies has permitted both the determination of primary structure and chromosomal localization of complement genes (Figure 1) and the capacity to elucidate the molecular basis of complement deficiency disorders.

Chromosome Mapping↗
PubMed1992

Tissue-specific regulation of inflammation.

Proteins of the complement system are important effectors and modulators of inflammation. The complement cascade is triggered by microbes, tissue debris, and specific antibodies. Serum complement proteins are derived primarily from liver, but extrahepatic complement synthesis is important in homeostasis and in local host defenses. Tissue-specific regulation of expression of complement genes is governed by mechanisms similar to those that regulate other "acute phase reactants." That is, tissue injury or infection elicit changes in expression of these acute phase proteins, which, although variable in kinetics, magnitude, and direction, are a consequence of an elaborate system of cell-to-cell communication. This communication is mediated via a complex network of cytokines, including the interferons, interleukins, several growth factors, and sex hormones. The cell biological and molecular biological details of these mechanisms are now under active investigation. An understanding in molecular terms of the balance between proinflammatory and counterregulatory forces on complement gene expression should provide new insight into the functions of complement and the design of novel therapies for disorders of inflammation.

Acute-Phase Proteins↗
PubMed1990

Murine complement C2 and factor B genomic and cDNA cloning reveals different mechanisms for multiple transcripts of C2 and B.

Murine genomic and cDNA clones were isolated to ascertain the mechanisms accounting for previously recognized multiple forms of complement C2 and factor B mRNA and to analyze structural similarities with the corresponding human gene products (C2, 74% and B, 85%, amino acid identity). Like the human Bf gene, murine Bf and C2 each consist of 18 exons with similar intron-exon organizations. The murine C2 gene (20 kilobases) is more than three times the size of Bf (6 kilobases) due to the presence of large intronic segments separating the exons encoding the NH2-terminal binding and central (von Willebrand factor) domains. Evidence from cDNA clones shows that the two C2 transcripts are generated by an alternative splice at the donor site of exon 14 producing long and short C2 mRNA species that differ by 21 base pairs encoding a region within the binding pocket (amino acids 636-642 (GSTCKDH)) of the serine proteinase domain. Tissue-specific multiple factor B transcripts are generated by alternative transcriptional initiation. From the structure of these transcripts the predicted regulation of expression and rates of translation of B programmed by the short and long mRNA species may differ, but the polypeptides are identical. These data indicate that different molecular mechanisms account for the multiple forms of C2 and factor B mRNA and that the structure of the different transcripts predicts differences in function and expression of the respective gene products.

Amino Acid Sequence↗
PubMed1990

Counterregulatory effects of interferon-gamma and endotoxin on expression of the human C4 genes.

Susceptibility to autoimmune disease is associated with null alleles at one of the two genetic loci encoding complement protein C4. These two genetic loci, C4A and C4B, are highly homologous in primary structure but encode proteins with different functional activities. Expression of C4A and C4B genes is regulated by IFN-gamma in human hepatoma cells and in murine fibroblasts transformed with the respective genes. In these cell lines, IFN-gamma has a significantly greater and longer-lasting effect on expression of C4A than that of C4B. In this study we examined synthesis and regulation of C4A and C4B in peripheral blood monocytes from normal, C4A-null, and C4B-null individuals. Synthesis of C4 in human peripheral blood monocytes decreases during time in culture. IFN-gamma mediates a concentration- and time-dependent increase in steady-state levels of C4 mRNA and a corresponding increase in synthesis of C4 in normal human monocytes. LPS decreases monocyte C4 expression and completely abrogates the effect of IFN-gamma on the expression of this gene. In contrast, LPS and IFN-gamma have a synergistic effect in upregulating expression of another class III MHC gene product, complement protein factor B. The effect of LPS on constitutive and IFN-gamma-regulated C4 synthesis is probably not mediated via release of endogenous monokines IL-1 beta, TNF-alpha, or IL-6. Synthesis of C4, and regulation of its synthesis by IFN-gamma and LPS, are similar in normal, C4A-, and C4B-null individuals. These results demonstrate the synthesis of C4 at extrahepatic sites and tissue-specific regulation of C4 gene expression.

Complement C4↗
PubMed1990

Molecular basis of complement C3 deficiency in guinea pigs.

In experiments to ascertain the biochemical basis of a genetically determined deficiency of the third component of complement (C3) in guinea pigs, we found that C3-deficient liver and peritoneal macrophages contain C3 messenger RNA of normal size (approximately 5 kb) and amounts, that this mRNA programs synthesis of pro-C3 in oocytes primed with liver RNA and in primary macrophage cultures. In each instance, heterodimeric native C3 protein was secreted with normal kinetics but the C3 protein product of the deficient cells failed to undergo autolytic cleavage and was unusually susceptible to proteolysis. These data and a selective failure of C3 in plasma of deficient animals to incorporate [14C]methylamine suggested either a mutation in primary structure of the C3 protein or a selective defect in co- or postsynthetic processing affecting the thiolester bridge, a structure important for C3 function. A mutation in the primary structure of C3 was ruled out by comparison of direct sequence analysis of C3 cDNA generated from two C3 deficient and two C3 sufficient guinea pig liver libraries. Three base pair differences, none resulting in derived amino acid sequence differences were identified. Finally, restriction fragment length polymorphisms were identified in the C3 gene that are independent of the deficiency phenotype. This marker of the C3 gene permits testing of these hypotheses using molecular biological and classical genetic methods.

Amino Acid Sequence↗
PubMed1990

Complement gene expression in hepatic and extrahepatic tissues of NZB and NZB x W (F1) mouse strains.

To study the role of local production of complement proteins during the evolution of a naturally occurring immune complex disease, C3, C4, C2 and Factor B mRNA expression was assessed in several tissues of the inbred mouse strains NZB and (NZB x W) F1 hybrid. In the NZB/W F1 hybrid strain, coincident with the development of glomerulonephritis a marked increase in kidney C3 and C4 mRNA was observed; Factor B mRNA, which is expressed as a doublet in kidney and intestine, showed an increase in expression of the smaller transcript. This alteration of kidney C3, C4 and Factor B mRNA is identical to that noted in association with lupus nephritis in the MRL lpr/lpr strain and following in vivo administration of endotoxin to the BALB/c strain. The development of systemic lupus erythematosis (SLE) in the NZB/W F1 was not associated with a marked change in hepatic complement gene expression. These findings support the hypothesis that local production of complement may play a role in the pathogenesis of glomerulonephritis and other tissue injury in SLE.

Animals↗
PubMed1979

Genetic defect in secretion of complement C5 in mice.

A genetic deficiency of the fifth (C5) component of complement1-3, a serum glycoprotein of molecular weight (MW) 220,000 (ref. 4), has been found in 39% of inbred strains of mice3. Sera of deficient mice lack detectable C5 activity and protein2,3. In addition deficient mice produce antibody to mouse C5 when injected with sera from C5 sufficient (normal) strains. Levy et al.5 showed that somatic cell hybrids between C5 deficient (B10.D2/old line) macrophages and either C5 sufficient (B10.D2/new line) mouse kidney or chicken erythroblasts secreted haemolytically active mouse C5 in vitro. Several possible molecular mechanisms to account for the findings were considered, but insufficient direct data were available to choose among them. We recently reported that mouse (CD.1 strain) peritoneal cells in culture synthesise and secrete a single chain precursor, pro-C5 (MW approximately 210,000), of the two-chain (alpha chain, 125,000 and beta chain 83,000 MW) C5 protein6. Radiolabelled precursor C5 was contained within the cells and was secreted into the tissue culture media. Using similar methods, we now find that C5 deficiency in each of five different mouse strains (AKR, SWR, DBA/2J8 A/HeJ and B10.D2/old line) is due to a failure in secretion of C5 protein and not to a failure in biosynthesis of pro-C5.

Animals↗
PubMed1979

Cutaneous necrotizing venulitis in patients with cystic fibrosis.

Palpable purpura was noted to occur late in the course of some patients with cystic fibrosis. Skin biopsy specimens showed necrotizing venulitis characterized by a perivenular infiltrate composed of neutrophilic leukocytes, fibrin, hypogranulated mast cells, and endothelial cell necrosis. Circulating immune complexes were detected. Recurrent pulmonary infections and the chronic administration of therapeutic agents provide sources of potential antigens.

Adolescent↗
PubMed1979

Complement-dependent histaminase release from human granulocytes.

The role of particle-bound complement proteins in the induction of noncytotoxic enzyme release from human granulocytes was investigated with the use of sera genetically deficient in complement and highly purified complement components. Release of histaminase, one of two important histamine catabolizing enzymes, and beta-glucuronidase from polymorphonuclear leukocytes was solely dependent on particle-bound C3b (the larger cleavage product of the third component of complement) when fluid-phase complement was excluded. The extent of enzyme release was a function of particle-bound C3b input, was reduced by exposing the particles to C3b inactivator, and was blocked by fluid-phase C3b. Phagocytosis of the C3b-coated particles was not required for enzyme release from neutrophils. In contrast, phagocytosis of "opsonized" particles was required for noncytotoxic release of histaminase and arylsulfatase from eosinophils; other proteins, as well as C3b, were able to opsonize particles for induction of enzyme release from eosinophils. These studies suggest a dual role for complement (particularly C3) in modulating vascular permeability phenomena, i.e., release of vasoactive mediators by the action of C3a and C5a, and release of the corresponding enzymes that inactivate the mediators by C3b.

Amine Oxidase (Copper-Containing)↗
PubMed1979

Cyclophosphamide and cortisone acetate inhibit complement biosynthesis by guinea pig bronchoalveolar macrophages.

To explore mechanisms of drug-induced alterations in local host defenses in the lung, the capacity of guinea pig alveolar macrophages to synthesize the second (C2) and fourth (C4) components of complement was studied following treatment with cyclophosphamide and cortisone acetate. Administration of either drug significantly inhibited biosynthesis of C2 and C4 within 1 day. After 1 week of treatment, local complement synthesis was inhibited approximately 80%, although serum levels of the corresponding proteins were normal.

Animals↗
PubMed1979

Biosynthesis and post-synthetic modification of a precursor (pro-C5) of the fifth component of mouse complement (C5).

Mouse peritoneal macrophages synthesized and secreted a precursor (pro-C5) of the fifth component of serum complement (C5) in short-term tissue culture. Approximately 0.2% of the newly synthesized intracellular protein and 0.8% of secreted protein were precipitable with antiserum to mouse C5. The precursor is similar in size to the native serum protein (210,000 daltons), but consists of a single polypeptide chain. In contrast, serum C5 consists of two polypeptide chains (m.w. 125,000 and 83,000) linked by disulfide bridges. An electrophoretic variant of pro-C5 distinct from intracellular C5 was detected in medium from macrophage cultures.

Animals↗