Search PubMed⌕ Search

Biomedical subjects

H R Colten

Publications and source records attributed to H R Colten.

At least 109 records · Page 6Linked to original sources
PubMed1984

Expression of the MHC class III genes.

The second (C2) and fourth (C4) components of complement and factor B (B) are coded for by genes within the major histocompatibility complex (MHC). These proteins are synthesized in liver and in extrahepatic mononuclear phagocytes. The isolation of complementary DNA probes corresponding to each of these proteins now permits analysis of the molecular mechanisms controlling expression of the class III MHC genes. Genetic control of C4 gene expression has been examined in two model systems. A defect in post transcriptional processing of C4-specific RNA accounts for a failure to generate mature C4 mRNA in homozygous deficients of a C4 deficient guinea-pig strain. On the other hand, a quantitative difference in the amounts of mature C4 liver mRNA accounts for the genetic variation in C4 levels observed among several mouse strains. The maturation of monocytes to macrophages results in changes in biosynthesis of the MHC class III products; for example, a significant increase in rate of secretion of C2 and B is noted in human monocytes during the first 3 d in culture and the proportion of C2-producing cells is greater in freshly isolated macrophages than in monocytes. Macrophages demonstrate selective increases in factor B and C2 mRNA characteristic of specific tissues. In the guinea-pig macrophage, C4 gene expression is regulated by a selective feedback mechanism induced by extracellular native C4. The C4 binds to the macrophage cell surface mediating a change in transcription or, less likely, a change in stability of C4 mRNA. Regulation of C4 synthesis in the mouse macrophage is accomplished by mechanisms that are independent of this feedback control but the murine cells also display separate mechanisms for regulation of C4 and factor B-specific mRNA levels. Resident and elicited macrophages from either mouse or guinea-pig differ with respect to expression of the class III MHC gene products. These studies form the basis for evaluating the molecular regulation of inflammation, maturation of mononuclear phagocytes and the genetic variants and deficiencies of complement proteins.

Animals↗
PubMed1984

Distinct primary translation products from human liver mRNA give rise to secreted and cell-associated forms of complement protein C2.

The second component of complement (C2), is a class III major histocompatibility complex gene product and a glycoprotein in the classical complement activating system. Synthesis in the human hepatoma-derived cell line HepG2 results in three intracellular forms: an 84-kDa form secreted in 1-2 h; 79-kDa and 70-kDa forms that remain cell-associated for intervals up to 12 h. All three forms are C2 polypeptides as demonstrated by inhibition of immunoprecipitation with unlabeled C2 and the presence of common major peptide fragments following chymotryptic digestion. The cell-associated forms of C2 are not products of proteolysis as demonstrated by experiments with multiple proteinase inhibitors and by observations of the kinetics of synthesis. Inhibition of core glycosylation by tunicamycin and deglycosylation by acid hydrolysis indicate that the three intracellular C2 polypeptides are glycosylated to a similar extent. Although the 84-kDa form of C2 is susceptible to C1s cleavage, the two cell-associated forms are not. Cell-free biosynthesis by mRNA from HepG2 or human liver results in three primary translation products corresponding to the three unglycosylated forms of C2. These results indicate that HepG2 cells synthesize C2 protein in both secreted and cell-associated forms and that each form is derived from a separate primary translation product.

Carcinoma, Hepatocellular↗
PubMed1984

Molecular mechanism for feedback regulation of C4 biosynthesis in guinea pig peritoneal macrophage.

Previous reports have shown that regulation of local extrahepatic production of complement may not reflect the regulation of plasma concentrations of the corresponding proteins and, further, that alteration of the tissue microenvironment can affect local macrophage protein synthesis. This report describes the molecular basis for control of the biosynthesis and secretion of a class III major histocompatibility complex gene product, the fourth component of complement (C4), from guinea pig macrophages by extracellular native C4 protein. The effect is specific for C4 synthesis, since production of C2 and total secreted protein was unaffected by fluid phase C4. C4 synthesis by extracellular C4 is regulated at a pretranslational level, without an effect on posttranslational proteolytic cleavage, glycosylation, or secretion. Specific C4 and factor B cDNA probes were used to demonstrate, by dot hybridization and Northern blot analysis, a decrease in messenger RNA coding for C4 that paralleled the inhibition of C4 biosynthesis, while the amount of total RNA and mRNA specific for factor B remained constant. Inhibition of C4 biosynthesis and the disappearance of mRNA encoding C4 occurred between 4 and 6 h after exposure of the macrophages to biologically active or methylamine-inactivated C4 protein. These data demonstrate that regulation of C4 biosynthesis by guinea pig macrophages serves as a model for the study of the molecular mechanisms of macrophage activation as well as the control of production of a component of the inflammatory response.

Animals↗
PubMed1984

Biosynthesis and postsynthetic processing of human C3b/C4b inactivator (factor I) in three hepatoma cell lines.

Human factor I is a two-chain plasma glycoprotein composed of disulfide-linked 50,000- and 38,000-dalton subunits. Analysis of its biosynthesis and postsynthetic processing demonstrated that factor I is synthesized as a single chain precursor (pro-I) that undergoes glycosylation and limited proteolysis to generate the native protein. One of three human hepatoma cell lines, HepG2 , secreted factor I predominantly (70-90%) in a single chain pro-I form. The other cell lines secrete factor I predominantly in its two chain native form. The defect in conversion of pro-I to I in HepG2 was protein specific since other multichain proteins, derived from single chain precursors, the third, fourth, and fifth components of complement were processed normally. Further analysis of the inefficient pro-I to I conversion by HepG2 revealed that Xenopus oocytes injected with HepG2 mRNA secreted factor I in a predominantly two-chain form. In addition, the apparent sizes of native factor I, transferrin, and alpha-1-antitrypsin secreted by the three hepatoma lines differed due to differences in postsynthetic processing.

Animals↗
PubMed1984

DNA polymorphism of the C4 genes. A new marker for analysis of the major histocompatibility complex.

Polymorphisms of the proteins encoded by genes that lie within the major histocompatibility complex (MHC) have served as useful markers for organ transplantation and in genetic analysis of a large number of MHC-linked diseases. To extend the range of MHC polymorphic markers, we used a complementary-DNA probe specific for the fourth component of human complement (C4) to identify a new variant within the MHC. Polymorphic variants at the DNA level were detected among subjects with identical phenotypes of the corresponding protein. C4 genomic polymorphisms are inherited with the segment of the short arm of chromosome 6 that carries the HLA-DR and complement loci. The autosomal codominant mode of inheritance of this genetic marker and its utility for evaluation of 21-hydroxylase-deficiency congenital adrenal hyperplasia, one of the many MHC-linked diseases, were established.

Adrenal Hyperplasia, Congenital↗
PubMed1984

Subdivision of the S region of the mouse major histocompatibility complex by identification of genomic polymorphisms of the class III genes.

The S region of the mouse major histocompatibility complex (MHC) encodes the class III proteins, the second (C2) and fourth (C4) components of complement, and factor B. Previously, the assignment of S-region haplotypes was based on analysis of protein polymorphisms. The recent availability of C2, C4, and factor B cDNA probes prompted a search for restriction fragment length polymorphisms which would serve as additional genetic markers for these loci. DNA was isolated from livers of mice of all standard inbred H-2 haplotypes and of haplotypes pz and bs. These DNA samples were digested with restriction endonucleases and analyzed by Southern blot. By the pattern of restriction fragment length polymorphism observed, specific markers have been identified in factor B of haplotypes f, u, z, bs, r, and v, and in C4 of haplotypes b,q,f,j,p,s,pz,r, and v. These genetic markers were used in the analysis of S-region composition in strains B10.TFR5(H-2ap5) and C3H.LG(H-2dx), and a possible intra-S-region recombinant was revealed in the H-2dx haplotype. The genetic markers identified here subdivide the S region and will be of value in defining further the composition of the complement gene complex of the mouse MHC.

Alleles↗
PubMed1984

Expression of hemolytically active murine fourth component of complement in transfected L cells.

The S region of the murine major histocompatibility complex contains two related genes (C4-X and C4-Y), one of which encodes the fourth component of complement (C4). We have introduced cloned C4-X and C4-Y genes into L cells. C4-X transfectants contained no detectable C4-related RNA, whereas C4-Y transfectants contained large amounts of C4-related RNA. Culture supernatants of C4-Y transfectants contained processed C4 which was indistinguishable from macrophage-derived C4. The L-cell-derived C4 restored hemolytic activity to C4-deficient serum, served as a substrate for C1s, and underwent autolytic cleavage suggesting the presence of an intramolecular thiolester bond. These experiments identify C4-Y as the functional C4 gene in H-2d mice and indicate that L cells can process pro-C4 correctly.

Amino Acid Sequence↗
PubMed1984

Complement C3 receptors: structure and function.

Significant progress has been made during the past few years toward elucidating the structure and function of various C3 receptors. Better understanding of the biochemical basis of the immune adherence phenomenon has been achieved. The availability of polyclonal and monoclonal antibodies to C3 receptors allowed more accurate studies of the cellular distribution of these receptors. Finally, advances in our knowledge of the functional properties of the C3 receptors and their relationship to specific cell surface molecules was facilitated by recognition of a patient deficient in various receptor-mediated functions. All together these developments will soon make it possible to describe in detail the molecular events involved in the modulation of complement receptor coupled cell functions.

Animals↗
PubMed1984

Progression of cystic fibrosis lung disease as a function of serum immunoglobulin G levels: a 5-year longitudinal study.

Seventy children with cystic fibrosis were studied over a 5-year period to assess the relationship between serum immunoglobulin G levels and progression of cystic fibrosis lung disease. Patients were grouped according to their serum IgG values (low, normal, or high) and evaluated with serial pulmonary function testing, radiographic and immunologic studies, and clinical observation. The children with persistent hypogammaglobulinemia G showed significantly better lung function, better weight for age, fewer hospitalizations for pulmonary exacerbations, less colonization with Pseudomonas aeruginosa, and slower decline in pulmonary functions than did age-matched patients with normal or high IgG levels. Death occurred in five of eight (63%) patients with hypergammaglobulinemia, three of 30 (10%) with normogammaglobulinemia, and one of 32 (3%) with hypogammaglobulinemia. No deaths occurred in the 15 patients with persistent hypogammaglobulinemia. These data indicate that children with cystic fibrosis and hypogammaglobulinemia G have milder lung disease and slower deterioration in pulmonary function than do age-matched patients with normal or elevated immunoglobulin G values. The mechanisms accounting for this finding are unclear.

Agammaglobulinemia↗
PubMed1984

Synthesis of complement by guinea pig bronchoalveolar macrophages. Effect of acute and chronic infection with Pseudomonas aeruginosa.

In order to assess the potential role of local production of complement in pulmonary host defenses against bacterial infection, this aspect of bronchoalveolar macrophage function was studied in guinea pigs challenged with Pseudomonas aeruginosa in an acute and chronic infection model. Acute infection resulted in an increase in bronchoalveolar macrophage cell number and an increase in synthesis and secretion rates for the second (C2) and fourth (C4) complement components per macrophage. Manipulation of the airway without introduction of Pseudomonas also increased synthesis of both C2 and C4 when studied 60 h after control solutions were administered. Pseudomonas aeruginosa delivered in agar beads to induce chronic inflammation resulted in specific stimulation of C2 and C4 synthesis at 2 wk and to a lesser extent at 4 wk postchallenge. This increase in local complement synthesis by bronchoalveolar macrophages, in addition to enhancing the local inflammatory response, may serve to facilitate recruitment of intravascular cellular and humoral mediators of host defense against bacterial infection.

Animals↗
PubMed1984

Isolation of a complementary DNA clone for the human complement protein C2 and its use in the identification of a restriction fragment length polymorphism.

Complementary DNA (cDNA) clones corresponding to the major histocompatibility (MHC) class III antigen, complement protein C2, have been isolated from human liver cDNA libraries with the use of a complex mixture of synthetic oligonucleotides (17 mer) that contains 576 different oligonucleotide sequences. The C2 cDNA were used to identify a DNA restriction enzyme fragment length polymorphism that provides a genetic marker within the MHC that was not detectable at the protein level. An extensive search for genomic polymorphisms using a cDNA clone for another MHC class III gene, factor B, failed to reveal any DNA variants. The genomic variants detected with the C2 cDNA probe provide an additional genetic marker for analysis of MHC-linked diseases.

Amino Acid Sequence↗
PubMed1984

Molecular regulation of MHC class III (C4 and factor B) gene expression in mouse peritoneal macrophages.

To study the molecular regulation of C4 and factor B synthesis in mouse peritoneal macrophages, mouse C4 cDNA clones isolated from an H-2d haplotype liver cDNA library, and a previously described mouse factor B cDNA clone, pBmB2 (9), were used to assess quantitative and qualitative differences in C4 and factor B mRNA in resident and elicited cells. The C4 clones that were isolated, pBmS2 (1 Kb) and pBmS10 (0.9 Kb), overlap and together span a 1.5 Kb coding region of mouse pro-C4, extending from the alpha-chain through the gamma-chain; four nucleotide substitutions are evident in comparing 316 bp of the sequence of clone pBmS10 to that of a previously described mouse C4 clone, pMLC4/w7-2 (23). By using these probes, Northern blot analysis of total cellular RNA revealed similar C4 mRNA levels in resident peritoneal macrophages from high-C4 (B10.A) and low-C4 (C3HeB) strains. Pulse and pulse-chase studies of C4 and factor B synthesis were performed on resident, starch-elicited, and thioglycollate-elicited peritoneal macrophages at two culture time periods, 0 to 9 and 24 to 33 hr, and total cellular RNA was isolated from each population at 4.5 and 28.5 hr of culture for Northern blot analysis of C4 and factor B mRNA content. The data demonstrate that as previously reported, C4 production decreases in elicited compared with resident macrophages and decreases with time in culture; however, factor B synthesis does not differ among resident and elicited cells and it increases with time in culture. The variations in C4 and factor B production by mouse peritoneal macrophages are not associated with alterations in C4 and factor B protein processing, catabolism, or secretion; rather, they are a function of differences in net amounts of C4 and factor B mRNA. These data provide direct evidence that the regulation of expression of these class III MHC genes in mouse peritoneal macrophages is a pretranslational event.

Animals↗
PubMed1983

Phylogenetic conservation of a class III major histocompatibility complex antigen, factor B. Isolation and nucleotide sequencing of mouse factor B cDNA clones.

The complement protein factor B is a novel serine protease which is encoded within the major histocompatibility complex in man, guinea pig, and mouse. To determine the structure of mouse factor B, cDNA clones were isolated from mouse strains of two different major histocompatibility complex haplotypes, H-2k and H-2d, and clones of 0.9 and 1.5 kilobases, respectively, were sequenced. The H-2d clone includes the H-2k clone sequence and spans 94% of the Bb-coding sequence. No differences in sequence or in restriction enzyme sites were observed between the H-2k and H-2d clones. The H-2d clone displays 83% nucleotide homology and 83% (derived) amino acid homology with that of human factor B; there are no insertions or deletions. Comparison of the mouse and human factor B sequence reveals extensive regional homology at the catalytic residues and in the NH2-terminal portion of the Bb fragment.

Amino Acid Sequence↗
PubMed1983

Isolation of human C-reactive protein complementary DNA and localization of the gene to chromosome 1.

With a synthetic oligonucleotide mixture as probe, complementary DNA clones of C-reactive protein were isolated from an adult human liver complementary DNA library. The clones ranged in size from 700 to 1100 base pairs and were identified by partial DNA sequence analysis. One complementary DNA clone was used as a probe for hybridization with human-rodent DNA's isolated from somatic cell hybrids and bound to nitrocellulose filters (Southern blot analysis) to assign the human C-reactive protein gene to chromosome 1.

Animals↗
PubMed1983

Use of a cDNA clone for the fourth component of human complement (C4) for analysis of a genetic deficiency of C4 in guinea pig.

A cDNA clone for the fourth complement component (C4), pC4AL1, has been isolated from a human adult liver cDNA library by using a synthetic oligonucleotide mixture containing all 384 possible sequences coding for residues 14-21 of the C4 gamma-chain amino acid sequence. This clone spans the entire C4 gamma-chain coding sequence and includes a short 3' untranslated region, a poly(A) recognition site, and 16 nucleotides of the poly(A) tail. The 5' end of the clone begins 18 nucleotides upstream from the amino terminus of the C4 gamma chain and codes for Arg-Asn-Arg-Arg-Arg-Arg, a highly charged proteolytic cleavage site involved in the processing of pro-C4 to native C4. Liver mRNA preparations from C4-deficient guinea pigs were incapable of directing synthesis of pro-C4 or C4 peptides in cell-free translation experiments. Southern blot analysis using pC4AL1 as a hybridization probe of C4-deficient guinea pig DNA established that the deficiency is not the result of deletion of the entire C4 gene. RNA blot analysis using pC4AL1 as a hybridization probe of normal guinea pig liver mRNA revealed a C4 mRNA of 5.0 kilobases (kb). No such mRNA species was observed in C4-deficient guinea pig liver mRNA; however, a 7.0-kb RNA was detected, indicating the presence of a C4 precursor RNA. These results suggest that the basis of C4 deficiency in the guinea pig is a post-transcriptional defect in the processing of C4 precursor RNA to mature C4 mRNA.

Amino Acid Sequence↗
PubMed1983

Molecular map of the murine S region.

Eighteen overlapping cosmid clones spanning 240 kilobases and encoding the gene for factor B and two genes related to the fourth component of complement (C4) were isolated from a murine H-2d genomic library. Cosmid clones were identified by hybridization to human cDNA probes for factor B and C4 and were linked by chromosomal walking procedures. The cluster of clones contains two regions with sequences homologous to the C4 cDNA probe, both in the same orientation, representing a direct duplication of at least 55 kilobases of chromosomal DNA, separated by a shorter (less than 25 kilobases) segment of nonduplicated DNA. Restriction fragment-length polymorphism seen by using C4 probes maps these sequences to the S region of the major histocompatibility complex. 5' to the two C4-like sequences is an approximately equal to 40-kilobase-long region of chromosomal DNA remarkable for its lack of restriction fragment-length polymorphism, containing sequences homologous to the human factor B cDNA probe. These experiments demonstrate that the structural gene for factor B is located in the S region of the murine major histocompatibility complex and that this region contains an extensive direct duplication that contains the structural gene for mouse C4 and, we presume, for the sex-limited protein variant, Slp. RNA transfer blot analysis of total liver RNA from high C4- and low C4-producing strains showed that steady-state levels of C4-hybridizing RNA were much greater in high C4-producing strains. Regulation of circulating C4 levels in high C4 and low C4 strains is at least partly at the level of mRNA transcription, processing, or degradation.

Animals↗
PubMed1983

Inhibition of phagocytosis of complement C3- or immunoglobulin G-coated particles and of C3bi binding by monoclonal antibodies to a monocyte-granulocyte membrane glycoprotein (Mol).

Events that lead to phagocytosis of complement (C3)- or IgG-coated particles after their interaction with specific cell surface receptors are poorly understood. Two mouse monoclonal antibodies (an IgM and an IgG2a) to a human granulocyte-monocyte surface membrane differentiation antigen (Mol) inhibited ingestion by granulocytes both of oil Red O particles opsonized with normal human serum or with IgG and of sheep erythrocytes sensitized with IgG. In addition, they specifically inhibited rosetting between phagocytes and sheep erythrocytes coated with C3bi, a fragment of the complement component C3, generated by cleaving C3b with C3b inactivator and beta IH protein. These monoclonal anti-Mol antibodies did not inhibit IgG Fc, C3b or C3d receptor-mediated binding of erythrocytes coated with the respective proteins. The Fab fragment of the IgG2a monoclonal antibody inhibited noncytotoxic enzyme release from granulocytes when these cells were stimulated with zymosan coated with C3bi. Electrophoretic transfer of polymorphonuclear leukocyte detergent lysates to nitrocellulose, followed by immunofixation with monoclonal antibody, showed that these antibodies were directed to a 155,000-mol wt glycoprotein. This surface membrane structure appears to be involved in Fc and C3 receptor-dependent phagocytosis and closely associated with the C3bi receptor.

Antibodies, Monoclonal↗