Biomedical subjects

D P Vik

Publications and source records attributed to D P Vik.

11 recordsLinked to original sources

PubMed1992

Complete structure of the murine C4b-binding protein gene and regulation of its expression by dexamethasone.

C4b-binding protein (C4BP) is involved in the fluid-phase regulation of the classical pathway of complement. A murine genomic library was screened, and five clones were selected that covered the remaining four exons in the 5'-region of the C4BP gene. Together with previous work (Barnum, S. R., Kristensen, T., Chaplin, D. D., Seldin, M. F., and Tack, B. F. (1989) Biochemistry 28, 8312-8317), the entire C4BP gene has now been shown to be 23 kilobases (kb) long and comprised of 10 exons ranging in size from 86 to 442 base pairs (bp). Primer extension analysis revealed the major transcription start site to be 46 bp upstream of the published cDNA start site. Northern blot analysis of RNA isolated from several mouse tissues demonstrated that the C4BP gene is expressed in a liver-specific manner. Several regions homologous to known response elements were identified upstream of the C4BP gene including a strong hepatocyte nuclear factor 1 binding site and four putative glucocorticoid response elements. Furthermore, dexamethasone increased C4BP mRNA and protein levels in the mouse liver cell line, NMuLi. The stimulation of C4BP gene expression was rapid and independent of protein synthesis. These results suggest dexamethasone induction of the C4BP gene is a primary response and therefore a transcriptional effect. Inhibition of the dexamethasone effect on C4BP by actinomycin D supports this theory. These studies also provide evidence that, for optimal induction of the C4BP gene, the glucocorticoid receptor complex may cooperatively interact with accessory transcription factors. It is likely that stimulation of C4BP gene expression by dexamethasone may allude to a mechanism by which glucocorticoids exert their anti-inflammatory effects.

Animals↗

PubMed1991

Structural aspects of the human C5 gene. Intron/exon organization, 5'-flanking region features, and characterization of two truncated cDNA clones.

Human C5 cDNA fragments were used to identify five overlapping cosmid clones that spanned the entire C5 gene. Partial sequencing and Southern analysis of the clones were performed to identify intron/exon boundaries and to map intron size. The human C5 gene is 79 kilobases in length and is comprised of 41 exons. Comparison of C5 with the homologous family members C3 and C4 revealed striking similarities in exon size and number. Less, although significant similarities were also observed with the family member alpha 2-macroglobulin. The transcriptional start site for the C5 gene was observed as a doublet at positions 29 and 28 nucleotides upstream of the ATG start codon. The 5'-flanking region of the gene contains sequences homologous with several known responsive elements, including interferon, interleukin-6, glucocorticoid, estrogen, NF-kappa B, and HNF-1. Two previously identified truncated cDNAs, pHC5A and pHC5B, contain 21 and 16 exons, respectively. The last exon in pHC5A, designated exon 21a, is a product of alternative splicing and is not present in the major full-length transcript. Truncation of pHC5A is the result of an alternative polyadenylation signal located in exon 21a. In pHC5B, exon 16 is extended on the 3' end by additional flanking genomic sequence that also contains an alternative polyadenylation signal.

Animals↗

PubMed1991

Structural features of the human C3 gene: intron/exon organization, transcriptional start site, and promoter region sequence.

The third component of human complement (C3) is a key molecule in the activation of the complement cascade. C3 cDNA fragments were used to identify seven cosmid clones that covered all but 1 kilobase pair (kb) of the C3 gene. The remainder of the gene was cloned by using the polymerase chain reaction. These clones were used to identify the intron/exon boundaries and to map the gene. The C3 gene is 42 kb in length and comprises 41 exons ranging in size from 52 to 213 base pairs (bp). The transcription start site was identified by primer extension, and approximately 1 kb of DNA upstream of this site was sequenced. Putative TATA and CAAT boxes were identified along with a number of regions that shared homology with known regulatory sequences. These include responsive elements for interferon-gamma, interleukin-6, nuclear factor kappa B, estrogen, glucocorticoids and thyroid hormone. Several of these agents have been shown to affect C3 synthesis and mRNA levels. The sizes of the exons in C3 were compared to those of C4 and alpha 2-macroglobulin (alpha 2M). Thirty-nine of 41 exons in C4 were found to be of similar size to the analogous ones in C3, and two-thirds of those in alpha 2M were also similarly sized, supporting the hypothesis that these genes arose from a common ancestor.

Amino Acid Sequence↗

PubMed1990

Mapping of a retinoic acid-responsive element in the promoter region of the complement factor H gene.

Retinoic acid receptors are members of the steroid/thyroid hormone receptor superfamily. Pursuant to the discovery that dexamethasone increases complement factor H expression, we examined the effects of retinoic acid on this gene. Both H mRNA and protein levels are increased by retinoic acid in L cells. Using the luciferase reporter gene system we have identified a region of the H promoter required for the retinoic acid response. This region contains an imperfect palindrome of the TGACC motif, present in thyroid hormone and estrogen-responsive elements. We demonstrate specific binding of the retinoic acid receptor beta to this sequence of the H gene by DNA-protein gel retardation assay. Therefore, these studies extend the sphere of influence of the retinoids to complement, an intrinsic component of the humoral immune system.

Animals↗

PubMed1990

Identification and sequence analysis of four complement factor H-related transcripts in mouse liver.

Mouse liver RNA analyzed by Northern blotting with a full-length complement factor H cDNA probe demonstrates the 4.4-kilobase (kb) H mRNA as well as three additional hybridizing species of 3.5, 2.8, and 1.8 kb, respectively. Further characterization of these alternative transcripts was pursued by isolation of additional cDNAs from a liver library using a full-length H probe. Twelve clones homologous to but distinct from H were isolated, analyzed by restriction mapping, and divided into four classes, A, B, C, and D, based on their sequences. Clones from classes A, B, and C all contained nearly identical 5'-untranslated regions and leader sequences that differed from H at more than 50% of their nucleotide positions. The 5'-untranslated and leader sequences of the class D clone were unrelated to the corresponding regions of H or the class A, B, or C clones. The remaining portions of the H-related cDNAs were made up of short consensus repeats, 7 in class A, 4 in class B, 13 in class C, and 5 in class D. To determine the relationship between the H-related transcripts and the cDNA clones, Northern blots of liver RNA were analyzed by hybridization with two probes, one specific for the class D cDNA and the other reacting specifically with the class A, B, and C cDNAs. The class A/B/C probe detected transcripts of 3.5, 2.8, and 1.8 kb in liver RNA, and the class D probe hybridized to a distinct 1.8-kb message. Additionally, a cosmid genomic library was screened with H cDNA, and nine H-related clones were isolated. They spanned a region of approximately 120 kb, defining at least two discrete H-related gene loci. These results identify new members of the super-family of C3b/C4b binding protein genes.

Amino Acid Sequence↗

PubMed1990

Factor H.

While the mouse and human H proteins are structurally and functionally similar, they differ in their genetics. Whereas there is no evidence in humans for more than one gene; in mice the H locus is complex. Based on cDNA sequence and hybridization analysis of genomic cosmid clones, there are at least three distinct genes, all highly related to one another. The consensus repeating unit that comprises this molecule has obviously been duplicated numerous times, since it is present in many other molecules. Thus, it is not surprising to discover that there are several genes related to H in the mouse. A similar case has been described for two other members of this family. In humans, CR1 cDNA hybridizes to two distinct genomic clusters in the CR1 locus (Wong et al. 1989), and in mice, mCRY hybridizes to two regions in the genome, one on chromosome 1 and another on chromosome 8 (Aegerter-Shaw et al. 1987). It will be of interest to see if any other members of this family display as complex a genetic locus as murine H.

Animals↗

PubMed1989

Analysis of complement factor H mRNA expression: dexamethasone and IFN-gamma increase the level of H in L cells.

Murine complement protein H is encoded by a 100-kb gene on chromosome 1. A 3.2-kb fragment of the 5' flanking region of the H gene was sequenced, and two transcription start sites for this gene were identified by RNase protection and S1 nuclease analyses, each of which had upstream TATA and CAAT boxes. This region shares sequence homology with known regulatory elements, including the SV40 enhancer consensus, the Sp1 binding site, and two glucocorticoid-responsive core elements (GRE). Tissue and cell-line specificity has been examined by Northern analysis, and the 4.4-kb full-length H messenger RNA was identified in liver, kidney, spleen, thymus, liver cell line 1469, and L cells. IFN-gamma did not induce H mRNA expression in the macrophage cell line P388D.1 but had a positive effect on both the mRNA and protein levels of H in L cells. PMA, LPS, and vitamin D did not increase H mRNA levels in L cells. Pursuant to the discovery of two GRE in the 5' regulatory region of the H gene, we examined the effects of glucocorticoids on H mRNA expression. Dexamethasone (10(-7) M) was found to increase markedly the levels of H mRNA and protein after 24 h of incubation, and the effect on the mRNA was detectable by 30 min. The fact that H is a down-regulator of complement activation is consistent with the known immunosuppressive role of glucocorticoids. To our knowledge, this is the first time that dexamethasone has been shown to increase the levels of a complement protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Genetic organization of complement receptor-related genes in the mouse.

Using an interspecific cross, gene linkage relationships among members of the murine complement receptor-related genes, C4bp, Cfh, Mcry, and Mcr2, were analyzed by segregation of RFLP in 200 mice. The human homologues of these genes are tightly linked, composing the RCA locus, which maps to human chromosome (Chr.)1q32, within a large linkage group conserved between human Chr.1q21-32 and mouse Chr.1. RFLP associated with C4bp and Cfh map within this conserved linkage group; Cfh is located 9 cM telomeric to C4bp, which is consistent with linkage data for their human homologues. Mcry and Mcr2, while tightly linked, are located outside the conserved group, 40 cM telomeric to C4bp. These data suggest that a translocation or inversion occurred within the RCA family during the evolution of the mouse, defining a breakpoint of this large conserved linkage group.

Animals↗

PubMed1988

Structure of the murine complement factor H gene.

Factor H is a regulatory protein of the alternative pathway of complement activation comprised of 20 tandem repeating units of 60 amino acids each. A factor H cDNA clone was used to identify 17 genomic clones from a cosmid library. Four clones were selected for analysis of intron/exon junctions and 5' and 3' regions of the gene and for mapping of the exons. The factor H gene was found to be comprised of 22 exons. Each repeating unit is encoded by one exon, except the second repeat, which is coded by two exons; the leader sequence is encoded by a separate exon. The exons range in size from 77 to 210 base pairs (bp) and average 178 bp. They span a region of approximately 100 kilobases (kb) on chromosome 1. The leader sequence exon is 26 kb upstream of the first repeat exon, representing the largest intron. The other introns range in size from 86 bp to 12.9 kb, and the average intron size is 4.7 kb. Analysis of the genomic organization of the factor H gene has provided insight into the protein structure and will enable the construction of deletion mutants for functional studies.

Animals↗

PubMed1987

Cellular distribution of complement receptor type 4 (CR4): expression on human platelets.

Neutrophils have been shown to express a receptor for C3dg that is distinct from CR2 and is termed complement receptor type 4 (CR4). In the present study, other peripheral blood cell types were examined by indirect immunofluorescence and flow cytometry for the presence of C3dg binding activity. Specific uptake of C3dg occurred with neutrophils, platelets, and B lymphocytes, but not with eosinophils or T lymphocytes. Monocytes, contained within a mixed cell population of peripheral blood mononuclear cells and platelets, also bound C3dg, whereas purified monocytes did not. Binding of 125I-labeled glutaraldehyde-cross-linked C3dg to platelets was saturable, with an average of 1940 C3dg molecules bound per platelet at saturation (n = 8), ranging in number from 660 to 3930 molecules bound. Activation of platelets with thrombin did not consistently cause an increase in the expression of CR4 sites. 125I-C3dg binding to platelets was competitively inhibited equally well by unlabeled C3dg and iC3b, and approximately fourfold less well by C3b. The addition of platelets to elutriated monocytes generated C3dg binding activity on these cells by the formation of platelet-monocyte complexes. Thus, the CR4 on platelets accounted for the C3dg binding activity initially observed with partially purified monocytes. The adherent property of platelets may enable them to confer on certain other cell types the ability to localize C3dg-coated immune complexes or particles.

Blood Platelets↗

PubMed1985

Neutrophils express a receptor for iC3b, C3dg, and C3d that is distinct from CR1, CR2, and CR3.

In the present study we examined human neutrophils for the expression of a receptor capable of binding C3dg and defined the relationship of this receptor to those that have been previously described, namely CR1, CR2, and CR3. C3dg was isolated from serum depleted of plasminogen, supplemented with 20 mM Mg++, and incubated at 37 degrees C for 6 to 8 days. The purified protein was homogeneous when analyzed by polyacrylamide gel electrophoresis and exhibited an apparent m.w. of 41,000. C3dg was polymerized by treatment with dimethyl suberimidate, and the dimer was isolated by gel filtration. Binding of both monomeric and dimeric 125I-labeled C3dg to neutrophils was saturable, and the latter ligand bound to an average of 12,400 sites/cell among nine normal individuals. At 4 degrees C, bound monomeric C3dg dissociated from neutrophils with an average t1/2 of 30 min, whereas dimeric C3dg dissociated with a t1/2 in excess of 120 min. Specific binding of multimeric C3dg was cation independent and was competitively inhibited by molar concentrations of iC3b and C3d that were equivalent to the inhibitory concentrations of unlabeled C3dg; C3b was less able to compete with C3dg for binding to these sites. The capacity of this neutrophil receptor to bind iC3b, C3dg, and C3d suggested its possible identity as CR2 or CR3. However, no specific binding to neutrophils of 125I-labeled HB-5 monoclonal anti-CR2 was detected. Furthermore, uptake of 125I-labeled C3dg was not inhibited by saturating concentrations of rabbit anti-CR1, anti-Mac-1, or OKM10. Thus, a receptor resides on neutrophils that binds the C3d region of iC3b and C3dg and is distinct from CR1, CR2, and CR3.

Animals↗